Similarly, as a strategy for repressing innate immunity, *Shigella flexneri* has evolved the capacity to precisely modulate host cell epigenetic "information", interfering with MAPKs and NF-κB pathways at several points. This is mainly drived by the effector protein OspF. OspF is remarkable not only for its biochemistry but also for the fact that is one of the few bacterial effectors that is known to translocate to the host-cell nucleus. At the cytosol level, the protein binds to the ubiquitylated form of the E2 ubiquitin-conjugating enzyme UBCH5B, and independently of IκB phosphorylation, prevents the transfer of ubiquitin to IκB by an E3 ubiquitin–protein ligase (Kim et al., 2005). Additionally, OspF dephosphorylates ERK and p38 MAPKs by either phosphatase (Arbibe et al., 2007) or phosphothreonine lyase (Li et al., 2007) activities. Recent data showed that this protein also manipulates the physical and spatial context of DNA encoding NF-κB-responsive genes (Arbibe et al., 2007). At the hostcell nucleus, OspF dephosphorylates the MAPK ERK2, impairing the activation of mitogenand stress-activated kinase 1 (MSK1) and MSK2. This prevents subsequent histone phosphorylation, which is necessary for NF-κB-dependent transcription. Therefore, several innate immune-related genes under control of NF-κB remain silent, allowing *S. flexneri* to

The mechanisms used by microorganisms to modulate NF-κB signaling are diverse and, as exemplified above, a common strategy is to target the steps that lead to IκB degradation. However, several pathogens, such *Toxoplasma gondii* and *Leishmania spp* have evolved distinct processes to block this central signaling pathway. Infection by *T. gondii* provides potent signals for IL-12 production and for induction of strong Th1 immunity, being NF-κB an important player in this process (Caamano & Hunter, 2002). However, at early times of infection (up to 24h) the parasite impairs in macrophages, the NF-κB signaling, limiting the production of IL-12, TNF-α and NO. This blockage was shown to occur independently of infection-induced IKK-dependent degradation of IκB-α, resulting in specific impairment of NF-κB nuclear translocation. The termination of NF-κB signaling was therefore associated with reduced phosphorylation of p65/RelA subunit, an event involved in the ability of NFκB to translocate to the nucleus and to bind DNA (Shapira et al., 2005).

Regarding *Leishmania*, the infection by this protozoan parasite has long been regarded as the paradigm of a Th2 immune response. Extensive studies have been conducted to disclose the molecular mechanism by which *Leishmania* modulate intracellular signaling events in infected macrophages and dendritic cells. Obtained data indicate that the parasite use an extensive "arsenal" of strategies and virulence factors to alter the host cell signaling, favoring its survival. Infection of macrophages with *L. donovani* promastigotes was shown to increase intracellular ceramide content causing a downregulation of classical PKC activity, up-regulation of calcium independent atypical PKC-zeta and dephosphorylation of ERK. Downregulation of ERK signaling was subsequently found to be associated with the inhibition of activated protein 1 (AP-1) and NF-κB transactivation (Ghosh et al., 2002). Other studies whit the same infection model showed that *Leishmania* alters signal transduction upstream of c-Fos and c-Jun, by inhibiting ERK, JNK and p38 MAP Kinases, resulting in a reduction of AP-1 nuclear translocation (Prive & Descoteaux, 2000). Until recently, little was known about the intervenients and molecular mechanisms behind these immunosuppressive abilities of *Leishmania*. In macrophages infected with *Leishmania mexicana* amastigotes, Cameron and co-workers showed that cysteine peptidase B (CPB) is the virulence factor responsible for proteolytic degradation of NF-κB, ERK and JNK (Cameron et al., 2004). Additionally, CPB is also involved in the activation of host protein tyrosine phosphatase 1B (PTP-1B), inhibition of AP-1 and cleavage of STAT-1α (Abu-Dayyeh et al., 2010). Another *Leishmania* virulence factor, the surface metalloprotease GP63, was shown to cleave host protein tyrosine phosphatases PTP-1B, TCPTP, and SHP-1, resulting in the stimulation of their phosphatase activity and consequent dephosphorylation of key kinases, such as JAK/STAT, IRAK-1 and MAPKs (Gomez et al., 2009). Moreover, GP63 is also responsible for the observed cleavage of NF-κB p65RelA subunit in *L. mexicana* and *L.infantum* -infected macrophages and dendritic cells (Gregory et al., 2008; Neves et al., 2010). From this cleavage results a fragment of approximately 35 kDa that is rapidly translocated into the nucleus where it has some transcriptional activity. It was postulated that the resulting p35RelA fragment may represent an important mediator by which *Leishmania* promastigotes induce several chemokines without inducing other NF-κB-regulated genes, such as iNOS and IL-12 that are detrimental for parasite survival.

Recently, the metalloprotease GP63 was shown to be involved in the decreased general translation observed in macrophages infected with *L.major* (Jaramillo et al., 2011). The parasite protease cleaves the serine/threonine kinase mammalian/ mechanistic target of rapamycin (mTOR), impairing the formation of mTOR complex 1 (mTORC1) and the downstream phosphorylation of translational repressor 4E-binding protein 1/2 (4E-BP1/2). The activity of the translational repressors 4E-BPs is controlled through their phosphorylation state and, in normal conditions, mTORC1 formation leads to hyperphosphorylation of 4E-binding proteins (4E-BPs), causing their dissociation from eukaryotic initiation factor 4F, facilitating this way the translation of mRNA (Gingras et al., 1999). mTORC1, through its downstream targets p70 ribosomal S6 protein kinases 1 and 2 (S6K1/2) and 4E-BPs controls the translation of key innate immune effector molecules, such as type I IFN (Cao et al., 2008; Costa-Mattioli &

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 149

used by the virus to control PPRs signaling, providing a molecular basis for viral persistence. In viral infections, recognition of pathogen associated molecular patterns by TLRs and RLRs leads, through independent signaling cascades, to the activation of transcription factors, such as IRF1, IRF3, IRF5, IRF7 and NF-κB. The activity of these transcription factors is crucial for an effective antiviral innate immune response, given that they control the expression of interferon-stimulated genes (ISGs) and type I interferons. Hepatitis C virus (HCV) has evolved to disrupt RLRs signaling by impairing RIG-I pathway, through NS3/4A-mediated cleavage of IPS-1 (Malmgaard, 2004). NS3/4A is formed by a complex of the NS3 and NS4A HCV proteins and has been shown to be an essential viral protein with serine protease activity (Brass et al., 2008). In HCV infection, cleavage of IPS-1 by NS3/4A impairs downstream activation of IRF-3 and NF-κB, blocking the production of IFN-β, as well as the expression of ISGs (Li et al., 2005). This results in a strongly compromised

Millenary host–microbe co-evolution has resulted in the development of ingenious strategies by pathogens in order to successfully evade host immune response. Besides the manipulation of host-cell cytoskeleton to gain entry and/or to gain motility in the cell, immune-cell signaling pathways are frequent targets of invading pathogens. Within the past decades, remarkable progress has been made in our understanding on how immune cells sense microorganisms and how microbial effectors counteract innate immune responses. Recognition of conserved microorganism patterns by PRRs activates, in immune cells, an intricate signaling network that culminates in the expression of effector molecules, such as cytokines, chemokines and reactive oxygen species, crucial elements to mount an adequate immune response. A common strategy of pathogens is to disrupt these signaling cascades, by promoting contradictory signals through engagement of distinct PRRs and/or by directly target intermediate components of these signaling pathways. Therefore, understanding the molecular mechanisms used by pathogens to exploit the host signaling networks is of crucial importance for the development of rational interventions in which host response will

Abu-Dayyeh, I., K. Hassani, E. R. Westra, J. C. Mottram & M. Olivier (2010). "Comparative

Ahren, I. L., E. Eriksson, A. Egesten & K. Riesbeck (2003). "Nontypeable Haemophilus

Akira, S. (2009). "Pathogen recognition by innate immunity and its signaling." *Proc Jpn Acad*

Akira, S., K. Takeda & T. Kaisho (2001). "Toll-like receptors: critical proteins linking innate

*Cell Mol Biol* Vol.29, No 5, (Nov), pp. 598-605, ISSN 1044-1549

*Ser B Phys Biol Sci* Vol.85, No 4, 143-56, ISSN 1349-2896

study of the ability of Leishmania mexicana promastigotes and amastigotes to alter macrophage signaling and functions." *Infect Immun* Vol.78, No 6, (Jun), pp. 2438-45,

influenzae activates human eosinophils through beta-glucan receptors." *Am J Respir*

and acquired immunity." *Nat Immunol* Vol.2, No 8, (Aug), pp. 675-80, ISSN 1529-

innate immune response, potentiating the propagation of chronic HCV infection.

**4. Conclusions**

**5. References**

be redirect to achieve protective immunity.

ISSN 1098-5522

2908

Sonenberg, 2008). This cleavage of mTOR by Leishamania GP63 represents, therefore, a survival mechanism where the parasite directly targets the host translational machinery. This strategy is also a common feature of several human viruses. Lytic viruses, such members of the picornavirus group (enterovirus, rhinovirus and aphtovirus) inhibit overall host cellular translation, redirecting the translational apparatus to viral protein synthesis. This effect was shown to be due to the poliovirus 2A protease-mediated cleavage of the translation initiation factor eIF4G (Borman et al., 1997).

*Bacillus anthracis*, *Chlamydia* and *Escherichia coli* are examples of other human pathogens that directly cleave intermediate molecules from NF-κB and MAPKs signaling cascades. *Bacillus anthracis*, a spore-forming encapsulated gram-positive bacterium kwon to cause anthrax disease, produces innumerous virulence factors critical for the establishment of infection and pathogenesis (Turnbull, 2002). Among these factors, the plasmid-encoded enzymes lethal factor (LF) and oedema factor (OF) are of major importance for the evasion abilities of *B. anthracis*. LF is a particularly selective metalloproteinase that cleaves MKKs at specific sites outside of their catalytic domains, impairing the downstream MAPK activation (Duesbery et al., 1998). In addition, it blocks the p38 MAPK-dependent activation of IRF3 (Dang et al., 2004) and, although not directly affecting NF-κB activity, it causes the downregulation of NF-κB target genes that simultaneously require p38 activity for induction (Park et al., 2002). Consequently, macrophage production of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, is severely impaired. In turn, OF is an active Ca2+ and calmodulin-dependent adenylate cyclase that increases cAMP in the cytosol of host cells (Drum et al., 2002). Raised intracellular levels of cAMP activate PKA, causing downstream inhibition of ERK and JNK pathways, as well as a decreased NADPH oxidase activity, resulting in impaired TNF-α and microbicidal superoxide production (Hoover et al., 1994).

The obligate intracellular bacterial parasite *Chlamydia* is the leading cause of preventable blindness worldwide and urogenital tract infection remains the most prevalent cause of sexually transmitted diseases in developed countries. The parasite avoids host inflammatory response, partially by disrupting the NF-κB signal resultant from the PPR recognition of bacterial component such LPS. This blockage was shown to result from the selective cleavage of the p65RelA subunit of NF-κB by the chlamydial protease-like activity factor (CPAF) (Christian et al., 2010). Similarly, *E.coli* decreases production of proinflammatory cytokines and reduces macrophage bactericidal activity, by targeting NF-κB signal transduction at multiple points. Infection by *E coli* induces a host caspase 3-mediated cleavage of p65RelA, by a mechanism not completely defined, but thought to be mediated through the mitochondrial pathway of apoptosis (Albee & Perlman, 2006). In addition, several studies have recently demonstrated that *E. coli* also downregulates NF-κB-mediated gene expression by injecting into host-cells several non-LEE encoded (Nle) effector proteins, such as NleB, NleC and NleE. NleB and NleE prevent IKKβ activation and consequently the degradation of IκB-α, thus limiting p65 translocation to the nucleus (Nadler et al., 2010; Newton et al., 2010) while the zinc-dependent metalloprotease NleC was shown to enzymatically degrade p65RelA and JNK (Yen et al., ; Baruch et al., 2010).

Although more frequent in bacteria, the shutdown of PPR signaling by direct cleavage of cascade intermediates is also a strategy used by some relevant human viral pathogens. As an example, hepatitis C virus-host interactions have revealed several evasion mechanisms used by the virus to control PPRs signaling, providing a molecular basis for viral persistence. In viral infections, recognition of pathogen associated molecular patterns by TLRs and RLRs leads, through independent signaling cascades, to the activation of transcription factors, such as IRF1, IRF3, IRF5, IRF7 and NF-κB. The activity of these transcription factors is crucial for an effective antiviral innate immune response, given that they control the expression of interferon-stimulated genes (ISGs) and type I interferons. Hepatitis C virus (HCV) has evolved to disrupt RLRs signaling by impairing RIG-I pathway, through NS3/4A-mediated cleavage of IPS-1 (Malmgaard, 2004). NS3/4A is formed by a complex of the NS3 and NS4A HCV proteins and has been shown to be an essential viral protein with serine protease activity (Brass et al., 2008). In HCV infection, cleavage of IPS-1 by NS3/4A impairs downstream activation of IRF-3 and NF-κB, blocking the production of IFN-β, as well as the expression of ISGs (Li et al., 2005). This results in a strongly compromised innate immune response, potentiating the propagation of chronic HCV infection.

#### **4. Conclusions**

148 Protein Kinases

Sonenberg, 2008). This cleavage of mTOR by Leishamania GP63 represents, therefore, a survival mechanism where the parasite directly targets the host translational machinery. This strategy is also a common feature of several human viruses. Lytic viruses, such members of the picornavirus group (enterovirus, rhinovirus and aphtovirus) inhibit overall host cellular translation, redirecting the translational apparatus to viral protein synthesis. This effect was shown to be due to the poliovirus 2A protease-mediated cleavage of the translation initiation

*Bacillus anthracis*, *Chlamydia* and *Escherichia coli* are examples of other human pathogens that directly cleave intermediate molecules from NF-κB and MAPKs signaling cascades. *Bacillus anthracis*, a spore-forming encapsulated gram-positive bacterium kwon to cause anthrax disease, produces innumerous virulence factors critical for the establishment of infection and pathogenesis (Turnbull, 2002). Among these factors, the plasmid-encoded enzymes lethal factor (LF) and oedema factor (OF) are of major importance for the evasion abilities of *B. anthracis*. LF is a particularly selective metalloproteinase that cleaves MKKs at specific sites outside of their catalytic domains, impairing the downstream MAPK activation (Duesbery et al., 1998). In addition, it blocks the p38 MAPK-dependent activation of IRF3 (Dang et al., 2004) and, although not directly affecting NF-κB activity, it causes the downregulation of NF-κB target genes that simultaneously require p38 activity for induction (Park et al., 2002). Consequently, macrophage production of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, is severely impaired. In turn, OF is an active Ca2+ and calmodulin-dependent adenylate cyclase that increases cAMP in the cytosol of host cells (Drum et al., 2002). Raised intracellular levels of cAMP activate PKA, causing downstream inhibition of ERK and JNK pathways, as well as a decreased NADPH oxidase activity, resulting in impaired TNF-α and microbicidal superoxide production (Hoover et al., 1994). The obligate intracellular bacterial parasite *Chlamydia* is the leading cause of preventable blindness worldwide and urogenital tract infection remains the most prevalent cause of sexually transmitted diseases in developed countries. The parasite avoids host inflammatory response, partially by disrupting the NF-κB signal resultant from the PPR recognition of bacterial component such LPS. This blockage was shown to result from the selective cleavage of the p65RelA subunit of NF-κB by the chlamydial protease-like activity factor (CPAF) (Christian et al., 2010). Similarly, *E.coli* decreases production of proinflammatory cytokines and reduces macrophage bactericidal activity, by targeting NF-κB signal transduction at multiple points. Infection by *E coli* induces a host caspase 3-mediated cleavage of p65RelA, by a mechanism not completely defined, but thought to be mediated through the mitochondrial pathway of apoptosis (Albee & Perlman, 2006). In addition, several studies have recently demonstrated that *E. coli* also downregulates NF-κB-mediated gene expression by injecting into host-cells several non-LEE encoded (Nle) effector proteins, such as NleB, NleC and NleE. NleB and NleE prevent IKKβ activation and consequently the degradation of IκB-α, thus limiting p65 translocation to the nucleus (Nadler et al., 2010; Newton et al., 2010) while the zinc-dependent metalloprotease NleC was shown to

enzymatically degrade p65RelA and JNK (Yen et al., ; Baruch et al., 2010).

Although more frequent in bacteria, the shutdown of PPR signaling by direct cleavage of cascade intermediates is also a strategy used by some relevant human viral pathogens. As an example, hepatitis C virus-host interactions have revealed several evasion mechanisms

factor eIF4G (Borman et al., 1997).

Millenary host–microbe co-evolution has resulted in the development of ingenious strategies by pathogens in order to successfully evade host immune response. Besides the manipulation of host-cell cytoskeleton to gain entry and/or to gain motility in the cell, immune-cell signaling pathways are frequent targets of invading pathogens. Within the past decades, remarkable progress has been made in our understanding on how immune cells sense microorganisms and how microbial effectors counteract innate immune responses. Recognition of conserved microorganism patterns by PRRs activates, in immune cells, an intricate signaling network that culminates in the expression of effector molecules, such as cytokines, chemokines and reactive oxygen species, crucial elements to mount an adequate immune response. A common strategy of pathogens is to disrupt these signaling cascades, by promoting contradictory signals through engagement of distinct PRRs and/or by directly target intermediate components of these signaling pathways. Therefore, understanding the molecular mechanisms used by pathogens to exploit the host signaling networks is of crucial importance for the development of rational interventions in which host response will be redirect to achieve protective immunity.

#### **5. References**

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 151

Bogoevska, V., A. Horst, B. Klampe, L. Lucka, C. Wagener & P. Nollau (2006). "CEACAM1,

Borman, A. M., R. Kirchweger, E. Ziegler, R. E. Rhoads, T. Skern & K. M. Kean (1997).

Brass, V., J. M. Berke, R. Montserret, H. E. Blum, F. Penin & D. Moradpour (2008).

Braun, L., B. Ghebrehiwet & P. Cossart (2000). "gC1q-R/p32, a C1q-binding protein, is a

Brown, G. D. (2006). "Dectin-1: a signalling non-TLR pattern-recognition receptor." *Nat Rev*

Brown, G. D. & S. Gordon (2001). "Immune recognition. A new receptor for beta-glucans."

Buchholz, K. R. & R. S. Stephens (2008). "The cytosolic pattern recognition receptor NOD1

Bugarcic, A., K. Hitchens, A. G. Beckhouse, C. A. Wells, R. B. Ashman & H. Blanchard

Cameron, P., A. McGachy, M. Anderson, A. Paul, G. H. Coombs, J. C. Mottram, J. Alexander

Cao, W., S. Manicassamy, H. Tang, S. P. Kasturi, A. Pirani, N. Murthy & B. Pulendran (2008).

Chamaillard, M., M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab, Y. Ogura, A.

Chen, G., M. H. Shaw, Y. G. Kim & G. Nunez (2009). "NOD-like receptors: role in innate

*Nat Immunol* Vol.9, No 10, (Oct), pp. 1157-64, ISSN 1529-2916

Vol.16, No 3, (Mar), pp. 197-209, ISSN 0959-6658

(Sep 23), pp. 14545-50, ISSN 1091-6490

3, (Jul), pp. 414-29, ISSN 0893-8512

(Jul), pp. 702-7, ISSN 1529-2908

304, ISSN 0022-1767

1553-4014

No 7, (Apr 3), pp. 1458-66, ISSN 0261-4189

*Immunol* Vol.6, No 1, (Jan), pp. 33-43, ISSN 1474-1733

*Immun* Vol.76, No 7, (Jul), pp. 3150-5, ISSN 1098-5522

*Nature* Vol.413, No 6851, (Sep 6), pp. 36-7, ISSN 0028-0836

186-96, ISSN 1355-8382

an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues." *Glycobiology*

"elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs." *RNA* Vol.3, No 2, (Feb), pp.

"Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex." *Proc Natl Acad Sci U S A* Vol.105, No 38,

receptor for the InlB invasion protein of Listeria monocytogenes." *EMBO J* Vol.19,

induces inflammatory interleukin-8 during Chlamydia trachomatis infection." *Infect*

(2008). "Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans." *Glycobiology* Vol.18, No 9, (Sep), pp. 679-85, ISSN 1460-2423 Caamano, J. & C. A. Hunter (2002). "NF-kappaB family of transcription factors: central

regulators of innate and adaptive immune functions." *Clin Microbiol Rev* Vol.15, No

& R. Plevin (2004). "Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-kappaB signaling pathway." *J Immunol* Vol.173, No 5, (Sep 1), pp. 3297-

"Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway."

Kawasaki, K. Fukase, S. Kusumoto, M. A. Valvano, S. J. Foster, T. W. Mak, G. Nunez & N. Inohara (2003). "An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid." *Nat Immunol* Vol.4, No 7,

immunity and inflammatory disease." *Annu Rev Pathol* Vol.4, No, 365-98, ISSN

Akira, S., S. Uematsu & O. Takeuchi (2006). "Pathogen recognition and innate immunity."

Albee, L. & H. Perlman (2006). "E. coli infection induces caspase dependent degradation of

Aoki, N., Y. Kimura, S. Kimura, T. Nagato, M. Azumi, H. Kobayashi, K. Sato & M. Tateno

lineage cells." *J Leukoc Biol* Vol.85, No 3, (Mar), pp. 508-17, ISSN 1938-3673 Arbibe, L., D. W. Kim, E. Batsche, T. Pedron, B. Mateescu, C. Muchardt, C. Parsot & P. J.

Bakker, A. B., E. Baker, G. R. Sutherland, J. H. Phillips & L. L. Lanier (1999). "Myeloid

Balboa, L., M. M. Romero, N. Yokobori, P. Schierloh, L. Geffner, J. I. Basile, R. M. Musella, E.

profile." *Immunol Cell Biol* Vol.88, No 7, (Oct), pp. 716-26, ISSN 1440-1711 Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran & K.

Banchereau, J. & R. M. Steinman (1998). "Dendritic cells and the control of immunity."

Barton, G. M. & J. C. Kagan (2009). "A cell biological view of Toll-like receptor function:

Baruch, K., L. Gur-Arie, C. Nadler, S. Koby, G. Yerushalmi, Y. Ben-Neriah, O. Yogev, E.

Bates, E. E., N. Fournier, E. Garcia, J. Valladeau, I. Durand, J. J. Pin, S. M. Zurawski, S. Patel,

*Nature* Vol.392, No 6673, (Mar 19), pp. 245-52, ISSN 0028-0836

*Exp Med* Vol.200, No 8, (Oct 18), pp. 979-90, ISSN 0022-1007

NF-kappaB and reduces the inflammatory response in macrophages." *Inflamm Res*

(2009). "Expression and functional role of MDL-1 (CLEC5A) in mouse myeloid

Sansonetti (2007). "An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses." *Nat Immunol* Vol.8, No 1, (Jan), pp. 47-56, ISSN 1529-2908 Ariizumi, K., G. L. Shen, S. Shikano, R. Ritter, 3rd, P. Zukas, D. Edelbaum, A. Morita & A.

Takashima (2000). "Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms." *J Biol Chem* Vol.275, No 16, (Apr

DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells." *Proc Natl Acad Sci U S A* Vol.96, No 17, (Aug 17), pp.

Abbate, S. de la Barrera, M. C. Sasiain & M. Aleman (2010). "Mycobacterium tuberculosis impairs dendritic cell response by altering CD1b, DC-SIGN and MR

Palucka (2000). "Immunobiology of dendritic cells." *Annu Rev Immunol* Vol.18, No,

regulation through compartmentalization." *Nat Rev Immunol* Vol.9, No 8, (Aug), pp.

Shaulian, C. Guttman, R. Zarivach & I. Rosenshine (2010). "Metalloprotease type III effectors that specifically cleave JNK and NF-kappaB." *EMBO J* Vol.30, No 1, (Jan

J. S. Abrams, S. Lebecque, P. Garrone & S. Saeland (1999). "APCs express DCIR, a novel C-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif." *J Immunol* Vol.163, No 4, (Aug 15), pp. 1973-83, ISSN 0022-1767 Bergman, M. P., A. Engering, H. H. Smits, S. J. van Vliet, A. A. van Bodegraven, H. P. Wirth,

M. L. Kapsenberg, C. M. Vandenbroucke-Grauls, Y. van Kooyk & B. J. Appelmelk (2004). "Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN." *J*

*Cell* Vol.124, No 4, (Feb 24), pp. 783-801, ISSN 0092-8674

Vol.55, No 1, (Jan), pp. 2-9, ISSN 1023-3830

21), pp. 11957-63, ISSN 0021-9258

9792-6, ISSN 0027-8424

767-811, ISSN 0732-0582

535-42, ISSN 1474-1741

5), pp. 221-31, ISSN 1460-2075

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 153

El Kasmi, K. C., J. E. Qualls, J. T. Pesce, A. M. Smith, R. W. Thompson, M. Henao-Tamayo, R.

Fenton, M. J. & M. W. Vermeulen (1996). "Immunopathology of tuberculosis: roles of

Fernandes, M. J., A. A. Finnegan, L. D. Siracusa, C. Brenner, N. N. Iscove & B. Calabretta

Franchi, L., N. Warner, K. Viani & G. Nunez (2009). "Function of Nod-like receptors in

Fratti, R. A., J. M. Backer, J. Gruenberg, S. Corvera & V. Deretic (2001). "Role of

Fratti, R. A., J. Chua, I. Vergne & V. Deretic (2003). "Mycobacterium tuberculosis

Gantner, B. N., R. M. Simmons, S. J. Canavera, S. Akira & D. M. Underhill (2003).

receptor 2." *J Exp Med* Vol.197, No 9, (May 5), pp. 1107-17, ISSN 0022-1007 Gazi, U. & L. Martinez-Pomares (2009). "Influence of the mannose receptor in host immune responses." *Immunobiology* Vol.214, No 7, (Jul), pp. 554-61, ISSN 1878-3279 Geijtenbeek, T. B., D. J. Krooshoop, D. A. Bleijs, S. J. van Vliet, G. C. van Duijnhoven, V.

Geijtenbeek, T. B., D. S. Kwon, R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, J. Middel,

Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, G. J. Adema, Y. van

*Acad Sci U S A* Vol.100, No 9, (Apr 29), pp. 5437-42, ISSN 0027-8424

cells." *Cancer Res* Vol.59, No 11, (Jun 1), pp. 2709-17, ISSN 0008-5472 Fitzgerald, K. A., E. M. Palsson-McDermott, A. G. Bowie, C. A. Jefferies, A. S. Mansell, G.

1399-406, ISSN 1529-2916

0019-9567

ISSN 0028-0836

28, ISSN 1600-065X

631-44, ISSN 0021-9525

353-7, ISSN 1529-2908

No 5, (Mar 3), pp. 575-85, ISSN 0092-8674

0092-8674

J. Basaraba, T. Konig, U. Schleicher, M. S. Koo, G. Kaplan, K. A. Fitzgerald, E. I. Tuomanen, I. M. Orme, T. D. Kanneganti, C. Bogdan, T. A. Wynn & P. J. Murray (2008). "Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens." *Nat Immunol* Vol.9, No 12, (Dec), pp.

macrophages and monocytes." *Infect Immun* Vol.64, No 3, (Mar), pp. 683-90, ISSN

(1999). "Characterization of a novel receptor that maps near the natural killer gene complex: demonstration of carbohydrate binding and expression in hematopoietic

Brady, E. Brint, A. Dunne, P. Gray, M. T. Harte, D. McMurray, D. E. Smith, J. E. Sims, T. A. Bird & L. A. O'Neill (2001). "Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction." *Nature* Vol.413, No 6851, (Sep 6), pp. 78-83,

microbial recognition and host defense." *Immunol Rev* Vol.227, No 1, (Jan), pp. 106-

phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest." *J Cell Biol* Vol.154, No 3, (Aug 6), pp.

glycosylated phosphatidylinositol causes phagosome maturation arrest." *Proc Natl*

"Collaborative induction of inflammatory responses by dectin-1 and Toll-like

Grabovsky, R. Alon, C. G. Figdor & Y. van Kooyk (2000). "DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking." *Nat Immunol* Vol.1, No 4, (Oct), pp.

I. L. Cornelissen, H. S. Nottet, V. N. KewalRamani, D. R. Littman, C. G. Figdor & Y. van Kooyk (2000). "DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells." *Cell* Vol.100, No 5, (Mar 3), pp. 587-97, ISSN

Kooyk & C. G. Figdor (2000). "Identification of DC-SIGN, a novel dendritic cellspecific ICAM-3 receptor that supports primary immune responses." *Cell* Vol.100,

Chen, S. T., Y. L. Lin, M. T. Huang, M. F. Wu, S. C. Cheng, H. Y. Lei, C. K. Lee, T. W. Chiou,

lethal disease." *Nature* Vol.453, No 7195, (May 29), pp. 672-6, ISSN 1476-4687 Christian, J., J. Vier, S. A. Paschen & G. Hacker (2010). "Cleavage of the NF-kappaB family

Costa-Mattioli, M. & N. Sonenberg (2008). "RAPping production of type I interferon in pDCs through mTOR." *Nat Immunol* Vol.9, No 10, (Oct), pp. 1097-9, ISSN 1529-2916 Dang, O., L. Navarro, K. Anderson & M. David (2004). "Cutting edge: anthrax lethal toxin

Demangel, C. & W. J. Britton (2000). "Interaction of dendritic cells with mycobacteria: where

Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart & Z. J.

Diebold, S. S. (2009). "Activation of dendritic cells by toll-like receptors and C-type lectins."

Dixit, E., S. Boulant, Y. Zhang, A. S. Lee, C. Odendall, B. Shum, N. Hacohen, Z. J. Chen, S. P.

Dolganiuc, A., S. Chang, K. Kodys, P. Mandrekar, G. Bakis, M. Cormier & G. Szabo (2006).

Drum, C. L., S. Z. Yan, J. Bard, Y. Q. Shen, D. Lu, S. Soelaiman, Z. Grabarek, A. Bohm & W. J.

Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Gunther, I.

East, L. & C. M. Isacke (2002). "The mannose receptor family." *Biochim Biophys Acta* Vol.1572,

*J Immunol* Vol.177, No 10, (Nov 15), pp. 6758-68, ISSN 0022-1767

No 53, (Dec 31), pp. 41320-7, ISSN 1083-351X

Vol.172, No 2, (Jan 15), pp. 747-51, ISSN 0022-1767

*Cell* Vol.103, No 2, (Oct 13), pp. 351-61, ISSN 0092-8674

*Handb Exp Pharmacol*, No 188, 3-30, ISSN 0171-2004

Vol.280, No 5364, (May 1), pp. 734-7, ISSN 0036-8075

12, (Dec 17), pp. 1823-34, ISSN 0022-1007

No 2-3, (Sep 19), pp. 364-86, ISSN 0006-3002

No 4, (May 14), pp. 668-81, ISSN 1097-4172

No 7, (Jul), pp. 1149-57, ISSN 1432-0851

C. H. Wong & S. L. Hsieh (2008). "CLEC5A is critical for dengue-virus-induced

protein p65/RelA by the chlamydial protease-like activity factor (CPAF) impairs proinflammatory signaling in cells infected with Chlamydiae." *J Biol Chem* Vol.285,

inhibits activation of IFN-regulatory factor 3 by lipopolysaccharide." *J Immunol*

the action starts." *Immunol Cell Biol* Vol.78, No 4, (Aug), pp. 318-24, ISSN 0818-9641 den Dunnen, J., S. I. Gringhuis & T. B. Geijtenbeek (2009). "Innate signaling by the C-type

lectin DC-SIGN dictates immune responses." *Cancer Immunol Immunother* Vol.58,

Chen (2000). "Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain."

Whelan, M. Fransen, M. L. Nibert, G. Superti-Furga & J. C. Kagan (2010). "Peroxisomes are signaling platforms for antiviral innate immunity." *Cell* Vol.141,

"Hepatitis C virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-alpha and plasmacytoid dendritic cell loss in chronic HCV infection."

Tang (2002). "Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin." *Nature* Vol.415, No 6870, (Jan 24), pp. 396-402, ISSN 0028-0836 Duesbery, N. S., C. P. Webb, S. H. Leppla, V. M. Gordon, K. R. Klimpel, T. D. Copeland, N.

G. Ahn, M. K. Oskarsson, K. Fukasawa, K. D. Paull & G. F. Vande Woude (1998). "Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor." *Science*

Johnston, A. Lanzavecchia, T. Nagasaka, T. Okada, W. Vermi, G. Winkels, T. Yamamoto, M. Zysk, Y. Yamaguchi & J. Schmitz (2001). "BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction." *J Exp Med* Vol.194, No

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 155

Hajishengallis, G. & J. D. Lambris (2011). "Microbial manipulation of receptor crosstalk in

Hajishengallis, G., M. A. Shakhatreh, M. Wang & S. Liang (2007). "Complement receptor 3

Hasegawa, M., Y. Fujimoto, P. C. Lucas, H. Nakano, K. Fukase, G. Nunez & N. Inohara

Henderson, R. A., S. C. Watkins & J. L. Flynn (1997). "Activation of human dendritic cells

Hmama, Z., K. Sendide, A. Talal, R. Garcia, K. Dobos & N. E. Reiner (2004). "Quantitative

Hodges, A., K. Sharrocks, M. Edelmann, D. Baban, A. Moris, O. Schwartz, H. Drakesmith, K.

Hoebe, K., E. Janssen & B. Beutler (2004). "The interface between innate and adaptive immunity." *Nat Immunol* Vol.5, No 10, (Oct), pp. 971-4, ISSN 1529-2908 Hoover, D. L., A. M. Friedlander, L. C. Rogers, I. K. Yoon, R. L. Warren & A. S. Cross (1994).

Horng, T., G. M. Barton & R. Medzhitov (2001). "TIRAP: an adapter molecule in the Toll signaling pathway." *Nat Immunol* Vol.2, No 9, (Sep), pp. 835-41, ISSN 1529-2908 Hovius, J. W., M. A. de Jong, J. den Dunnen, M. Litjens, E. Fikrig, T. van der Poll, S. I.

Inohara, N., T. Koseki, J. Lin, L. del Peso, P. C. Lucas, F. F. Chen, Y. Ogura & G. Nunez

*PLoS Pathog* Vol.4, No 2, (Feb 8), pp. e31, ISSN 1553-7374

Vol.22, No 4, (Apr), pp. 415-26, ISSN 1074-7613

15), pp. 635-43, ISSN 0022-1767

40, ISSN 0021-9533

ISSN 1529-2908

ISSN 0019-9567

ISSN 0021-9258

1741

0022-1767

innate immunity." *Nat Rev Immunol* Vol.11, No 3, (Mar), pp. 187-200, ISSN 1474-

blockade promotes IL-12-mediated clearance of Porphyromonas gingivalis and negates its virulence in vivo." *J Immunol* Vol.179, No 4, (Aug 15), pp. 2359-67, ISSN

(2008). "A critical role of RICK/RIP2 polyubiquitination in Nod-induced NFkappaB activation." *EMBO J* Vol.27, No 2, (Jan 23), pp. 373-83, ISSN 1460-2075 Hashimoto, C., K. L. Hudson & K. V. Anderson (1988). "The Toll gene of Drosophila,

required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein." *Cell* Vol.52, No 2, (Jan 29), pp. 269-79, ISSN 0092-8674 Hawlisch, H., Y. Belkaid, R. Baelder, D. Hildeman, C. Gerard & J. Kohl (2005). "C5a

negatively regulates toll-like receptor 4-induced immune responses." *Immunity*

following infection with Mycobacterium tuberculosis." *J Immunol* Vol.159, No 2, (Jul

analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3 phosphoinositide 3-kinase pathway." *J Cell Sci* Vol.117, No Pt 10, (Apr 15), pp. 2131-

Davies, B. Kessler, A. McMichael & A. Simmons (2007). "Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication." *Nat Immunol* Vol.8, No 6, (Jun), pp. 569-77,

"Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP." *Infect Immun* Vol.62, No 10, (Oct), pp. 4432-9,

Gringhuis & T. B. Geijtenbeek (2008). "Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization."

(2000). "An induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways." *J Biol Chem* Vol.275, No 36, (Sep 8), pp. 27823-31,

Geijtenbeek, T. B., S. J. Van Vliet, E. A. Koppel, M. Sanchez-Hernandez, C. M.

Ghosh, S., S. Bhattacharyya, M. Sirkar, G. S. Sa, T. Das, D. Majumdar, S. Roy & S. Majumdar

Gilchrist, M., V. Thorsson, B. Li, A. G. Rust, M. Korb, J. C. Roach, K. Kennedy, T. Hai, H.

Gingras, A. C., B. Raught & N. Sonenberg (1999). "eIF4 initiation factors: effectors of mRNA

Gomez, M. A., I. Contreras, M. Halle, M. L. Tremblay, R. W. McMaster & M. Olivier (2009).

Goodridge, H. S., R. M. Simmons & D. M. Underhill (2007). "Dectin-1 stimulation by

expression." *Eur J Immunol* Vol.38, No 4, (Apr), pp. 1071-81, ISSN 0014-2980 Gringhuis, S. I., J. den Dunnen, M. Litjens, M. van der Vlist & T. B. Geijtenbeek (2009).

Gringhuis, S. I., J. den Dunnen, M. Litjens, M. van der Vlist, B. Wevers, S. C. Bruijns & T. B.

Gringhuis, S. I., J. den Dunnen, M. Litjens, B. van Het Hof, Y. van Kooyk & T. B. Geijtenbeek

Gross, O., A. Gewies, K. Finger, M. Schafer, T. Sparwasser, C. Peschel, I. Forster & J. Ruland

host defence." *Nature* Vol.459, No 7245, (May 21), pp. 433-6, ISSN 1476-4687

immunity." *Nature* Vol.442, No 7103, (Aug 10), pp. 651-6, ISSN 1476-4687 Gross, O., H. Poeck, M. Bscheider, C. Dostert, N. Hannesschlager, S. Endres, G. Hartmann,

phosphatases." *Sci Signal* Vol.2, No 90, ra58, ISSN 1937-9145

*Immunol* Vol.10, No 10, (Oct), pp. 1081-8, ISSN 1529-2916

No 2, (Feb), pp. 203-13, ISSN 1529-2916

No 5, (May), pp. 605-16, ISSN 1074-7613

7-17, ISSN 0022-1007

38, ISSN 0019-9567

173-8, ISSN 1476-4687

No, 913-63, ISSN 0066-4154

Vandenbroucke-Grauls, B. Appelmelk & Y. Van Kooyk (2003). "Mycobacteria target DC-SIGN to suppress dendritic cell function." *J Exp Med* Vol.197, No 1, (Jan 6), pp.

(2002). "Leishmania donovani suppresses activated protein 1 and NF-kappaB activation in host macrophages via ceramide generation: involvement of extracellular signal-regulated kinase." *Infect Immun* Vol.70, No 12, (Dec), pp. 6828-

Bolouri & A. Aderem (2006). "Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4." *Nature* Vol.441, No 7090, (May 11), pp.

recruitment to ribosomes and regulators of translation." *Annu Rev Biochem* Vol.68,

"Leishmania GP63 alters host signaling through cleavage-activated protein tyrosine

Candida albicans yeast or zymosan triggers NFAT activation in macrophages and dendritic cells." *J Immunol* Vol.178, No 5, (Mar 1), pp. 3107-15, ISSN 0022-1767 Gregory, D. J., M. Godbout, I. Contreras, G. Forget & M. Olivier (2008). "A novel form of NF-

kappaB is induced by Leishmania infection: involvement in macrophage gene

"Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori." *Nat*

Geijtenbeek (2009). "Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk." *Nat Immunol* Vol.10,

(2007). "C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB." *Immunity* Vol.26,

(2006). "Card9 controls a non-TLR signalling pathway for innate anti-fungal

A. Tardivel, E. Schweighoffer, V. Tybulewicz, A. Mocsai, J. Tschopp & J. Ruland (2009). "Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 157

Kawai, T. & S. Akira (2010)."The role of pattern-recognition receptors in innate immunity:

Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii, O. Takeuchi & S.

Kim, D. W., G. Lenzen, A. L. Page, P. Legrain, P. J. Sansonetti & C. Parsot (2005). "The

Komuro, A. & C. M. Horvath (2006). "RNA- and virus-independent inhibition of antiviral

Kumar, H., T. Kawai & S. Akira (2011). "Pathogen recognition by the innate immune system." *Int Rev Immunol* Vol.30, No 1, (Feb), pp. 16-34, ISSN 1563-5244 Kumar, H., Y. Kumagai, T. Tsuchida, P. A. Koenig, T. Satoh, Z. Guo, M. H. Jang, T. Saitoh, S.

Lambert, A. A., C. Gilbert, M. Richard, A. D. Beaulieu & M. J. Tremblay (2008). "The C-type

Lamkanfi, M., T. D. Kanneganti, L. Franchi & G. Nunez (2007). "Caspase-1 inflammasomes

Le Negrate, G., B. Faustin, K. Welsh, M. Loeffler, M. Krajewska, P. Hasegawa, S. Mukherjee,

LeibundGut-Landmann, S., O. Gross, M. J. Robinson, F. Osorio, E. C. Slack, S. V. Tsoni, E.

Lemaitre, B., E. Nicolas, L. Michaut, J. M. Reichhart & J. A. Hoffmann (1996). "The

*J Immunol* Vol.180, No 7, (Apr 1), pp. 5045-56, ISSN 0022-1767

1529-2916

0022-538X

0741-5400

ISSN 1529-2908

ISSN 0092-8674

No 1, (Mar), pp. 335-52, ISSN 1600-065X

(Sep 27), pp. 14046-51, ISSN 0027-8424

12, (Dec 15), pp. 8061-7, ISSN 1550-6606

(Aug 15), pp. 1299-307, ISSN 1528-0020

update on Toll-like receptors." *Nat Immunol* Vol.11, No 5, (May), pp. 373-84, ISSN

Akira (2005). "IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction." *Nat Immunol* Vol.6, No 10, (Oct), pp. 981-8, ISSN 1529-2908 Kelly, E. K., L. Wang & L. B. Ivashkiv (2010). "Calcium-activated pathways and oxidative

burst mediate zymosan-induced signaling and IL-10 production in human macrophages." *J Immunol* Vol.184, No 10, (May 15), pp. 5545-52, ISSN 1550-6606 Kerrigan, A. M. & G. D. Brown (2010). "Syk-coupled C-type lectin receptors that mediate

cellular activation via single tyrosine based activation motifs." *Immunol Rev* Vol.234,

Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes." *Proc Natl Acad Sci U S A* Vol.102, No 39,

signaling by RNA helicase LGP2." *J Virol* Vol.80, No 24, (Dec), pp. 12332-42, ISSN

Akira & T. Kawai (2009). "Involvement of the NLRP3 inflammasome in innate and humoral adaptive immune responses to fungal beta-glucan." *J Immunol* Vol.183, No

lectin surface receptor DCIR acts as a new attachment factor for HIV-1 in dendritic cells and contributes to trans- and cis-infection pathways." *Blood* Vol.112, No 4,

in infection and inflammation." *J Leukoc Biol* Vol.82, No 2, (Aug), pp. 220-5, ISSN

K. Orth, S. Krajewski, A. Godzik, D. G. Guiney & J. C. Reed (2008). "Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses."

Schweighoffer, V. Tybulewicz, G. D. Brown, J. Ruland & C. Reis e Sousa (2007). "Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17." *Nat Immunol* Vol.8, No 6, (Jun), pp. 630-8,

dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults." *Cell* Vol.86, No 6, (Sep 20), pp. 973-83,

Inui, M., Y. Kikuchi, N. Aoki, S. Endo, T. Maeda, A. Sugahara-Tobinai, S. Fujimura, A.

Iwasaki, A. & R. Medzhitov (2004). "Toll-like receptor control of the adaptive immune responses." *Nat Immunol* Vol.5, No 10, (Oct), pp. 987-95, ISSN 1529-2908 Iwasaki, A. & R. Medzhitov (2010). "Regulation of adaptive immunity by the innate immune system." *Science* Vol.327, No 5963, (Jan 15), pp. 291-5, ISSN 1095-9203 Jahn, P. S., K. S. Zanker, J. Schmitz & A. Dzionek (2010). "BDCA-2 signaling inhibits TLR-9-

Jaramillo, M., M. A. Gomez, O. Larsson, M. T. Shio, I. Topisirovic, I. Contreras, R.

Jones, R. M., H. Wu, C. Wentworth, L. Luo, L. Collier-Hyams & A. S. Neish (2008).

Kanazawa, N., T. Okazaki, H. Nishimura, K. Tashiro, K. Inaba & Y. Miyachi (2002). "DCIR

biogenesis." *J Exp Med* Vol.202, No 7, (Oct 3), pp. 987-99, ISSN 0022-1007 Karp, C. L., M. Wysocka, L. M. Wahl, J. M. Ahearn, P. J. Cuomo, B. Sherry, G. Trinchieri &

measles virus." *Science* Vol.273, No 5272, (Jul 12), pp. 228-31, ISSN 0036-8075 Kato, H., O. Takeuchi, E. Mikamo-Satoh, R. Hirai, T. Kawai, K. Matsushita, A. Hiiragi, T. S.

Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. Uematsu, A. Jung,

13, (Dec 21), pp. 2879-88, ISSN 1540-9538

4, (Apr 21), pp. 331-41, ISSN 1934-6069

233-44, ISSN 1934-6069

ISSN 1540-9538

4), pp. 101-5, ISSN 1476-4687

*Cell Immunol* Vol.265, No 1, 15-22, ISSN 1090-2163

Nakamura, A. Kumanogoh, M. Colonna & T. Takai (2009). "Signal adaptor DAP10 associates with MDL-1 and triggers osteoclastogenesis in cooperation with DAP12." *Proc Natl Acad Sci U S A* Vol.106, No 12, (Mar 24), pp. 4816-21, ISSN 1091-6490 Ishikawa, E., T. Ishikawa, Y. S. Morita, K. Toyonaga, H. Yamada, O. Takeuchi, T. Kinoshita,

S. Akira, Y. Yoshikai & S. Yamasaki (2009). "Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle." *J Exp Med* Vol.206, No

agonist-induced plasmacytoid dendritic cell activation and antigen presentation."

Luxenburg, A. Rosenfeld, R. Colina, R. W. McMaster, M. Olivier, M. Costa-Mattioli & N. Sonenberg (2011). "Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection." *Cell Host Microbe* Vol.9, No

"Salmonella AvrA Coordinates Suppression of Host Immune and Apoptotic Defenses via JNK Pathway Blockade." *Cell Host Microbe* Vol.3, No 4, (Apr 17), pp.

acts as an inhibitory receptor depending on its immunoreceptor tyrosine-based inhibitory motif." *J Invest Dermatol* Vol.118, No 2, (Feb), pp. 261-6, ISSN 0022-202X Kang, P. B., A. K. Azad, J. B. Torrelles, T. M. Kaufman, A. Beharka, E. Tibesar, L. E.

DesJardin & L. S. Schlesinger (2005). "The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome

D. E. Griffin (1996). "Mechanism of suppression of cell-mediated immunity by

Dermody, T. Fujita & S. Akira (2008). "Length-dependent recognition of doublestranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5." *J Exp Med* Vol.205, No 7, (Jul 7), pp. 1601-10,

T. Kawai, K. J. Ishii, O. Yamaguchi, K. Otsu, T. Tsujimura, C. S. Koh, C. Reis e Sousa, Y. Matsuura, T. Fujita & S. Akira (2006). "Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses." *Nature* Vol.441, No 7089, (May

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 159

Meyer-Wentrup, F., A. Cambi, B. Joosten, M. W. Looman, I. J. de Vries, C. G. Figdor & G. J.

Mittal, R., S. Y. Peak-Chew & H. T. McMahon (2006). "Acetylation of MEK2 and I kappa B

Murli, S., R. O. Watson & J. E. Galan (2001). "Role of tyrosine kinases and the tyrosine

Nadler, C., K. Baruch, S. Kobi, E. Mills, G. Haviv, M. Farago, I. Alkalay, S. Bartfeld, T. F.

Netea, M. G., R. Sutmuller, C. Hermann, C. A. Van der Graaf, J. W. Van der Meer, J. H. van

Newton, H. J., J. S. Pearson, L. Badea, M. Kelly, M. Lucas, G. Holloway, K. M. Wagstaff, M.

Nguyen, T., B. Ghebrehiwet & E. I. Peerschke (2000). "Staphylococcus aureus protein A

with platelets." *Infect Immun* Vol.68, No 4, (Apr), pp. 2061-8, ISSN 0019-9567 Olynych, T. J., D. L. Jakeman & J. S. Marshall (2006). "Fungal zymosan induces leukotriene

*Allergy Clin Immunol* Vol.118, No 4, (Oct), pp. 837-43, ISSN 0091-6749 Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa & T. Seya (2003). "TICAM-1, an

induction." *Nat Immunol* Vol.4, No 2, (Feb), pp. 161-7, ISSN 1529-2908 Oshiumi, H., M. Sasai, K. Shida, T. Fujita, M. Matsumoto & T. Seya (2003). "TIR-containing

*Immunol* Vol.38, No 12, (Dec), pp. 3274-81, ISSN 1521-4141

Osorio, F., S. LeibundGut-Landmann, M. Lochner, K. Lahl, T. Sparwasser, G. Eberl & C. Reis

*U S A* Vol.103, No 49, (Dec 5), pp. 18574-9, ISSN 0027-8424

Vol.3, No 12, (Dec), pp. 795-810, ISSN 1462-5814

(Dec), pp. 2898-911, ISSN 1525-2191

e1000898, ISSN 1553-7374

49751-62, ISSN 0021-9258

1938-3673

1553-7374

Adema (2009). "DCIR is endocytosed into human dendritic cells and inhibits TLR8 mediated cytokine production." *J Leukoc Biol* Vol.85, No 3, (Mar), pp. 518-25, ISSN

kinase (IKK) activation loop residues by YopJ inhibits signaling." *Proc Natl Acad Sci*

phosphatase SptP in the interaction of Salmonella with host cells." *Cell Microbiol*

Meyer, Y. Ben-Neriah & I. Rosenshine (2010). "The type III secretion effector NleE inhibits NF-kappaB activation." *PLoS Pathog* Vol.6, No 1, (Jan), pp. e1000743, ISSN

Krieken, T. Hartung, G. Adema & B. J. Kullberg (2004). "Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells." *J Immunol* Vol.172, No 6, (Mar 15), pp. 3712-8, ISSN 0022-1767 Neves, B. M., R. Silvestre, M. Resende, A. Ouaissi, J. Cunha, J. Tavares, I. Loureiro, N.

Santarem, A. M. Silva, M. C. Lopes, M. T. Cruz & A. Cordeiro da Silva (2010). "Activation of phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum-infected dendritic cells." *Am J Pathol* Vol.177, No 6,

A. Dunstone, J. Sloan, J. C. Whisstock, J. B. Kaper, R. M. Robins-Browne, D. A. Jans, G. Frankel, A. D. Phillips, B. S. Coulson & E. L. Hartland (2010). "The type III effectors NleE and NleB from enteropathogenic E. coli and OspZ from Shigella block nuclear translocation of NF-kappaB p65." *PLoS Pathog* Vol.6, No 5, (May), pp.

recognizes platelet gC1qR/p33: a novel mechanism for staphylococcal interactions

production by human mast cells through a dectin-1-dependent mechanism." *J*

adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta

adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta." *J Biol Chem* Vol.278, No 50, (Dec 12), pp.

e Sousa (2008). "DC activated via dectin-1 convert Treg into IL-17 producers." *Eur J*

Li, H., H. Xu, Y. Zhou, J. Zhang, C. Long, S. Li, S. Chen, J. M. Zhou & F. Shao (2007). "The

Li, X. D., L. Sun, R. B. Seth, G. Pineda & Z. J. Chen (2005). "Hepatitis C virus protease

Lin, S. L., T. X. Le & D. S. Cowen (2003). "SptP, a Salmonella typhimurium type III-secreted

Malik, Z. A., C. R. Thompson, S. Hashimi, B. Porter, S. S. Iyer & D. J. Kusner (2003). "Cutting

Malmgaard, L. (2004). "Induction and regulation of IFNs during viral infections." *J Interferon*

Marth, T. & B. L. Kelsall (1997). "Regulation of interleukin-12 by complement receptor 3 signaling." *J Exp Med* Vol.185, No 11, (Jun 2), pp. 1987-95, ISSN 0022-1007 Masumoto, J., K. Yang, S. Varambally, M. Hasegawa, S. A. Tomlins, S. Qiu, Y. Fujimoto, A.

Mazurkiewicz, P., J. Thomas, J. A. Thompson, M. Liu, L. Arbibe, P. Sansonetti & D. W.

McDonald, C., N. Inohara & G. Nunez (2005). "Peptidoglycan signaling in innate immunity

McGettrick, A. F. & L. A. O'Neill "Localisation and trafficking of Toll-like receptors: an

McGhie, E. J., L. C. Brawn, P. J. Hume, D. Humphreys & V. Koronakis (2009). "Salmonella

Medzhitov, R. (2007). "Recognition of microorganisms and activation of the immune response." *Nature* Vol.449, No 7164, (Oct 18), pp. 819-26, ISSN 1476-4687 Meyer-Wentrup, F., D. Benitez-Ribas, P. J. Tacken, C. J. Punt, C. G. Figdor, I. J. de Vries & G.

*Immunol* Vol.170, No 6, (Mar 15), pp. 2811-5, ISSN 0022-1767

*Cytokine Res* Vol.24, No 8, (Aug), pp. 439-54, ISSN 1079-9907

activation." *Cell Microbiol* Vol.5, No 4, (Apr), pp. 267-75, ISSN 1462-5814 Lu, C., H. Xu, C. T. Ranjith-Kumar, M. T. Brooks, T. Y. Hou, F. Hu, A. B. Herr, R. K. Strong,

Vol.315, No 5814, (Feb 16), pp. 1000-3, ISSN 1095-9203

17717-22, ISSN 0027-8424

pp. 1032-43, ISSN 1878-4186

pp. 203-13, ISSN 0022-1007

1371-83, ISSN 1365-2958

No 1, (Feb), pp. 117-24, ISSN 1879-0364

15), pp. 4245-53, ISSN 0006-4971

0021-9258

ISSN 1879-0372

phosphothreonine lyase activity of a bacterial type III effector family." *Science*

NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity." *Proc Natl Acad Sci U S A* Vol.102, No 49, (Dec 6), pp.

protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf

C. C. Kao & P. Li (2010). "The structural basis of 5' triphosphate double-stranded RNA recognition by RIG-I C-terminal domain." *Structure* Vol.18, No 8, (Aug 11),

edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase." *J*

Kawasaki, S. J. Foster, Y. Horie, T. W. Mak, G. Nunez, A. M. Chinnaiyan, K. Fukase & N. Inohara (2006). "Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo." *J Exp Med* Vol.203, No 1, (Jan 23),

Holden (2008). "SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases." *Mol Microbiol* Vol.67, No 6, (Mar), pp.

and inflammatory disease." *J Biol Chem* Vol.280, No 21, (May 27), pp. 20177-80, ISSN

important mode of regulation." *Curr Opin Immunol* Vol.22, No 1, (Feb), pp. 20-7,

takes control: effector-driven manipulation of the host." *Curr Opin Microbiol* Vol.12,

J. Adema (2008). "Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-alpha production." *Blood* Vol.111, No 8, (Apr

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 161

Saito, T., R. Hirai, Y. M. Loo, D. Owen, C. L. Johnson, S. C. Sinha, S. Akira, T. Fujita & M.

Sato, K., X. L. Yang, T. Yudate, J. S. Chung, J. Wu, K. Luby-Phelps, R. P. Kimberly, D.

Satoh, T., H. Kato, Y. Kumagai, M. Yoneyama, S. Sato, K. Matsushita, T. Tsujimura, T. Fujita,

Schlee, M., A. Roth, V. Hornung, C. A. Hagmann, V. Wimmenauer, W. Barchet, C. Coch, M.

Shapira, S., O. S. Harb, J. Margarit, M. Matrajt, J. Han, A. Hoffmann, B. Freedman, M. J. May,

Shim, J. H., C. Xiao, A. E. Paschal, S. T. Bailey, P. Rao, M. S. Hayden, K. Y. Lee, C. Bussey, M.

Slack, E. C., M. J. Robinson, P. Hernanz-Falcon, G. D. Brown, D. L. Williams, E.

Smeekens, S. P., F. L. van de Veerdonk, J. W. van der Meer, B. J. Kullberg, L. A. Joosten & M.

Sporri, R. & C. Reis e Sousa (2005). "Inflammatory mediators are insufficient for full

helper function." *Nat Immunol* Vol.6, No 2, (Feb), pp. 163-70, ISSN 1529-2908

*Eur J Immunol* Vol.37, No 6, (Jun), pp. 1600-12, ISSN 0014-2980

vivo." *Genes Dev* Vol.19, No 22, (Nov 15), pp. 2668-81, ISSN 0890-9369 Simmons, D. P., D. H. Canaday, Y. Liu, Q. Li, A. Huang, W. H. Boom & C. V. Harding

*Immunity* Vol.31, No 1, (Jul 17), pp. 25-34, ISSN 1097-4180

Vol.118, No Pt 15, (Aug 1), pp. 3501-8, ISSN 0021-9533

(Aug 15), pp. 2405-15, ISSN 1550-6606

ISSN 1460-2377

28), pp. 681-91, ISSN 1097-4180

7, ISSN 0027-8424

1512-7, ISSN 1091-6490

9258

"Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albicans." *Immunity* Vol.32, No 5, (May

Gale, Jr. (2007). "Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2." *Proc Natl Acad Sci U S A* Vol.104, No 2, (Jan 9), pp. 582-

Underhill, P. D. Cruz, Jr. & K. Ariizumi (2006). "Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor gamma chain to induce innate immune responses." *J Biol Chem* Vol.281, No 50, (Dec 15), pp. 38854-66, ISSN 0021-

S. Akira & O. Takeuchi (2010). "LGP2 is a positive regulator of RIG-I- and MDA5 mediated antiviral responses." *Proc Natl Acad Sci U S A* Vol.107, No 4, (Jan 26), pp.

Janke, A. Mihailovic, G. Wardle, S. Juranek, H. Kato, T. Kawai, H. Poeck, K. A. Fitzgerald, O. Takeuchi, S. Akira, T. Tuschl, E. Latz, J. Ludwig & G. Hartmann (2009). "Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus."

D. S. Roos & C. A. Hunter (2005). "Initiation and termination of NF-kappaB signaling by the intracellular protozoan parasite Toxoplasma gondii." *J Cell Sci*

Steckel, N. Tanaka, G. Yamada, S. Akira, K. Matsumoto & S. Ghosh (2005). "TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in

(2010). "Mycobacterium tuberculosis and TLR2 agonists inhibit induction of type I IFN and class I MHC antigen cross processing by TLR9." *J Immunol* Vol.185, No 4,

Schweighoffer, V. L. Tybulewicz & C. Reis e Sousa (2007). "Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan."

G. Netea (2010). "The Candida Th17 response is dependent on mannan- and betaglucan-induced prostaglandin E2." *Int Immunol* Vol.22, No 11, (Nov), pp. 889-95,

dendritic cell activation and promote expansion of CD4+ T cell populations lacking

Park, J. M., F. R. Greten, Z. W. Li & M. Karin (2002). "Macrophage apoptosis by anthrax

Pedra, J. H., S. L. Cassel & F. S. Sutterwala (2009). "Sensing pathogens and danger signals by

Pichlmair, A., O. Schulz, C. P. Tan, T. I. Naslund, P. Liljestrom, F. Weber & C. Reis e Sousa

Qualls, J. E., G. Neale, A. M. Smith, M. S. Koo, A. A. DeFreitas, H. Zhang, G. Kaplan, S. S.

Richard, M., N. Thibault, P. Veilleux, G. Gareau-Page & A. D. Beaulieu (2006). "Granulocyte

Ritter, M., O. Gross, S. Kays, J. Ruland, F. Nimmerjahn, S. Saijo, J. Tschopp, L. E. Layland &

*Natl Acad Sci U S A* Vol.107, No 47, (Nov 23), pp. 20459-64, ISSN 1091-6490 Robinson, M. J., F. Osorio, M. Rosas, R. P. Freitas, E. Schweighoffer, O. Gross, J. S. Verbeek, J.

Rogers, N. C., E. C. Slack, A. D. Edwards, M. A. Nolte, O. Schulz, E. Schweighoffer, D. L.

Rothenfusser, S., N. Goutagny, G. DiPerna, M. Gong, B. G. Monks, A. Schoenemeyer, M.

Said-Sadier, N., E. Padilla, G. Langsley & D. M. Ojcius (2010). "Aspergillus fumigatus

Saijo, S., S. Ikeda, K. Yamabe, S. Kakuta, H. Ishigame, A. Akitsu, N. Fujikado, T. Kusaka, S.

*Immunol* Vol.175, No 8, (Oct 15), pp. 5260-8, ISSN 0022-1767

pp. 2048-51, ISSN 1095-9203

No 135, ra62, ISSN 1937-9145

Vol.30, No 8, (Aug), pp. 2235-44, ISSN 0014-2980

Vol.43, No 10, (Apr), pp. 1716-21, ISSN 0161-5890

0372

1540-9538

7613

6203

lethal factor through p38 MAP kinase inhibition." *Science* Vol.297, No 5589, (Sep 20),

the inflammasome." *Curr Opin Immunol* Vol.21, No 1, (Feb), pp. 10-6, ISSN 1879-

(2006). "RIG-I-mediated antiviral responses to single-stranded RNA bearing 5' phosphates." *Science* Vol.314, No 5801, (Nov 10), pp. 997-1001, ISSN 1095-9203 Prive, C. & A. Descoteaux (2000). "Leishmania donovani promastigotes evade the activation

of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages." *Eur J Immunol*

Watowich & P. J. Murray (2010). "Arginine usage in mycobacteria-infected macrophages depends on autocrine-paracrine cytokine signaling." *Sci Signal* Vol.3,

macrophage-colony stimulating factor reduces the affinity of SHP-2 for the ITIM of CLECSF6 in neutrophils: a new mechanism of action for SHP-2." *Mol Immunol*

C. Prazeres da Costa (2010). "Schistosoma mansoni triggers Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune responses." *Proc*

Ruland, V. Tybulewicz, G. D. Brown, L. F. Moita, P. R. Taylor & C. Reis e Sousa (2009). "Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection." *J Exp Med* Vol.206, No 9, (Aug 31), pp. 2037-51, ISSN

Williams, S. Gordon, V. L. Tybulewicz, G. D. Brown & C. Reis e Sousa (2005). "Sykdependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins." *Immunity* Vol.22, No 4, (Apr), pp. 507-17, ISSN 1074-

Yamamoto, S. Akira & K. A. Fitzgerald (2005). "The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I." *J*

stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase." *PLoS One* Vol.5, No 4, e10008, ISSN 1932-

Kubo, S. H. Chung, R. Komatsu, N. Miura, Y. Adachi, N. Ohno, K. Shibuya, N. Yamamoto, K. Kawakami, S. Yamasaki, T. Saito, S. Akira & Y. Iwakura (2010). "Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albicans." *Immunity* Vol.32, No 5, (May 28), pp. 681-91, ISSN 1097-4180

Pathogen Strategies to Evade Innate Immune Response: A Signaling Point of View 163

van Gisbergen, K. P., M. Sanchez-Hernandez, T. B. Geijtenbeek & Y. van Kooyk (2005).

van Kooyk, Y. & T. B. Geijtenbeek (2003). "DC-SIGN: escape mechanism for pathogens." *Nat*

van Vliet, S. J., J. J. Garcia-Vallejo & Y. van Kooyk (2008). "Dendritic cells and C-type lectin

Viboud, G. I. & J. B. Bliska (2005). "Yersinia outer proteins: role in modulation of host cell

Waggoner, S. N., C. H. Hall & Y. S. Hahn (2007). "HCV core protein interaction with gC1q

Wang, C., L. Deng, M. Hong, G. R. Akkaraju, J. Inoue & Z. J. Chen (2001). "TAK1 is a

Watson, A. A., A. A. Lebedev, B. A. Hall, A. E. Fenton-May, A. A. Vagin, W. Dejnirattisai, J.

Weis, W. I., M. E. Taylor & K. Drickamer (1998). "The C-type lectin superfamily in the immune system." *Immunol Rev* Vol.163, No, (Jun), pp. 19-34, ISSN 0105-2896 Wells, C. A., J. A. Salvage-Jones, X. Li, K. Hitchens, S. Butcher, R. Z. Murray, A. G.

Werninghaus, K., A. Babiak, O. Gross, C. Holscher, H. Dietrich, E. M. Agger, J. Mages, A.

Werts, C., L. le Bourhis, J. Liu, J. G. Magalhaes, L. A. Carneiro, J. H. Fritz, S. Stockinger, V.

Wilkins, C. & M. Gale, Jr. (2010). "Recognition of viruses by cytoplasmic sensors." *Curr Opin*

Xu, S., J. Huo, K. G. Lee, T. Kurosaki & K. P. Lam (2009). "Phospholipase Cgamma2 is critical

*Biol Chem* Vol.286, No 27, (Jul 8), pp. 24208-18, ISSN 1083-351X

No 11, (Jun 1), pp. 7404-13, ISSN 0022-1767

Vol.206, No 1, (Jan 16), pp. 89-97, ISSN 1540-9538

*Immunol* Vol.37, No 9, (Sep), pp. 2499-508, ISSN 0014-2980

*Chem* Vol.284, No 11, (Mar 13), pp. 7038-46, ISSN 0021-9258

*Immunol* Vol.22, No 1, (Feb), pp. 41-7, ISSN 1879-0372

*Rev Immunol* Vol.3, No 9, (Sep), pp. 697-709, ISSN 1474-1733

(Apr 18), pp. 1281-92, ISSN 0022-1007

No 7, (Oct), pp. 580-7, ISSN 0818-9641

pp. 346-51, ISSN 0028-0836

0066-4227

"Neutrophils mediate immune modulation of dendritic cells through glycosylationdependent interactions between Mac-1 and DC-SIGN." *J Exp Med* Vol.201, No 8,

receptors: coupling innate to adaptive immune responses." *Immunol Cell Biol* Vol.86,

signaling responses and pathogenesis." *Annu Rev Microbiol* Vol.59, No, 69-89, ISSN

receptor inhibits Th1 differentiation of CD4+ T cells via suppression of dendritic cell IL-12 production." *J Leukoc Biol* Vol.82, No 6, (Dec), pp. 1407-19, ISSN 0741-5400

ubiquitin-dependent kinase of MKK and IKK." *Nature* Vol.412, No 6844, (Jul 19),

Felce, J. Mongkolsapaya, A. S. Palma, Y. Liu, T. Feizi, G. R. Screaton, G. N. Murshudov & C. A. O'Callaghan (2011). "Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling." *J*

Beckhouse, Y. L. Lo, S. Manzanero, C. Cobbold, K. Schroder, B. Ma, S. Orr, L. Stewart, D. Lebus, P. Sobieszczuk, D. A. Hume, J. Stow, H. Blanchard & R. B. Ashman (2008). "The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans." *J Immunol* Vol.180,

Mocsai, H. Schoenen, K. Finger, F. Nimmerjahn, G. D. Brown, C. Kirschning, A. Heit, P. Andersen, H. Wagner, J. Ruland & R. Lang (2009). "Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRgamma-Syk-Card9-dependent innate immune activation." *J Exp Med*

Balloy, M. Chignard, T. Decker, D. J. Philpott, X. Ma & S. E. Girardin (2007). "Nod1 and Nod2 induce CCL5/RANTES through the NF-kappaB pathway." *Eur J*

for Dectin-1-mediated Ca2+ flux and cytokine production in dendritic cells." *J Biol*

Steinman, R. M. (2006). "Linking innate to adaptive immunity through dendritic cells." *Novartis Found Symp* Vol.279, No, 101-9; discussion 109-13, 216-9, ISSN 1528-2511 Suram, S., G. D. Brown, M. Ghosh, S. Gordon, R. Loper, P. R. Taylor, S. Akira, S. Uematsu,

receptor." *J Biol Chem* Vol.281, No 9, (Mar 3), pp. 5506-14, ISSN 0021-9258 Suzuki, N., S. Suzuki, G. S. Duncan, D. G. Millar, T. Wada, C. Mirtsos, H. Takada, A.

Swantek, J. L., M. F. Tsen, M. H. Cobb & J. A. Thomas (2000). "IL-1 receptor-associated

Takahashi, K., T. Kawai, H. Kumar, S. Sato, S. Yonehara & S. Akira (2006). "Roles of caspase-

Takahasi, K., M. Yoneyama, T. Nishihori, R. Hirai, H. Kumeta, R. Narita, M. Gale, Jr., F.

Takeda, K. & S. Akira (2004). "TLR signaling pathways." *Semin Immunol* Vol.16, No 1, (Feb),

Takeuchi, O. & S. Akira (2010). "Pattern recognition receptors and inflammation." *Cell*

Taniguchi, T., K. Ogasawara, A. Takaoka & N. Tanaka (2001). "IRF family of transcription

Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. Martinez-Pomares, S. Gordon & S.

Taylor, P. R., S. V. Tsoni, J. A. Willment, K. M. Dennehy, M. Rosas, H. Findon, K. Haynes, C.

Trosky, J. E., Y. Li, S. Mukherjee, G. Keitany, H. Ball & K. Orth (2007). "VopA inhibits ATP

Tsuji, S., M. Matsumoto, O. Takeuchi, S. Akira, I. Azuma, A. Hayashi, K. Toyoshima & T.

Turnbull, P. C. (2002). "Introduction: anthrax history, disease and ecology." *Curr Top*

0836

15), pp. 4301-6, ISSN 0022-1767

40, ISSN 1097-4164

0732-0582

pp. 3-9, ISSN 1044-5323

pp. 31-8, ISSN 1529-2908

47, (Nov 23), pp. 34299-305, ISSN 0021-9258

Vol.176, No 8, (Apr 15), pp. 4520-4, ISSN 0022-1767

Vol.140, No 6, (Mar 19), pp. 805-20, ISSN 1097-4172

*Immunol* Vol.169, No 7, (Oct 1), pp. 3876-82, ISSN 0022-1767

*Infect Immun* Vol.68, No 12, (Dec), pp. 6883-90, ISSN 0019-9567

*Microbiol Immunol* Vol.271, No, 1-19, ISSN 0070-217X

D. L. Williams & C. C. Leslie (2006). "Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan

Wakeham, A. Itie, S. Li, J. M. Penninger, H. Wesche, P. S. Ohashi, T. W. Mak & W. C. Yeh (2002). "Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4." *Nature* Vol.416, No 6882, (Apr 18), pp. 750-6, ISSN 0028-

kinase modulates host responsiveness to endotoxin." *J Immunol* Vol.164, No 8, (Apr

8 and caspase-10 in innate immune responses to double-stranded RNA." *J Immunol*

Inagaki & T. Fujita (2008). "Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses." *Mol Cell* Vol.29, No 4, (Feb 29), pp. 428-

factors as regulators of host defense." *Annu Rev Immunol* Vol.19, No, 623-55, ISSN

Y. Wong (2002). "The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages." *J*

Steele, M. Botto, S. Gordon & G. D. Brown (2007). "Dectin-1 is required for betaglucan recognition and control of fungal infection." *Nat Immunol* Vol.8, No 1, (Jan),

binding by acetylating the catalytic loop of MAPK kinases." *J Biol Chem* Vol.282, No

Seya (2000). "Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin: involvement of toll-like receptors."

**7**

*USA*

**Phosphorylation-Regulated Cell Surface**

To maintain its functional integrity, a cell senses and reacts to the acute or chronic changes in the environment under physiological and pathological conditions. This typically involves cell surface membrane proteins such as receptors, ion channels, and structural proteins, whose surface expression level is regulated at multiple different steps of their biosynthesis and trafficking. Protein trafficking is mediated by a series of dynamic interactions between the sorting motifs of cargo proteins and the cellular machineries that recognize these motifs. While the constitutive trafficking of many cargo proteins relies on intrinsic sorting signals, post-translational modification of cargo proteins often serves as a key switch that enables the spatio-temporal regulation of their trafficking. Protein phosphorylation is one of the most intensively studied post-translational modifications that control the membrane trafficking. However, molecular mechanisms by which phosphorylation signal regulates the protein localization are diverse and remain not fully understood. The 14-3-3 proteins had been identified to specifically recognize phosphorylated serine or threonine residues, and thus represents one of the most distinct effector molecules that function downstream of the phosphorylation signal by kinases. This chapter will focus on the emerging role of 14-3-3 proteins in the phosphorylation-dependent control of cell surface membrane protein

**2. Control of cell surface expression by phosphorylation signal**

A typical mechanism by which phosphorylation signal controls protein trafficking is that phosphorylation of cargo proteins creates docking sites for the interacting proteins. A well studied example is the internalization of G protein-coupled receptors (GPCRs), where ligand binding induces the conformational change of the receptor and subsequent recruitment of GPCR kinases (GRKs) to the receptor. Receptor phosphorylation by GRKs recruits arrestin that couples the receptor to the adaptor protein of clathrin coat, thereby initiating the internalization of the cargo vesicles (Drake *et al.*, 2006; Tobin, 2008). This way phosphorylation signal leads to the desensitization of ligand stimulus by reducing the cell surface density of GPCRs. On the other hand, phosphorylation signalling can also regulate localization of membrane proteins by attenuating the sorting signal activity. In the neuron, ligand stimulation of N-methyl-D-aspartate (NMDA) receptor leads to receptor phosphorylation by casein kinase II (CK2) at the serine residue within the C-terminal PDZ

**1. Introduction**

trafficking.

**Expression of Membrane Proteins**

Yukari Okamoto and Sojin Shikano

*University of Illinois at Chicago,*

### **Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins**

Yukari Okamoto and Sojin Shikano *University of Illinois at Chicago, USA*

#### **1. Introduction**

164 Protein Kinases

Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M.

Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho, K. Hoshino, O.

Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda & S. Akira (2002).

Yamamoto, M., K. Takeda & S. Akira (2004). "TIR domain-containing adaptors define the

Yamasaki, S., M. Matsumoto, O. Takeuchi, T. Matsuzawa, E. Ishikawa, M. Sakuma, H.

*Natl Acad Sci U S A* Vol.106, No 6, (Feb 10), pp. 1897-902, ISSN 1091-6490 Ye, Z., E. O. Petrof, D. Boone, E. C. Claud & J. Sun (2007). "Salmonella effector AvrA

Yen, H., T. Ooka, A. Iguchi, T. Hayashi, N. Sugimoto & T. Tobe "NleC, a type III secretion

Yoneyama, M., M. Kikuchi, K. Matsumoto, T. Imaizumi, M. Miyagishi, K. Taira, E. Foy, Y.

Zhang, D., G. Zhang, M. S. Hayden, M. B. Greenblatt, C. Bussey, R. A. Flavell & S. Ghosh

Zhang, X., L. Majlessi, E. Deriaud, C. Leclerc & R. Lo-Man (2009). "Coactivation of Syk

Zhou, H., D. M. Monack, N. Kayagaki, I. Wertz, J. Yin, B. Wolf & V. M. Dixit (2005).

activation." *J Exp Med* Vol.202, No 10, (Nov 21), pp. 1327-32, ISSN 0022-1007

*Science* Vol.303, No 5663, (Mar 5), pp. 1522-6, ISSN 1095-9203

5633, (Aug 1), pp. 640-3, ISSN 1095-9203

0161-5890

4180

Vol.420, No 6913, (Nov 21), pp. 324-9, ISSN 0028-0836

Vol.171, No 3, (Sep), pp. 882-92, ISSN 0002-9440

Vol.6, No 12, e1001231, ISSN 1553-7374

7, (Jul), pp. 730-7, ISSN 1529-2908

*Immunol* Vol.169, No 12, (Dec 15), pp. 6668-72, ISSN 0022-1767

Sugiyama, M. Okabe, K. Takeda & S. Akira (2003). "Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway." *Science* Vol.301, No

Takeuchi, M. Kobayashi, T. Fujita, K. Takeda & S. Akira (2002). "Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4." *Nature*

"Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling." *J*

specificity of TLR signaling." *Mol Immunol* Vol.40, No 12, (Feb), pp. 861-8, ISSN

Tateno, J. Uno, J. Hirabayashi, Y. Mikami, K. Takeda, S. Akira & T. Saito (2009). "Ctype lectin Mincle is an activating receptor for pathogenic fungus, Malassezia." *Proc*

regulation of colonic epithelial cell inflammation by deubiquitination." *Am J Pathol*

protease, compromises NF-kappaB activation by targeting p65/RelA." *PLoS Pathog*

M. Loo, M. Gale, Jr., S. Akira, S. Yonehara, A. Kato & T. Fujita (2005). "Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity." *J Immunol* Vol.175, No 5, (Sep 1), pp. 2851-8, ISSN 0022-1767 Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira,

S. Akira & T. Fujita (2004). "The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses." *Nat Immunol* Vol.5, No

(2004). "A toll-like receptor that prevents infection by uropathogenic bacteria."

kinase and MyD88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils." *Immunity* Vol.31, No 5, (Nov 20), pp. 761-71, ISSN 1097-

"Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kappa B

To maintain its functional integrity, a cell senses and reacts to the acute or chronic changes in the environment under physiological and pathological conditions. This typically involves cell surface membrane proteins such as receptors, ion channels, and structural proteins, whose surface expression level is regulated at multiple different steps of their biosynthesis and trafficking. Protein trafficking is mediated by a series of dynamic interactions between the sorting motifs of cargo proteins and the cellular machineries that recognize these motifs. While the constitutive trafficking of many cargo proteins relies on intrinsic sorting signals, post-translational modification of cargo proteins often serves as a key switch that enables the spatio-temporal regulation of their trafficking. Protein phosphorylation is one of the most intensively studied post-translational modifications that control the membrane trafficking. However, molecular mechanisms by which phosphorylation signal regulates the protein localization are diverse and remain not fully understood. The 14-3-3 proteins had been identified to specifically recognize phosphorylated serine or threonine residues, and thus represents one of the most distinct effector molecules that function downstream of the phosphorylation signal by kinases. This chapter will focus on the emerging role of 14-3-3 proteins in the phosphorylation-dependent control of cell surface membrane protein trafficking.

#### **2. Control of cell surface expression by phosphorylation signal**

A typical mechanism by which phosphorylation signal controls protein trafficking is that phosphorylation of cargo proteins creates docking sites for the interacting proteins. A well studied example is the internalization of G protein-coupled receptors (GPCRs), where ligand binding induces the conformational change of the receptor and subsequent recruitment of GPCR kinases (GRKs) to the receptor. Receptor phosphorylation by GRKs recruits arrestin that couples the receptor to the adaptor protein of clathrin coat, thereby initiating the internalization of the cargo vesicles (Drake *et al.*, 2006; Tobin, 2008). This way phosphorylation signal leads to the desensitization of ligand stimulus by reducing the cell surface density of GPCRs. On the other hand, phosphorylation signalling can also regulate localization of membrane proteins by attenuating the sorting signal activity. In the neuron, ligand stimulation of N-methyl-D-aspartate (NMDA) receptor leads to receptor phosphorylation by casein kinase II (CK2) at the serine residue within the C-terminal PDZ

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 167

Table 1. 14-3-3 binding sequences. Consensus mode I and mode II motifs and the reported C-terminal mode III binding sequences are shown. S/T: phosphorylated serine or threonine required for 14-3-3 binding, : aromatic residue, Reference#1: (Bodnar *et al.*, 1999) , #2: (Sliva *et al.*, 2000), #3 and #4: (O'Kelly *et al.*, 2002):, #5 and #6: (Shikano *et al.*, 2005), #7:(Rong *et al.*,

(Yaffe *et al.*, 1997) and by random C-terminal peptide selection in a cell-based genetic screen (Shikano *et al.*, 2005). Indeed, the majority of the so far identified C-terminal 14-3-3 binding sequences contain arginine residues upstream of the phosphorylated serine or threonine (Table 1). Recent mutagenesis study of the C-terminal 14-3-3 binding site in GPR15 demonstrated the importance of the upstream arginine residue for phosphorylationdependent 14-3-3 binding (Okamoto & Shikano, 2011). In a crystal structure with 14-3-3, a mode II peptide displayed an arginine in the -4 position from phosphorylated serine (*R*LYHpSLPA) that was looped back to interact with the phosphate on the peptide (Rittinger *et al.*, 1999). These lines of evidence support significant contribution of upstream arginine residues to the 14-3-3 affinity. Thus, Mode III would be better defined as RXXpS/pTX-COOH. For all three modes of 14-3-3 binding, phosphorylation is a prerequisite and arginine residues located upstream of the phospho-serine/threonine are also important for recognition by a number of kinases (Kobe *et al.*, 2005). Thus, the absence of an arginine residue in the C-terminal 14-3-3 binding sequence in plant H+-ATPase (QQXYpTV-COOH) suggests the possibility that plant and animal differ significantly in kinase recognition. As more C-terminal 14-3-3 binding proteins become available, it would be valuable to revisit the issue of upstream sequence requirements both in terms of 14-3-3 binding *per se* and in

Proteomic screens have identified over 200 phosphoproteins which interact with 14-3-3 (Chang *et al.*, 2009; Ichimura *et al.*, 2002; Kakiuchi *et al.*, 2007; Meek *et al.*, 2004; Pozuelo Rubio *et al.*, 2004). Understanding when and how 14-3-3 proteins impact on these targets

2007), #8: (Wurtele *et al.*, 2003), #9: (Ganguly *et al.*, 2005), #10: (Fujita *et al.*, 2003).

terms of kinase recognition.

**2.2 Protein kinases that phosphorylate 14-3-3 target sites**

[postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] binding motif (IESDV-COOH) of NMDA receptor subunit 2B (NR2B). CK2 phosphorylation disrupts the interaction of NR2B with the PDZ domains of PSD-95 and SAP102 and thereby decreases cell surface NR2B expression (Chung *et al.*, 2004). This represents the regulatory role of phosphorylation in excitatory synaptic function and plasticity.

In contrast to the downregulation of surface expression, the molecular basis for the role of phosphorylation in promoting cell surface trafficking has been less well understood. However, studies in the past decade have revealed that 14-3-3s are the key class of phosphosensing proteins which mediate cell surface trafficking of various membrane proteins. Here we will review recent findings on the emerging role of 14-3-3 in cell surface protein trafficking, with particular focus on their mechanisms of action and relevant kinases.

#### **2.1 14-3-3 proteins and 14-3-3 binding sites**

The 14-3-3 proteins were first discovered in 1967 as brain-rich, acidic protein (Moore & Perez, 1967). The name 14-3-3 refers to the elution and migration profile of these proteins on DEAEcellulose chromatography and starch gel electrophoresis. They are highly conserved and expressed in all eukaryotic cells, with seven isoforms in mammals (β, γ, ε, ζ, η, τ, σ) and two in yeast (Bmh1 and Bmh2). 14-3-3 proteins participate in fundamental biological processes such as signal transduction, metabolism, protein degradation, and trafficking (Tzivion & Avruch, 2002; van Hemert *et al.*, 2001). All the 14-3-3 proteins, except for the sigma isoform, are able to form stable homo- and heterodimers (Benzinger *et al.*, 2005; Gardino *et al.*, 2006; Wilker *et al.*, 2005). The dimeric structure of the 14-3-3 protein allows it to simultaneously bind two binding sites through an amphipathic ligand-binding groove present in each monomer. In the majority of cases, 14-3-3 proteins recognize phosphorylated peptides in their binding partners. Screening of phosphoserine-oriented peptide libraries has identified two consensus 14-3-3 binding motifs that are present in many of known 14-3-3 binding proteins (Yaffe *et al.*, 1997). These are R-[S/]-X-pS/pT-X-P (mode I) and R-X-[S/]-X-pS/pT-X-P (mode II) binding sites where pS/pT is phosphoserine or phosphothreonine, is an aromatic residue, and X is any residue (typically leucine, glutamate, alanine, and methionine). However, it should be noted that 14-3-3-binding sites in numerous proteins do not conform to these optimal motifs, presumably because other structural features also contribute to the interactions. For instance, proline located at position +2 of the phosphorylation site occurs in only about half of known 14-3-3 binding motifs in mammalian proteins (Johnson *et al.*, 2010).

The C-terminal 14-3-3 binding motifs have recently become a newly recognized group with a distinct mode of interaction (see Table 1). Based on the similarity between the C-terminal 14-3-3 binding motifs of the oAANAT (RRNpSDR-COOH) and H+-ATPase (QQXYpTV-COOH) proteins, a new mode III consensus for 14-3-3 binding (pSX1–2-COOH) had been proposed (Ganguly *et al.*, 2005). The focal points of this consensus are that the motif is at the extreme C-terminus in contrast to the canonical mode I and II internal binding sites, and that the binding is phosphorylation-dependent. Mode III sequences interact with the same ligand-binding groove of 14-3-3 as do the mode I and mode II motifs (Coblitz *et al.*, 2005). Amino acid selectivity upstream of the phosphorylated residue is conspicuously absent from the proposed mode III motif, presumably due to the discrepancy between the oAANAT and H+-ATPase motifs. However, upstream arginine residues are preferred for modes I and II 14-3-3 binding as determined by random synthetic peptide library screening

[postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] binding motif (IESDV-COOH) of NMDA receptor subunit 2B (NR2B). CK2 phosphorylation disrupts the interaction of NR2B with the PDZ domains of PSD-95 and SAP102 and thereby decreases cell surface NR2B expression (Chung *et al.*, 2004). This represents the regulatory role of

In contrast to the downregulation of surface expression, the molecular basis for the role of phosphorylation in promoting cell surface trafficking has been less well understood. However, studies in the past decade have revealed that 14-3-3s are the key class of phosphosensing proteins which mediate cell surface trafficking of various membrane proteins. Here we will review recent findings on the emerging role of 14-3-3 in cell surface protein

The 14-3-3 proteins were first discovered in 1967 as brain-rich, acidic protein (Moore & Perez, 1967). The name 14-3-3 refers to the elution and migration profile of these proteins on DEAEcellulose chromatography and starch gel electrophoresis. They are highly conserved and expressed in all eukaryotic cells, with seven isoforms in mammals (β, γ, ε, ζ, η, τ, σ) and two in yeast (Bmh1 and Bmh2). 14-3-3 proteins participate in fundamental biological processes such as signal transduction, metabolism, protein degradation, and trafficking (Tzivion & Avruch, 2002; van Hemert *et al.*, 2001). All the 14-3-3 proteins, except for the sigma isoform, are able to form stable homo- and heterodimers (Benzinger *et al.*, 2005; Gardino *et al.*, 2006; Wilker *et al.*, 2005). The dimeric structure of the 14-3-3 protein allows it to simultaneously bind two binding sites through an amphipathic ligand-binding groove present in each monomer. In the majority of cases, 14-3-3 proteins recognize phosphorylated peptides in their binding partners. Screening of phosphoserine-oriented peptide libraries has identified two consensus 14-3-3 binding motifs that are present in many of known 14-3-3 binding proteins (Yaffe *et al.*, 1997). These are R-[S/]-X-pS/pT-X-P (mode I) and R-X-[S/]-X-pS/pT-X-P (mode II) binding sites where pS/pT is phosphoserine or phosphothreonine, is an aromatic residue, and X is any residue (typically leucine, glutamate, alanine, and methionine). However, it should be noted that 14-3-3-binding sites in numerous proteins do not conform to these optimal motifs, presumably because other structural features also contribute to the interactions. For instance, proline located at position +2 of the phosphorylation site occurs in only about half of known

The C-terminal 14-3-3 binding motifs have recently become a newly recognized group with a distinct mode of interaction (see Table 1). Based on the similarity between the C-terminal 14-3-3 binding motifs of the oAANAT (RRNpSDR-COOH) and H+-ATPase (QQXYpTV-COOH) proteins, a new mode III consensus for 14-3-3 binding (pSX1–2-COOH) had been proposed (Ganguly *et al.*, 2005). The focal points of this consensus are that the motif is at the extreme C-terminus in contrast to the canonical mode I and II internal binding sites, and that the binding is phosphorylation-dependent. Mode III sequences interact with the same ligand-binding groove of 14-3-3 as do the mode I and mode II motifs (Coblitz *et al.*, 2005). Amino acid selectivity upstream of the phosphorylated residue is conspicuously absent from the proposed mode III motif, presumably due to the discrepancy between the oAANAT and H+-ATPase motifs. However, upstream arginine residues are preferred for modes I and II 14-3-3 binding as determined by random synthetic peptide library screening

trafficking, with particular focus on their mechanisms of action and relevant kinases.

phosphorylation in excitatory synaptic function and plasticity.

14-3-3 binding motifs in mammalian proteins (Johnson *et al.*, 2010).

**2.1 14-3-3 proteins and 14-3-3 binding sites**

Table 1. 14-3-3 binding sequences. Consensus mode I and mode II motifs and the reported C-terminal mode III binding sequences are shown. S/T: phosphorylated serine or threonine required for 14-3-3 binding, : aromatic residue, Reference#1: (Bodnar *et al.*, 1999) , #2: (Sliva *et al.*, 2000), #3 and #4: (O'Kelly *et al.*, 2002):, #5 and #6: (Shikano *et al.*, 2005), #7:(Rong *et al.*, 2007), #8: (Wurtele *et al.*, 2003), #9: (Ganguly *et al.*, 2005), #10: (Fujita *et al.*, 2003).

(Yaffe *et al.*, 1997) and by random C-terminal peptide selection in a cell-based genetic screen (Shikano *et al.*, 2005). Indeed, the majority of the so far identified C-terminal 14-3-3 binding sequences contain arginine residues upstream of the phosphorylated serine or threonine (Table 1). Recent mutagenesis study of the C-terminal 14-3-3 binding site in GPR15 demonstrated the importance of the upstream arginine residue for phosphorylationdependent 14-3-3 binding (Okamoto & Shikano, 2011). In a crystal structure with 14-3-3, a mode II peptide displayed an arginine in the -4 position from phosphorylated serine (*R*LYHpSLPA) that was looped back to interact with the phosphate on the peptide (Rittinger *et al.*, 1999). These lines of evidence support significant contribution of upstream arginine residues to the 14-3-3 affinity. Thus, Mode III would be better defined as RXXpS/pTX-COOH. For all three modes of 14-3-3 binding, phosphorylation is a prerequisite and arginine residues located upstream of the phospho-serine/threonine are also important for recognition by a number of kinases (Kobe *et al.*, 2005). Thus, the absence of an arginine residue in the C-terminal 14-3-3 binding sequence in plant H+-ATPase (QQXYpTV-COOH) suggests the possibility that plant and animal differ significantly in kinase recognition. As more C-terminal 14-3-3 binding proteins become available, it would be valuable to revisit the issue of upstream sequence requirements both in terms of 14-3-3 binding *per se* and in terms of kinase recognition.

#### **2.2 Protein kinases that phosphorylate 14-3-3 target sites**

Proteomic screens have identified over 200 phosphoproteins which interact with 14-3-3 (Chang *et al.*, 2009; Ichimura *et al.*, 2002; Kakiuchi *et al.*, 2007; Meek *et al.*, 2004; Pozuelo Rubio *et al.*, 2004). Understanding when and how 14-3-3 proteins impact on these targets

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 169

The phospho-binding ability of 14-3-3 proteins is reminiscent of other proteins carrying specific modules that recognize phosphorylated sites. Such modules include FHA (Durocher *et al.*, 2000), WD40 (Yaffe & Elia, 2001), Polo-box (Lowery *et al.*, 2005), and BRCT (BRCA-1 Cterminal) repeat domains (Manke *et al.*, 2003), which target serine and threonine phosphorylation, as well as SH2 (Src-homology 2) domains which target phosphorylated tyrosine residues in specific sequence contexts (Bradshaw & Waksman, 2002). These domains are found in a large number of proteins involved in a wide range of signaling processes. 14-3-3s are distinct from those proteins in that 14-3-3s are not modular components of other proteins. They are discrete binding proteins with no intrinsic enzyme activities, except for the nucleoside diphosphate (NDP) kinase-like activity (Yano *et al.*, 1997) and chaperonic activity toward selected substrates (Yano *et al.*, 2006). So, how do 14-3- 3s exert their effects? Several excellent reviews discuss different models of 14-3-3 action as masking, scaffolding, or clamping of proteins (Dougherty & Morrison, 2004; Mackintosh, 2004; Mrowiec & Schwappach, 2006). Recruitment of proteins may be regulated by masking of functional signals by 14-3-3 binding. Alternatively, the scaffolding model suggests that 14-3-3 proteins tether different molecules together and form a platform for complex assembly. Clamping describes the idea that 14-3-3 binding alters the functional property of the client protein by stabilizing a certain conformation (Figure 1). It should be noted that combinations of these 'masking', 'scaffolding', and 'clamping' types of 14-3-3 action may occur together. As far as membrane proteins are concerned, very few examples exist where the interactions of 14-3-3 with client proteins are sufficiently well understood to ascribe a particular mode of action. Nevertheless, 14-3-3s may be considered as general switch proteins, of which effect of binding depends on the client protein. In most cases, phosphorylation at a serine or threonine residue activates the switch, and the subsequent binding of 14-3-3 proteins is thought to prevent rapid dephosphorylation. Recent studies have firmly demonstrated that 14-3-3 proteins are involved in controlling cell surface expression level of various cargo membrane proteins. We will discuss the pertinent evidence and hypothetical molecular mechanisms explaining these observations, with attention to the

14-3-3s are implicated in regulating the subcellular localization of many phosphorylated target proteins. The majority of the cases seem to involve the mechanism where 14-3-3 binding blocks the access of other proteins to the sorting signal of target proteins. Such a 'masking' role of 14-3-3 was first implicated in the mitochondria-cytoplasm translocation of the pro-apoptotic protein BAD. BAD interferes with the anti-apoptotic function of Bcl-2 and Bcl-xL in the mitochondria by binding to those proteins via its BH3 domain (Zha *et al.*, 1996). BH3 domain is located immediately adjacent to the serine136, of which phosphoryaltion by Akt (Datta *et al.*, 1997) is required for the binding of BAD to 14-3-3 in the cytoplasm (Zha *et al.*, 1997). These results suggested that 14-3-3 binding obscures the BH3 domain and prevents the targeting of BAD to mitochondria. A similar mechanism was found for the nuclear-cytoplasmic shuttling of various proteins including tyrosine phosphatase Cdc25C (Kumagai & Dunphy, 1999), transcription factor FKHRL1 (Brunet *et al.*, 1999), glucocorticoidreceptor (Kino *et al.*, 2003), CDK (cyclin-dependent kinase) inhibitor p27

**2.3 Molecular mechanism for the 14-3-3 effects on membrane protein trafficking**

relevant kinases that regulate 14-3-3 binding.

**2.3.1 Masking**

offers a great opportunity to gain mechanistic insights into many phosphorylation-regulated biological pathways. Since 14-3-3 target sites in proteins must satisfy the specificity requirements for both 14-3-3s and the protein kinases that create the sites in the first place, identification of kinases that phosphorylate 14-3-3 target sites is crucial for elucidating the physiological roles of 14-3-3 binding. Unfortunately, in the majority of cases the identity of the physiologically relevant kinases that phosphorylate the mode I, II or III 14-3-3 binding motifs is still unknown. This is largely due to the high similarities between different serine/threonine protein kinase recognition sites (Table 2) and their likely redundant activities. One reasonable approach to overcome such obstacles and gain better understanding of the physiological kinases for 14-3-3 target proteins would be the global analysis of the actual 14-3-3-binding phosphoproteins. Based on the proteomics data and other available literature on 14-3-3, Johnson *et al*. have recently attempted to define 14-3-3 specificity and identify relevant protein kinases (Johnson *et al.*, 2010). This study points out several features that are distinctive of 14-3-3-binding sequences as compared with other protein phosphorylation sites. For instance, few reported 14-3-3-binding sites have a +1 (relative to phosphorylated serine/threonine) proline residue, which contrasts with phosphoproteomic studies of cell lysates and subcellular fractions where phosphoserineproline is the most commonly reported phosphorylation motif overall (Ubersax & Ferrell, 2007). This indicates that proline-directed kinases do not phosphorylate 14-3-3-binding sites. Similarly, no reported 14-3-3-binding sites conform to the canonical consensus site for casein kinase II (pS/pT-X-X-D/S/pS), which is probably the second most common type of motif in the entire mammalian phosphoproteome (Salvi *et al.*, 2009). Another interesting notion is that, while the optimal mode I (R-[S/]-X-pS/pT-X-P) and mode II (R-X-[S/]-X-pS/pT-X-P) 14-3-3-binding motifs were defined using phosphopeptides, many 14-3-3-binding sites in mammalian proteins (but not in plant 14-3-3-binding proteins) have basic residues in position -5 (and -4) in addition to -3. This creates a motif RXRXXS/T, which is a good target for the basophilic AGC kinase family (cAMP-dependent protein kinases A, cGMPdependent protein kinases G, and phospholipid-dependent protein kinases) and the calcium/calmodulin-dependent kinase (CaMK) family (Pearson & Kemp, 1991). Indeed, members of these kinase families, including protein kinase A (PKA), protein kinase C (PKC), CaMKI, checkpoint kinases 1 and 2 (Chk1 and 2), Akt/protein kinase B (PKB) and p90 ribosomal S6 kinase (p90Rsk), are all known to phosphorylate sites that mediate 14-3-3 binding (Dougherty & Morrison, 2004). Among these, Akt is one of the most well documented kinases in phosphorylating 14-3-3 client proteins (Mackintosh, 2004).

Table 2. Consensus recognition sequences of major serine/threonine kinases that are known to phosphorylate 14-3-3 binding site.

The phospho-binding ability of 14-3-3 proteins is reminiscent of other proteins carrying specific modules that recognize phosphorylated sites. Such modules include FHA (Durocher *et al.*, 2000), WD40 (Yaffe & Elia, 2001), Polo-box (Lowery *et al.*, 2005), and BRCT (BRCA-1 Cterminal) repeat domains (Manke *et al.*, 2003), which target serine and threonine phosphorylation, as well as SH2 (Src-homology 2) domains which target phosphorylated tyrosine residues in specific sequence contexts (Bradshaw & Waksman, 2002). These domains are found in a large number of proteins involved in a wide range of signaling processes. 14-3-3s are distinct from those proteins in that 14-3-3s are not modular components of other proteins. They are discrete binding proteins with no intrinsic enzyme activities, except for the nucleoside diphosphate (NDP) kinase-like activity (Yano *et al.*, 1997) and chaperonic activity toward selected substrates (Yano *et al.*, 2006). So, how do 14-3- 3s exert their effects? Several excellent reviews discuss different models of 14-3-3 action as masking, scaffolding, or clamping of proteins (Dougherty & Morrison, 2004; Mackintosh, 2004; Mrowiec & Schwappach, 2006). Recruitment of proteins may be regulated by masking of functional signals by 14-3-3 binding. Alternatively, the scaffolding model suggests that 14-3-3 proteins tether different molecules together and form a platform for complex assembly. Clamping describes the idea that 14-3-3 binding alters the functional property of the client protein by stabilizing a certain conformation (Figure 1). It should be noted that combinations of these 'masking', 'scaffolding', and 'clamping' types of 14-3-3 action may occur together. As far as membrane proteins are concerned, very few examples exist where the interactions of 14-3-3 with client proteins are sufficiently well understood to ascribe a particular mode of action. Nevertheless, 14-3-3s may be considered as general switch proteins, of which effect of binding depends on the client protein. In most cases, phosphorylation at a serine or threonine residue activates the switch, and the subsequent binding of 14-3-3 proteins is thought to prevent rapid dephosphorylation. Recent studies have firmly demonstrated that 14-3-3 proteins are involved in controlling cell surface expression level of various cargo membrane proteins. We will discuss the pertinent evidence and hypothetical molecular mechanisms explaining these observations, with attention to the relevant kinases that regulate 14-3-3 binding.

#### **2.3.1 Masking**

168 Protein Kinases

offers a great opportunity to gain mechanistic insights into many phosphorylation-regulated biological pathways. Since 14-3-3 target sites in proteins must satisfy the specificity requirements for both 14-3-3s and the protein kinases that create the sites in the first place, identification of kinases that phosphorylate 14-3-3 target sites is crucial for elucidating the physiological roles of 14-3-3 binding. Unfortunately, in the majority of cases the identity of the physiologically relevant kinases that phosphorylate the mode I, II or III 14-3-3 binding motifs is still unknown. This is largely due to the high similarities between different serine/threonine protein kinase recognition sites (Table 2) and their likely redundant activities. One reasonable approach to overcome such obstacles and gain better understanding of the physiological kinases for 14-3-3 target proteins would be the global analysis of the actual 14-3-3-binding phosphoproteins. Based on the proteomics data and other available literature on 14-3-3, Johnson *et al*. have recently attempted to define 14-3-3 specificity and identify relevant protein kinases (Johnson *et al.*, 2010). This study points out several features that are distinctive of 14-3-3-binding sequences as compared with other protein phosphorylation sites. For instance, few reported 14-3-3-binding sites have a +1 (relative to phosphorylated serine/threonine) proline residue, which contrasts with phosphoproteomic studies of cell lysates and subcellular fractions where phosphoserineproline is the most commonly reported phosphorylation motif overall (Ubersax & Ferrell, 2007). This indicates that proline-directed kinases do not phosphorylate 14-3-3-binding sites. Similarly, no reported 14-3-3-binding sites conform to the canonical consensus site for casein kinase II (pS/pT-X-X-D/S/pS), which is probably the second most common type of motif in the entire mammalian phosphoproteome (Salvi *et al.*, 2009). Another interesting notion is that, while the optimal mode I (R-[S/]-X-pS/pT-X-P) and mode II (R-X-[S/]-X-pS/pT-X-P) 14-3-3-binding motifs were defined using phosphopeptides, many 14-3-3-binding sites in mammalian proteins (but not in plant 14-3-3-binding proteins) have basic residues in position -5 (and -4) in addition to -3. This creates a motif RXRXXS/T, which is a good target for the basophilic AGC kinase family (cAMP-dependent protein kinases A, cGMPdependent protein kinases G, and phospholipid-dependent protein kinases) and the calcium/calmodulin-dependent kinase (CaMK) family (Pearson & Kemp, 1991). Indeed, members of these kinase families, including protein kinase A (PKA), protein kinase C (PKC), CaMKI, checkpoint kinases 1 and 2 (Chk1 and 2), Akt/protein kinase B (PKB) and p90 ribosomal S6 kinase (p90Rsk), are all known to phosphorylate sites that mediate 14-3-3 binding (Dougherty & Morrison, 2004). Among these, Akt is one of the most well

documented kinases in phosphorylating 14-3-3 client proteins (Mackintosh, 2004).

Table 2. Consensus recognition sequences of major serine/threonine kinases that are known

to phosphorylate 14-3-3 binding site.

14-3-3s are implicated in regulating the subcellular localization of many phosphorylated target proteins. The majority of the cases seem to involve the mechanism where 14-3-3 binding blocks the access of other proteins to the sorting signal of target proteins. Such a 'masking' role of 14-3-3 was first implicated in the mitochondria-cytoplasm translocation of the pro-apoptotic protein BAD. BAD interferes with the anti-apoptotic function of Bcl-2 and Bcl-xL in the mitochondria by binding to those proteins via its BH3 domain (Zha *et al.*, 1996). BH3 domain is located immediately adjacent to the serine136, of which phosphoryaltion by Akt (Datta *et al.*, 1997) is required for the binding of BAD to 14-3-3 in the cytoplasm (Zha *et al.*, 1997). These results suggested that 14-3-3 binding obscures the BH3 domain and prevents the targeting of BAD to mitochondria. A similar mechanism was found for the nuclear-cytoplasmic shuttling of various proteins including tyrosine phosphatase Cdc25C (Kumagai & Dunphy, 1999), transcription factor FKHRL1 (Brunet *et al.*, 1999), glucocorticoidreceptor (Kino *et al.*, 2003), CDK (cyclin-dependent kinase) inhibitor p27

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 171

the nucleus or other organelles. However, this model of 14-3-3 action is contradicted by the observation that 14-3-3 can also promote the nuclear localization of other binding partners. Seimiya *et al.* found that 14-3-3 binding to human TERT, which requires threonine1030, serine1037 and serine1041, lead to the nuclear localization of hTERT. This was attributed to the blocking of the nearby leucine-rich nuclear export sequence (NES) present at residues 970– 981 of hTERT, which is otherwise recognized by the nuclear export receptor CRM1 (Seimiya *et al.*, 2000). So, how does 14-3-3 regulate nuclear-cytoplasmic shuttling of different proteins in two opposing directions? One simple hypothesis may be that 14-3-3 itself bears no specific information about protein sorting and the effect of 14-3-3 binding on the subcellular localization of a client protein depends entirely on sorting signals encoded within the client protein and the proximity of those signals to a 14-3-3 binding motif. If so, 14-3-3 binding

Consistent with this idea, recent studies report a critical role of 14-3-3 in controlling the cell surface expression level of membrane proteins of different functions, including ion channels, receptors, and adhesion molecules. In many cases, the underlying mechanism seems to involve 'masking' of a short sequence motif, namely a di-arginine or di-basic (RXR) ER localization signal. The RXR-type ER-localisation signals were first identified in ATPsensitive potassium channels (KATP channels) (Zerangue *et al.*, 1999). These channels assemble as octameric complexes consisting of four Kir6 channel subunits and four sulphonylurea receptor (SUR) subunits, and each of these subunits carries an RXR signal in the cytoplasmic tail. It is believed that the ER-localisation activity of these motifs is mediated by the retrieval of cargo proteins from the post-ER compartments such as ER-Golgi intermediate compartment (ERGIC) and cis-Glogi through their interaction with the retrograde transport coatomer protein, COPI (Michelsen *et al.*, 2007; Zerangue *et al.*, 1999). Although the RXR motif resembles the C-terminal di-lysine (KKXX) motif which also mediates ER retrieval of membrane protein cargos through direct interaction with COPI (Michelsen *et al.*, 2007), RXR motif is distinct in that it is present almost exclusively in the multimeric cell surface membrane proteins (Michelsen *et al.*, 2005), while KKXX motif is found in the membrane proteins that are resident to the ER such as nucleotide sugar

Efficient cell surface transport of proteins harbouring RXR motifs will be allowed only when the motif becomes inaccessible to COPI probably by multiple different mechanisms including folding, subunit assembly, post-translational modification, and protein recruitment. 14-3-3 proteins have been implicated in the masking of RXR motif in several instances. The first evidence that 14-3-3 proteins control cell surface transport of membrane protein was shown for two-pore-domain potassium (K2P) channels TASK1 and TASK3 (O'Kelly *et al.*, 2002). These channels bound to 14-3-3 via a mode III C-terminal binding motif (RRSSV-COOH and RRKSV-COOH for TASK1 and TASK3, respectively), where phosphorylation of the penultimate serine and the upstream arginine residues were critically required. In the absence of phosphorylation on the penultimate serine, COPI proteins bind to the adjacent RXR-like sequence (KRR) which shares two arginine residues with the 14-3-3 binding motif. It is thought that phosphorylation switch allows 14-3-3 binding that occludes the partially overlapping RXR-like COPI binding motif. A very recent study by Mant *et al*. reports that PKA, which recognizes a consensus sequence of RXXS/T, phosphorylates the penultimate serine residue in TASK1 and TASK3 channels and promote their expression on cell surface,

could possibly affect any other protein sorting pathways.

transporters (Jackson *et al.*, 1990, 1993).

Fig. 1. Hypothetical models for the effects of 14-3-3 binding on the trafficking of membrane proteins. **(A)** *Masking*. Sorting signals (e.g., RXR motif) are physically masked by 14-3-3 binding to the nearby phosphorylated target site (shown as ○ P). This blocks the access of transport machineries (e.g., COPI proteins) to the sorting signal. Only the action of one 14-3- 3 monomer is drawn here. **(B)** *Scaffolding*. Dimeric 14-3-3 facilitates the interaction of cargo proteins with transport machineries (e.g., motor proteins and microtubules). Binding of 14- 3-3 dimer to two different targets might involve phosphorylation-independent and/or outer surface-mediated interaction (see main text). **(C)** *Clamping*. Binding of a 14-3-3 dimer induces a conformation that is unfavorable for sorting signals (e.g., RXR motif), which can be achieved by clustering of targets or relocation from active zones (e.g., proximity to the transmembrane region (see main text)). This could result in reduced accessibility by transport machineries (e.g., COPI proteins).

(Fujita *et al.*, 2003), and catalytic subunit of telomerase TERT (Seimiya *et al.*, 2000). In most cases, 14-3-3 binding promotes the cytoplasmic localization of target proteins. For instance, 14-3-3 binding to serine287 of Cdc25C leads to the cytoplasmic retention of Cdc25C. This seems to be due to the occlusion of the closely located bipartite nuclear localization sequence (NLS) at amino acids 298-316 of Cdc25C from importin-, a receptor for bipartite NLS (Kumagai & Dunphy, 1999). These observations are consistent with the predominant cytoplasmic localization of 14-3-3 proteins at the steady-state, which has led to the hypothesis that they might serve as a universal cytoplasmic anchor that blocks import into

Fig. 1. Hypothetical models for the effects of 14-3-3 binding on the trafficking of membrane proteins. **(A)** *Masking*. Sorting signals (e.g., RXR motif) are physically masked by 14-3-3

transport machineries (e.g., COPI proteins) to the sorting signal. Only the action of one 14-3- 3 monomer is drawn here. **(B)** *Scaffolding*. Dimeric 14-3-3 facilitates the interaction of cargo proteins with transport machineries (e.g., motor proteins and microtubules). Binding of 14- 3-3 dimer to two different targets might involve phosphorylation-independent and/or outer

(Fujita *et al.*, 2003), and catalytic subunit of telomerase TERT (Seimiya *et al.*, 2000). In most cases, 14-3-3 binding promotes the cytoplasmic localization of target proteins. For instance, 14-3-3 binding to serine287 of Cdc25C leads to the cytoplasmic retention of Cdc25C. This seems to be due to the occlusion of the closely located bipartite nuclear localization sequence (NLS) at amino acids 298-316 of Cdc25C from importin-, a receptor for bipartite NLS (Kumagai & Dunphy, 1999). These observations are consistent with the predominant cytoplasmic localization of 14-3-3 proteins at the steady-state, which has led to the hypothesis that they might serve as a universal cytoplasmic anchor that blocks import into

surface-mediated interaction (see main text). **(C)** *Clamping*. Binding of a 14-3-3 dimer induces a conformation that is unfavorable for sorting signals (e.g., RXR motif), which can be achieved by clustering of targets or relocation from active zones (e.g., proximity to the transmembrane region (see main text)). This could result in reduced accessibility by

P). This blocks the access of

binding to the nearby phosphorylated target site (shown as ○

transport machineries (e.g., COPI proteins).

the nucleus or other organelles. However, this model of 14-3-3 action is contradicted by the observation that 14-3-3 can also promote the nuclear localization of other binding partners. Seimiya *et al.* found that 14-3-3 binding to human TERT, which requires threonine1030, serine1037 and serine1041, lead to the nuclear localization of hTERT. This was attributed to the blocking of the nearby leucine-rich nuclear export sequence (NES) present at residues 970– 981 of hTERT, which is otherwise recognized by the nuclear export receptor CRM1 (Seimiya *et al.*, 2000). So, how does 14-3-3 regulate nuclear-cytoplasmic shuttling of different proteins in two opposing directions? One simple hypothesis may be that 14-3-3 itself bears no specific information about protein sorting and the effect of 14-3-3 binding on the subcellular localization of a client protein depends entirely on sorting signals encoded within the client protein and the proximity of those signals to a 14-3-3 binding motif. If so, 14-3-3 binding could possibly affect any other protein sorting pathways.

Consistent with this idea, recent studies report a critical role of 14-3-3 in controlling the cell surface expression level of membrane proteins of different functions, including ion channels, receptors, and adhesion molecules. In many cases, the underlying mechanism seems to involve 'masking' of a short sequence motif, namely a di-arginine or di-basic (RXR) ER localization signal. The RXR-type ER-localisation signals were first identified in ATPsensitive potassium channels (KATP channels) (Zerangue *et al.*, 1999). These channels assemble as octameric complexes consisting of four Kir6 channel subunits and four sulphonylurea receptor (SUR) subunits, and each of these subunits carries an RXR signal in the cytoplasmic tail. It is believed that the ER-localisation activity of these motifs is mediated by the retrieval of cargo proteins from the post-ER compartments such as ER-Golgi intermediate compartment (ERGIC) and cis-Glogi through their interaction with the retrograde transport coatomer protein, COPI (Michelsen *et al.*, 2007; Zerangue *et al.*, 1999). Although the RXR motif resembles the C-terminal di-lysine (KKXX) motif which also mediates ER retrieval of membrane protein cargos through direct interaction with COPI (Michelsen *et al.*, 2007), RXR motif is distinct in that it is present almost exclusively in the multimeric cell surface membrane proteins (Michelsen *et al.*, 2005), while KKXX motif is found in the membrane proteins that are resident to the ER such as nucleotide sugar transporters (Jackson *et al.*, 1990, 1993).

Efficient cell surface transport of proteins harbouring RXR motifs will be allowed only when the motif becomes inaccessible to COPI probably by multiple different mechanisms including folding, subunit assembly, post-translational modification, and protein recruitment. 14-3-3 proteins have been implicated in the masking of RXR motif in several instances. The first evidence that 14-3-3 proteins control cell surface transport of membrane protein was shown for two-pore-domain potassium (K2P) channels TASK1 and TASK3 (O'Kelly *et al.*, 2002). These channels bound to 14-3-3 via a mode III C-terminal binding motif (RRSSV-COOH and RRKSV-COOH for TASK1 and TASK3, respectively), where phosphorylation of the penultimate serine and the upstream arginine residues were critically required. In the absence of phosphorylation on the penultimate serine, COPI proteins bind to the adjacent RXR-like sequence (KRR) which shares two arginine residues with the 14-3-3 binding motif. It is thought that phosphorylation switch allows 14-3-3 binding that occludes the partially overlapping RXR-like COPI binding motif. A very recent study by Mant *et al*. reports that PKA, which recognizes a consensus sequence of RXXS/T, phosphorylates the penultimate serine residue in TASK1 and TASK3 channels and promote their expression on cell surface,

Phosphorylation-Regulated Cell Surface Expression of Membrane Proteins 173

demonstrate that, despite high similarity in the recognition sequences of basophilic serine/threonine protein kinases, specific kinase signaling can modulate membrane protein trafficking through 14-3-3 binding. Further search of the human protein database for this Cterminal 14-3-3-binding motif has identified several candidate membrane proteins that would interact with 14-3-3. These include GPR15, an orphan GPCR that serves for a coreceptor for human immunodeficiency virus (HIV) entry (Farzan *et al.*, 1997). GPR15, which has a C-terminal sequence of RRRKRSVSL-COOH, was indeed confirmed to bind 14-3-3 and this binding absolutely required phosphorylation of penultimate serine359. A recent study by Okamoto and Shikano reported that alanine mutation of the serine359 resulted in substantial ER localization of GPR15 and this was mediated by the upstream arginine residues at amino acids 352 and 354, which constitute a COPI-binding RXR motif (Okamoto & Shikano, 2011). These results suggested that mode III binding of 14-3-3 to the receptor C-terminus physically occludes the adjacent RXR motif from the access by COPI, similar to the cases for TASK channels. Thus, a non-biased screening has led to the identification of 14-3-3 function as a key switch that converts phosphorylation signal to the sorting of membrane proteins by

The masking effect of 14-3-3 on the cell surface transport is not restricted to the mode III Cterminal binding. The cytoplasmic tail of ADAM22, a member of ADAM (a disintegrin and metalloprotease domain) protein family, contains two internal 14-3-3 binding sites and three RXR-type ER-localization signals that overlap with both of the 14-3-3 protein binding sites. Mutations in both 14-3-3 binding sites inhibited surface expression of ADAM22, while deletion of both RXR motif and 14-3-3 binding sites restored the surface expression (Godde *et al.*, 2006). Although this study did not investigate COPI interaction with ADAM22, the results suggest the possibility that 14-3-3 binding inhibited the COPI-dependent ER

The cell surface transport of a gap junction protein connexin 43 is also promoted by 14-3-3 binding to its internal mode I binding site (RASSRP) (Park *et al.*, 2007). The authors have found that Akt phosphorylates the serine373 in this site in the epidermal growth factor (EGF) stimulated cell. Although not characterized yet, the existence of an overlapping RXR motif (RPR) suggests a possible masking effect of 14-3-3 on the cell surface transport of connexin 43. NMDA receptors are tetramers composed of homologous subunits (NR1; NR2A-D; NR3A-B (Cull-Candy & Leszkiewicz, 2004). There are multiple NR2 subunits, each with unique spatio-temporal expression patterns, ensuring functional diversity of NMDA receptors. Recently, cerebellar NR2C subunit was found to be directly phosphorylated by Akt at the sequence that conforms to mode I 14-3-3 binding motif (RPRHASLP) (Chen & Roche, 2009). The Akt phosphorylation induced by insulin growth factor (IGF-1) results in the recruitment of 14-3-3 and the increase of cell surface expression of NMDA receptors. Although the causative role of 14-3-3 in surface transport remains unclear in this study, the presence of ER localization signals (Horak & Wenthold, 2009) in the NR1 subunit and the obligatory assembly of NR2 with NR1 for functional NMDA receptor suggest a possible mechanism where Akt-induced 14-3-3 binding to NR2C attenuates the activity of ER localization signals

Interestingly, 14-3-3s have also been reported to bind directly to the RXR motif itself in a phosphorylation-independent manner. By using an artificial multimer of the distal C-

modulating the activity of an ER localization signal.

localization activity of RXR signal.

in NR1 by physically occluding them.

although it was not shown in the literature whether this phosphorylation actually promotes the binding of 14-3-3 proteins to these sites (Mant *et al.*, 2011).

A similar masking mechanism was suggested for the ER-export of Iip35 isoform of major histocompatibility complex (MHC) class-II-associated invariant chain (O'Kelly *et al.*, 2002). During their assembly in the ER, MHC class-II αβ dimers associate with preformed trimers of the invariant chains (Iip33, Iip35, Iip41, Iip43), to form nonameric (αβIi)3 oligomers. It had been known that phosphorylation of an N-terminal serine8, present exclusively in the Iip35 cytoplasmic tail (NH3-MHRRRSRS…), is a prerequisite for efficient ER exit and sorting of class-II/Iip35 complexes to the cell surface. Phosphorylation of serine8 leads to the 14-3-3 binding to Iip35 and the alanine mutation on this residue inhibits the ER exit of class-II/Iip35 complex (Kuwana *et al.*, 1998). Together with the fact that 14-3-3 and COPI bound to the N-terminal sequence of Iip35 in a mutually exclusive manner, it was concluded that the cell surface transport of MHC class-II complex is promoted by 'masking' effect of 14-3-3 (O'Kelly *et al.*, 2002). However, as Khalil *et al*. pointed out (Khalil *et al.*, 2005), this model seems to require further investigation, since it is not consistent with the fact that, even when associated with a 14-3-3 protein, Iip35 will not leave the ER in the absence of class-II molecules and that mutation of serine8 to asparagine prevents 14-3-3 protein binding but still allows ER export if class-II molecules are present (Kuwana *et al.*, 1998).

The effect of 14-3-3 binding on promoting cell surface transport was also found by a completely different approach. Shikano *et al*. screened a random peptide library to identify C-terminal peptide signals that would functionally override the ER localization activity of the RXR motif (Shikano *et al.*, 2005). The screening was based on the yeast growth complementation assay using a mutant *Saccharomyces cerevisiae* SGY1528 which cannot survive in low potassium media due to the lack of endogenous potassium uptake transporters. However, SGY1528 growth in low potassium media can be rescued by heterologous expression of mammalian inward rectifying potassium channel Kir2.1, but not when Kir2.1 was artificially fused with the RXR motif (RKR) due to the efficient retention of channel in the ER (Shikano *et al.*, 2005). In the screen, SGY1528 cells were transformed with Kir2.1 constructs where random peptide library of 8-mer sequences was placed at the extreme C-terminus, downstream of the implanted RXR motif. By selecting the transformed cells that survived in low potassium media, the authors searched for the sequence that were able to override the ER localization activity of the RXR motif and restored the surface expression of the chimeric Kir2.1 channel. The screen of about 2x106 clones yielded several sequences that showed robust surface expression of Kir2.1 as tested in mammalian cell. Those sequences shared a minimum consensus of RXXS/TX-COOH and showed strong binding to 14-3-3, which required penultimate serine or threonine and the upstream arginine residues (Shikano *et al.*, 2005). By using one of the identified C-terminal sequences, namely RGRSWTY-COOH, Chung *et al*. investigated the relevant kinases. Using *in vitro*  phosphorylation assay using recombinant proteins and *in vivo* studies using reporter Kir2.1 channel bearing both RXR motif and downstream C-terminal RGRSWTY sequence, the authors found that Akt, but not PKA or CamKII, is responsible for direct phosphorylation of the RGRSWTY sequence which recruits 14-3-3 proteins to this site (Chung *et al.*, 2009). Importantly, the extracellular stimulation that activates Akt pathway, such as insulin and platelet-derived growth factor (PDGF), enhanced 14-3-3 binding and promoted the cell surface transport of the reporter Kir2.1 channel (Chung *et al.*, 2009). These results

although it was not shown in the literature whether this phosphorylation actually promotes

A similar masking mechanism was suggested for the ER-export of Iip35 isoform of major histocompatibility complex (MHC) class-II-associated invariant chain (O'Kelly *et al.*, 2002). During their assembly in the ER, MHC class-II αβ dimers associate with preformed trimers of the invariant chains (Iip33, Iip35, Iip41, Iip43), to form nonameric (αβIi)3 oligomers. It had been known that phosphorylation of an N-terminal serine8, present exclusively in the Iip35 cytoplasmic tail (NH3-MHRRRSRS…), is a prerequisite for efficient ER exit and sorting of class-II/Iip35 complexes to the cell surface. Phosphorylation of serine8 leads to the 14-3-3 binding to Iip35 and the alanine mutation on this residue inhibits the ER exit of class-II/Iip35 complex (Kuwana *et al.*, 1998). Together with the fact that 14-3-3 and COPI bound to the N-terminal sequence of Iip35 in a mutually exclusive manner, it was concluded that the cell surface transport of MHC class-II complex is promoted by 'masking' effect of 14-3-3 (O'Kelly *et al.*, 2002). However, as Khalil *et al*. pointed out (Khalil *et al.*, 2005), this model seems to require further investigation, since it is not consistent with the fact that, even when associated with a 14-3-3 protein, Iip35 will not leave the ER in the absence of class-II molecules and that mutation of serine8 to asparagine prevents 14-3-3 protein binding but

The effect of 14-3-3 binding on promoting cell surface transport was also found by a completely different approach. Shikano *et al*. screened a random peptide library to identify C-terminal peptide signals that would functionally override the ER localization activity of the RXR motif (Shikano *et al.*, 2005). The screening was based on the yeast growth complementation assay using a mutant *Saccharomyces cerevisiae* SGY1528 which cannot survive in low potassium media due to the lack of endogenous potassium uptake transporters. However, SGY1528 growth in low potassium media can be rescued by heterologous expression of mammalian inward rectifying potassium channel Kir2.1, but not when Kir2.1 was artificially fused with the RXR motif (RKR) due to the efficient retention of channel in the ER (Shikano *et al.*, 2005). In the screen, SGY1528 cells were transformed with Kir2.1 constructs where random peptide library of 8-mer sequences was placed at the extreme C-terminus, downstream of the implanted RXR motif. By selecting the transformed cells that survived in low potassium media, the authors searched for the sequence that were able to override the ER localization activity of the RXR motif and restored the surface expression of the chimeric Kir2.1 channel. The screen of about 2x106 clones yielded several sequences that showed robust surface expression of Kir2.1 as tested in mammalian cell. Those sequences shared a minimum consensus of RXXS/TX-COOH and showed strong binding to 14-3-3, which required penultimate serine or threonine and the upstream arginine residues (Shikano *et al.*, 2005). By using one of the identified C-terminal sequences, namely RGRSWTY-COOH, Chung *et al*. investigated the relevant kinases. Using *in vitro*  phosphorylation assay using recombinant proteins and *in vivo* studies using reporter Kir2.1 channel bearing both RXR motif and downstream C-terminal RGRSWTY sequence, the authors found that Akt, but not PKA or CamKII, is responsible for direct phosphorylation of the RGRSWTY sequence which recruits 14-3-3 proteins to this site (Chung *et al.*, 2009). Importantly, the extracellular stimulation that activates Akt pathway, such as insulin and platelet-derived growth factor (PDGF), enhanced 14-3-3 binding and promoted the cell surface transport of the reporter Kir2.1 channel (Chung *et al.*, 2009). These results

the binding of 14-3-3 proteins to these sites (Mant *et al.*, 2011).

still allows ER export if class-II molecules are present (Kuwana *et al.*, 1998).