the actin cytoskeleton plays an essential role in numerous aspects of cell biology such as cell adhesion , cell morphology , cytokinesis , and migration .
cell migration machinery is regulated by signaling intermediates that can be activated by diverse stimuli and that can exert control over a large number of downstream target molecules , all with temporal and spatial specificity [ 1 , 2 ] .
pka , pkc , and ca - binding proteins are examples of cellular regulators that mediate diverse effects on cytoskeletal dynamics , cell adhesion , and cell migration [ 3 , 4 ] .
control of the subcellular localization of pka and pkc activities in a temporal manner by a - kinase - anchoring proteins ( akap ) has emerged as a pivotal mechanism to control cell migration .
for instance , akap12/ssecks / gravin ( akap12 ) is thought to control a number of cellular events by scaffolding key signaling molecules such as cyclin d1 , calmodulin , pka , and pkc ( figure 1 ) .
ssecks ( rodent akap12 ) , the src - suppressed - c - kinase - substrate , was originally identified in a screen for genes severely downregulated by v - src , but subsequently we and others showed that it is also downregulated by oncogenic forms of ras , myc and jun [ 7 , 8 ] and in sv40-transformed fibroblasts .
the gene encoding the human ssecks orthologue , gravin , is localized on chromosome 6q24 - 25.2 , a deletion hotspot in advanced prostate , ovarian , and breast cancer , implicating a role for the loss of akap12 in cancer progression .
importantly , akap12 orthologues have been identified in all vertebrate species , and , in humans and rodents , two major akap12 transcripts ,  and  , are expressed ubiquitously in the embryo and the adult as 305- or 290-kda products ( rodents : 290-kda or 280-kda ) , respectively .
akap12 transcript levels increase in confluent cultures of untransformed cells irrespective of the effects of serum growth factors [ 7 , 1012 ] , yet , in subconfluent cultures , they are unaffected by either serum deprivation or inhibition of dna synthesis .
although the coding sequence of akap12 contains several pest motifs linked to protein instability , akap12 is a long - lived protein under certain conditions .
newly synthesized akap12 in confluent cultures is not well phosphorylated , whereas serum addition to subconfluent cultures results in a rapid serine and tyrosine phosphorylation concurrent with g1 to s progression [ 13 , 15 ] .
akap12 is a major in vitro and in vivo substrate of pkc [ 9 , 16 ] .
both akap12 and pkc isozymes ( including conventional and novel ) contain phosphatidylserine ( ps ) binding sites , and although ps enhances akap12/pkc binding [ 9 , 17 ] , recent data identify two ps - independent pkc binding motifs in akap12 .
phosphorylation of akap12 in vitro with pkc decreases ps - mediated pkc binding , though it has not been shown that this phosphorylation inhibits ps binding itself .
interestingly , pkc - induced phosphorylation of akap12 causes it to translocate from plasma membrane and cytoskeletal sites to the perinucleus in fibroblasts , mesangial and epithelial cells [ 10 , 11 , 16 , 19 ] , suggesting that this event may play a role in the pkc - mediated reorganization of the actin cytoskeleton .
the functions of akap12 are based upon its ability to scaffold key signaling proteins in a spatiotemporal manner and specific scaffolding functions have been described for the control of ( i ) cell migration , ( ii ) maintenance of cytoskeletal architecture , ( iii ) cell proliferation , and ( iv ) cytokinesis .
one of major scaffolding roles for akap12 is as a critical regulator of cell migration .
for instance , akap12 reexpression is sufficient to inhibit src - induced anchorage - independence and matrigel invasiveness as well as to induce formation of normalized stress fibers and vinculin - associated adhesion plaques typical of those found in untransformed cells .
in addition , reexpression of akap12 in the rat metastatic prostate cancer cell line , mat - lylu , suppresses colony formation in soft agar , decreases refractility , and increases cell - cell interactions .
importantly , akap12 attenuates specialized motility , such as chemotaxis and invasiveness , rather than generic cell motility .
for instance , akap12 reexpression in mat - lylu cells has no effect on short- and long - term motility in monolayer wound - healing assays .
in contrast , akap12 inhibits chemotaxis via the attenuation of a pkc - raf / mek / erk pathway .
in addition , upregulated akap12 facilitates hgf - induced , c - met - dependent cell motility through the upregulation of pka activity and pka - induced genes , presumably through akap12 's scaffolding function .
interestingly , akap12 phosphorylation by pkc decreases akap12-pkc scaffolding but no change in akap12-pka binding   or agonist - induced pka activation .
it is likely that the differential pka - pkc control relates to an overlap between pkc binding and phosphorylation sites mapping to the n - terminus of akap12 , whereas the pka binding site maps to the c - terminus of akap12 lacking phosphorylation sites ( figure 1 ) .
the c - terminal domain of akap12 is required for akap12 to target pka to the cell periphery . however
, pkc activation by phorbol esters causes translocation of the akap12/pka complex to the perinucleus [ 23 , 24 ] . taken altogether ,
these data strengthen the notion that akap12 promotes the differential activation of pka and pkc in processes such as cell motility .
reexpression also enhances integrin - mediated adhesion and superinduces fak autophosphorylation levels , likely by physically disengaging src from fak complexes , which , in turn , leads to less focal adhesion turnover ( bing su , lingqiu gao , fanjie meng , li - wu guo , julian rothschild , irwin h. gelman , adhesion - mediated cytoskeletal remodeling is controlled by the direct scaffolding of src from fak complexes to lipid rafts by ssecks / akap12 .
in addition , mitogen - induced , fak - dependent tyrosine phosphorylation of akap12 modulates its binding to the actin - based cytoskeleton , suggesting a role for akap12 in mitogen - induced cytoskeletal reorganization .
recently we discovered that akap12 negatively regulates fak expression , implicating a role for akap12 in the control of fak levels .
although further work is required to address how akap12 controls fak expression , it is likely that akap12 affects both fak - mediated adhesion and motility pathways .
the ablation of akap12 expression in glomerular mesangial cells leads to the thickening and polarization of f - actin stress fibers , an increase in the number of transverse focal adhesion plaques , and an increase of phosphotyrosine staining in focal complexes .
consistent with this report , akap12-null mouse embryonic fibroblasts ( ko - mef ) exhibit robust stress fiber formation as well as increased numbers of focal adhesion plaques ( , figure 2 ) .
ablation of akap12 converts the stellate morphology of human mesangial cells and rodent fibroblasts to a spindle morphology . in actively dividing cells , akap12 associates with a cortical cytoskeleton and
overexpression of akap12 in untransformed nih3t3 cells causes g1-arrest marked by severe cell flattening , elaboration of an akap12-associated cytoskeleton , a transient loss of actin stress fibers and vinculin - associated adhesion plaques , and the production of filopodia and lamellipodia - like projections [ 25 , 28 ] .
ectopic expression of akap12 diminishes stress fiber formation likely through a direct binding to f - actin   ( figure 1 ) , suggesting that akap12 directly affects the formation or maintenance of stress fibers .
these observations suggest that akap12 plays a critical role in the dynamic reorganization of the actin cytoskeleton during the processes such as cell migration or maintenance of cell architecture .
another major scaffolding role for akap12 is as a negative regulator of g1 to s progression .
it is likely that akap12 controls cell cycle progression in nih3t3 cells by regulating cyclin d1 in two ways : ( i ) directly scaffolding cyclin d1 via cyclin d1 binding ( cy ) motifs , thereby facilitating contact - inhibition by sequestering cyclin d1 pools in the cytoplasm , and ( ii ) decreasing cyclin d1 expression by suppressing serum - inducible erk2 activity .
the cy domains overlap with major pkc phosphorylation sites on akap12 ( figure 1 ) .
the notion that akap12 scaffolding activity for cyclin d1 is antagonized after it is phosphorylated by pkc comes from the findings that ( i ) activation of pkc by phorbol esters induces cyclin d1 translocation to the nucleus , and ( ii ) phosphorylation of akap12 by pkc in vitro antagonizes its binding to cyclin d1 and cyclin e .
consistent with previous reports [ 16 , 23 , 24 ] , live cell imaging analysis demonstrates that localization of akap12 of resting cells is mainly in the cytoplasm , with some portion localizing to specific compartments such as plasma membrane and perinucleus ( s. akakura and i. h. gelman , unpublished observation ) .
although further work is required to elucidate the role of akap12 in cell cycle regulation , it is conceivable that akap12 localization and the timing of expression are critical for cell cycle progression .
we   and others [ 30 , 32 ] suggested that the loss of akap12 leads to cytokinesis defects .
a recent systems biology analysis implicates the existence of a mitotic protein complex containing akap12 and other molecules such as polo - like kinase 4 , apc , dynein and profilin ( http://www.mitocheck.org/cgi-bin/mtc?query=mcg_0000007 ) .
the furrow contains the actin - myosin ring whose pkc - rho gtpase - dependent contraction helps complete daughter chromosome separation , and , thus , it is conceivable that akap12 regulates cytokinesis via its ability to scaffold pkc and f - actin , and to attenuate rho gtpase activity .
since akap12 controls the activity of pkc , and pkc mediates the completion of cytokinesis , it is conceivable that akap12 normally scaffolds pkc and regulates actomyosin ring formation through controlling pkc-rhoa signaling during specific mitotic stages and structures .
despite the fact that akap12 is widely expressed throughout embryogenesis , akap12-null mice ( ko - mice ) are viable though they exhibit spontaneous prostatic hyperplasia .
dysplastic foci were observed less frequently but were associated with the loss of e - cadherin staining and the loss of basal cell markers , suggesting that the loss of akap12 causes a cancer - prone condition .
in fact , prostates of ko mice exhibit senescence associated -galactosidase expression , which can be used as a premalignant marker , implicating that the loss of akap12 causes a precancerous condition .
these cells are readily transformed by single oncogenes such as src or ras , suggesting that the loss of akap12 's tumor suppressor function renders the cell transformation - prone .
importantly , akap12 deficiency causes hyperactivation of pkc isozymes , leading to rb - dependent senescence involving pkc and  but not pkc. ko - mef are also more susceptible to immortalization in culture . immortalized
ko - mef have decreased levels of senescence associated -galactosidase staining and of the cyclin - kinase inhibitor , p16 , indicating that the cells override rb - dependent senescence . moreover
, expression levels of cyclin - dependent kinase 4 and lats / warts ( a mitotic kinase ) , which are downregulated in ko - mef , are upregulated in immortalized ko - mef .
these data suggest that akap12 facilitates pathways for continued proliferation through g1 and g2 phase arrest points found in senescent cells .
it is likely that akap12 controls senescence through a direct scaffolding of pkc isozymes because reexpression of full - length akap12 , but not akap12 deleted of its pkc - binding domains , suppresses senescence .
this suggests that akap12-null cells would be a unique tool to study the biological effects of pkc isozyme hyperactivation .
rhim et al . reported that the protein levels of akap12 are higher in senescent human diploid fibroblasts and in aging rat and human keratinocytes .
given our finding that ko - mef express higher levels of p47phox , a component of nadph oxidase , it is reasonable to speculate that akap12 suppresses the production of reactive oxygen species ( ros ) , ultimately serving as a protection against premature aging and spontaneous oncogenesis .
evidence is accumulating that akap12 expression is downregulated in many cancer types , either associated with gene deletion or epigenetic downregulation due to promoter hypermethylation or changes in chromatinization .
for instance , the expression level of akap12 is downregulated in breast cancer , leukemia , ovarian cancer , colorectal cancer , and hepatocellular carcinoma .
many microarray - based studies demonstrate significant reduction in relative akap12 mrna levels in many cancer types that cited in entrez geo ( gene expression omnibus ) or oncomine ( http://www.oncomine.org/ ) linking akap12 expression with tumor suppression .
in addition , we showed recently that akap12-null mice have increased susceptibility to papilloma and squamous cell carcinoma formation induced by dmba and tpa , well - known skin carcinogens .
interestingly , dermal layers in akap12-null mice are hyperplastic , and they show significant upregulation of fak , a known promoter of carcinogen - induced squamous cell carcinoma .
akap12 has been shown to function as a metastasis suppressor possibly by inhibiting the expression of vegf at distal sites , and by inhibiting oncogenic invasiveness .
reexpression of akap12 in mat - lylu cells causes a small decrease in primary subcutaneous tumor growth yet severely suppresses the formation of macroscopic lung metastasis .
in addition , multiple oncomine studies show significant decreases in akap12 expression in metastases compared to levels in primary tumors , suggesting a role for akap12 in suppressing metastasis . taken altogether
, these data suggest that akap12 is especially potent in regulating the metastatic process , a function likely relating to its ability to downregulate angiogenesis - controlling genes , such as vegf , and invasion - controlling genes , such as mmp-2 .
there is mounting evidence that akap12 scaffolding of pkc plays a role in regulation of mesangial cell differentiation and proliferation , and thus , glomerular function .
nelson et al . reported that akap12 mediates the control of the actin - based cytoskeletal architecture in mesangial cells by pkc .
reported that ssecks controls the localization and activity of cyclin d1 in glomerular parietal epithelial cells and influences response to proliferative injury in the glomerulus .
this paper demonstrates severely increased proliferative injury levels of glomerular parietal epithelial cells , leading to proteinurea in akap12-null versus wild type mice .
thus , akap12 plays a critical role in architectural maintenance of glomerular parietal epithelial cells , and akap12 deficiency increases the susceptibility to injury - induced glomerulonephritis .
agonist stimulation of the 2-adrenergic receptor leads to activation of kinases that are associated with akap12 .
pka - mediated phosphorylation of akap12 stabilizes the interaction between akap12 and the receptor , while pkc - mediated phosphorylation of akap12 causes it to translocate from the 2-adrenergic receptor .
prolonged agonist stimulation leads to degradation of the receptor and induces desensitization [ 48 , 49 ] .
akap12 is also critical in the regulation of blood - brain barrier ( bbb ) [ 45 , 50 ] .
akap12 attenuates neovascularization as well as barrier formation through the downregulation of proangiogenic genes such as hif1 or vegf [ 46 , 50 ] .
akap12 is upregulated during normoxic transition of the mouse embryo at birth , and akap12 is responsible for suppressing brain angiogenesis through a jnk - dependent downregulation of vegf and for inducing postnatal formation of the bbb by promoting tighter astrocyte / endothelial cell junctions .
reported that akap12 is essential for the integrity of the endothelium by maintaining the expression of pak2 and af6 , cell - cell adhesion regulators , during vascular development .
although akap12-null mice do not exhibit blood vessel issues , deficiency of akap12 causes hemorrhage in embryos of zebrafish and overexpression of pak2 and af6 is sufficient to rescue the abnormal hemorrhage in akap12-depleted zebrafish embryos . taken altogether , akap12 is essential for cellular architecture and is required for the integrity of cell - cell junction .
akap12 regulates cell cycle progression , cell motility , and cell morphology through its multiple scaffolding domains .
suppression of oncogenic proliferation , chemotaxis , and cellular senescence all involve attenuation of pkc activation through direct spatiotemporal scaffolding functions of akap12 .  
first , relating to its subcellular localization , it is still not clear whether akap12 plays a role in the nucleus , even though it contains at least six nuclear localization signals .
second , the molecular mechanisms by which akap12 differentially regulates the crosstalk between pka and pkc signaling pathways remain unclear .
lastly , other akap12 binding partners are likely to be found that either regulate akap12 functions or that are regulated by akap12 scaffolding .  
further studies are required to elucidate how the regulation of pkc and other molecules through scaffolding proteins such as akap12 maintains the integrity of cellular signaling and cytoskeletal control .
results from those studies would strongly suggest that targeting pkc - regulators such as akap12 should have therapeutic benefit for cancer patients .
 cellular dynamics are controlled by key signaling molecules such as camp - dependent protein kinase ( pka ) and protein kinase c ( pkc ) . 
 akap12/ssecks / gravin ( akap12 ) is a scaffold protein for pka and pkc which controls actin - cytoskeleton reorganization in a spatiotemporal manner . 
 akap12 also acts as a tumor suppressor which regulates cell - cycle progression and inhibits src - mediated oncogenic signaling and cytoskeletal pathways . 
 reexpression of akap12 causes cell flattening , reorganization of the actin cytoskeleton , and the production of normalized focal adhesion structures . 
 downregulation of akap12 induces the formation of thickened , longitudinal stress fibers and the proliferation of adhesion complexes . 
 akap12-null mouse embryonic fibroblasts exhibit hyperactivation of pkc , premature cellular senescence , and defects in cytokinesis , relating to the loss of pkc scaffolding activity by akap12 . 
 akap12-null mice exhibit increased cell senescence and increased susceptibility to carcinogen - induced oncogenesis . 
 the paper describes the regulatory and scaffolding functions of akap12 and how it regulates cell adhesion , signaling , and oncogenic suppression . 
1. Introduction
2. Role of AKAP12 on Cellular Architecture, Adhesion, and Migration
3. Role of AKAP12 on Cell Cycle Regulation and Cytokinesis
4. AKAP12 as a Tumor Suppressor and Metastasis Suppressor
5. Other Roles for AKAP12
6. Conclusion