signal transducing adaptor proteins are a group of intracellular and transmembrane molecules which are crucial supplementary factors of signaling pathways . they mediate interactions between different molecules and contribute to the formation of signaling complexes .
adaptor proteins lack enzymatic activity and interaction domains enable them to connect with other molecules ( e.g. , proteins , lipids ) .
src - like adaptor protein 1 ( slap-1 ) was cloned in a yeast two - hybrid screen with the cytoplasmic domain of the receptor tyrosine kinase ephrin type - a receptor 2 as decoy [ 1 , 2 ] . slap proteins are not identical in size ; slap-1 ( or often quoted as slap ) contains 276 amino acids while slap-2 consists of 261 amino acids ( figure 1 ) .
src protein tyrosine kinases ( ptks ) are protooncogenes that play a role in cell proliferation , survival , and morphology . unlike src ptks ,
in addition to the myristoylated amino - terminal and sh domains , slap family members contain unique carboxy - terminal sequences as well .
slap-1 has a longer carboxy - tail than slap-2 , but the n - terminal is longer in slap-2 . myristoylated part of the slap promotes the association with membranes , while isoforms without the myristoylated n - terminal are located in the nucleus ( figure 1 ) .
human slap is coded by a 64  kb intron of the thyroglobulin gene , on chromosome 8q24.23 in the candidate territory for a recessive demyelinating neuropathy .
sequence analysis could not find any mutations suggesting that this gene is not responsible for the disease .
sh domains are the most similar parts of the two slap molecules , in which the sequence homology is 59% , while the n - terminals are the most different parts ; they have only 19% identity .
this isoform contains only 210 amino acids due to the deletion of 50  bp from exon 6 , which results an alternative reading frame .
although expression of slap-1 and slap-2 mrnas has been most extensively studied in lymphocytes , they are also expressed by numerous human and murine tissues and cell lines [ 1 , 2 , 712 ] ( table 1 ) .
several proteins have been reported to interact with slap-1 and slap-2 [ 5 , 8 , 9 , 1315 ] ( figure 2 ) .
slaps are involved in a broad range of cellular processes , for example , lymphocyte development , neuronal excitotoxicity and platelet activation .
slap molecules may participate in several pathological conditions of the immune system as well . in the present review
, we will discuss the role of both slap proteins in different cell types and overview our current understanding regarding their relevance in pathological conditions .
tcr consists of a ligand binding  heterodimer and the cd3 complex that includes the  and  heterodimers and the  homodimer .
the expression of the -chains is about the 10% of the other subunits of the tcr complex .
slap-1 may interact with many molecules involved in the tcr signal transduction ( zap-70 , syk , lat , cd3 -chain , vav , and lck ) through the sh2 domain [ 9 , 16 ] .
slap-1 reduces the production of il-2 and the transcription of nfatc1 and ap-1 , thereafter functions as a negative regulator of the tcr signaling ; both sh2 and sh3 domains are necessary for this effect . however , upon ionomycin or pma activation of lymphocytes , these inhibitory effects are absent suggesting that slap-1 regulates the proximal part of the tcr pathway .
slap may associate with the n - terminal of the e3 ubiquitin ligase c - cbl in a tyrosine phosphorylation independent way .
the simultaneous expression of slap-1 and c - cbl promotes the ubiquitination and degradation of the -chain , consequently enhancing the recycling and preventing the accumulation of the receptor complexes .
by contrast , similarly to slap / lymphocytes , c - cbl / lymphocytes overexpress the -chain due to its abolished degradation .
for successful operation , slap-1 requires the phosphorylation of the cytoplasmic domains of the tcr -chains and the activation of lck , but not of zap70 .
it has been shown that the phosphorylated proportion of the -chains is slightly traceable in lck / lymphocytes [ 20 , 21 ] .
slap colocalizes with early endosomes according to confocal microscopy images   ( figure 3 ) .
it is noteworthy that downregulation of -chain of t - cells has been observed in many pathological conditions including rheumatoid arthritis ( ra ) , systemic lupus erythematosus ( sle ) , human immunodeficiency virus ( hiv ) infection , and various cancers [ 2228 ] .
the fate of the early lymphocytes depends on the tcr - mediated signals : both too strong and too week signals through the tcr lead to cell death ( during positive and negative selections , resp . ) .
it is expressed at low levels in cd4 cd8 cells and at high levels in cd4 + cd8 + thymocytes .
slap as a regulator of the tcr expression plays a pivotal role in the downregulation of tcr complexes of the developing lymphocytes .
moreover , the apoptosis of zap70 / cells was inhibited in the absence of slap .
it has been reported that the proline rich - sequence ( prs ) of cd3 act together with slap in the regulation of tcr expression in cd4 + cd8 + thymocytes .
cd3 prs deficient cells were unable to degrade the -chain . in double positive thymocytes ,
the chains of the tcr complex are constitutively ubiquitinated , but the ubiquitination is absent in mature cells .
it has been shown that cd3 prs , lck , c - cbl , and slap are required for the ubiquitination and degradation of the -chains . in the absence of ubiquitination , both the lysosomal sequestration and degradation are failed , and tcr chains are upregulated in cd4 + cd8 + lymphocytes .
in addition , modified tcr complex ubiquitination influences the formation of the immunological synapse and alters the selection of the immature cells .
the lack of slap increases the avidity of the tcr which leads to the negative selection of antigen specific cd8 + cells .
all these data suggest that slap plays a major role in the tcr repertoire configuration .
slap is associated with c - cbl in b lymphocytes , leading to bcr recycling   ( figure 4 ) .
slap deficiency increases the bcr levels of immature b cells in hel - specific md4- transgenic mice , which is a frequently used model to study nave b lymphocytes . in this model
the upregulated receptor complex levels lead to increased signal transduction . slap deficient mice have an increased number of splenic b cells , but the surface expression of bcr and igm of mature lymphocytes is decreased .
furthermore , the activation induced calcium flux is diminished in slap ko b cells . in virtue of the previous data slap
regulates the level of bcr s which is essential for the adequate development and function of b cells .
slap-2 has a negative regulatory role on the antigen receptor signaling of t and b lymphocytes .
the overexpression of slap-1 and slap-2 inhibits the upregulation of cd69 after antigen receptor cross - linking .
slap-2 suppresses the antigen binding induced calcium influx in t ( jurkat ) and b ( bjab ) cell lines .
slap-2 reduces the cd69 expression in jurkat cells ( 61% ) more significantly than in bjab cells ( 28% ) .
slap-2 is associated with ubiquitin ligase c - cbl similarly to slap-1 , deletion of the carboxy - terminal of slap-2 was reported to inhibit this connection . in activated jurkat cells
slap-2 binds to c - cbl , zap-70 , and cd3  in a phosphorylation independent manner .
the coexpression of slap-2 and zap-70 or syk in t - cell lines lead to the degradation of both kinases .
thus , slap-2 induces the c - cbl dependent degradation of tyrosine kinases and downregulates cd3 expression .
slap-2 is expressed in human monocytes and bone marrow cells [ 1 , 35 ] , but not in csf1 independent monocyte cell lines such as raw264.7 .
it was shown that slap-2 may bind to both c - fms and c - cbl , and several domains of slap-2 are involved in this interaction ( figure 5 ) .
the overexpression of slap-2 in murine bone marrow reduced the m - csf - induced tyrosine phosphorylation .
slap-2 downregulates the c - fms signaling through a c - cbl dependent internalization and degradation of the csf-1r , providing a negative feedback of the m - csf pathway .
in addition , c - fms stimulation induces the phosphorylation of the serine rich n - terminal domain of the slap-2 by a jnk dependent pathway .
the activation - induced gm - csfr downregulation is attenuated in slap-1 and slap-2 deficient bone marrow dendritic cells ( bm - dc ) , which is associated with enhanced map / erk and akt pathways upon gm - csf stimulation . the inhibited activation through gm - csfr impairs the bone marrow derived dendritic cell maturation .
slap-1 and slap-2 deficient bm - dc cells produce less il-12 and tnf- upon lipopolysaccharide ( lps ) stimulation and induce significantly less ifn- secretion of t - lymphocytes than the wild type cells .
these data suggest that slap proteins are necessary for monocyte and dendritic cell maturation and activation .
slap has a prominent role in the regulation of intracellular signal transduction in mast cells .
slap - specific small interfering rnas inhibit the effect of dexamethasone on the phosphorylation of plc  , lat , syk , and erk .
actinomycin d inhibits the transcription of slap following dexamethason treatment , suggesting that the upregulation for slap-1 upon glucocorticoid treatment occurs at the transcriptional level .
slap-1 plays a role in the negative regulation of antigen - stimulated mast cells as well . upon antigen stimulation of the rbl-2h3 mast cell line ,
elevated transcription of the sla gene and upregulation of the slap-1 ( but not slap-2 ) protein were reported .
an increased amount of slap-1 was detected after 60 minutes stimulation and it reached the maximum after 2 - 3 hours .
by contrast , after silencing of slap , increased il-3 and mcp-1 production was detected .
inhibiting the development and activation of osteoclasts is currently the gold standard therapeutic strategy in this disease .
although today several antiosteoporotic drugs are widely used in the clinical practice , the effect of these medications on increasing bone density and strength is moderate .
there is an inverse correlation between both slap-1 and slap-2 and the tartrate - resistant acid phosphatase ( osteoclastogenesis marker ) mrna expression .
the level of osteoclast - specific protein mrnas ( e.g. , catepsin k and mmp-9 ) are elevated in the slap-1 / preosteoclasts .
slap-1 has an inhibitory effect on the map kinase pathway which is initiated with the binding of m - csf to its receptor c - fms .
this receptor is a tyrosine kinase transmembrane protein which accumulates in lipid rafts where it associates with slap-1 .
the lack of slap enhances osteoclastogenesis without changing the resorptive function of the individual cells .
in addition , slap-1 deficiency increases the apoptosis of the mature polykariotic osteoclasts without altering the viability of the precursors . according to our current understanding
, slap-1 has a regulatory effect on osteoclastogenesis and mature cell survival through the m - csf pathway .
slap-2 is expressed in human platelets and may associate with syk , c - cbl , and lat .
after its activation , slap-2 promotes syk and c - cbl to approach their substrates .
slap-2 inhibits glycoprotein vi ( gpvi ) initiated signal transduction of platelets through the connection with c - cbl .
this inhibitory effect on thrombocyte activation is similar to those described previously in lymphocytes [ 1 , 5 ] .
slap may also associate with platelet derived growth factor receptor ( pdgfr ) in nih3t3 mouse embryonic fibroblast cell line .
overexpression of slap in nih3t3 cells inhibits pdgf - induced mitogenesis   suggesting that slap is a negative regulator of growth factor initiated signaling .
although in embryonic rat telencephalon sections slap-1 mrna is absent in migrating neurons , it is highly and selectively expressed in neurons which have reached their final location .
pyramid cells are typically located in the deeper layers of the cortex , and they have connection with the subcortical areas .
it is not yet clear whether slap-1 plays a role in the axon guidance during development but apparently has a remarkable expression .
western blot analysis and immunolabeling of rat brain extract showed that slap-1 is located in the postsynaptic membrane .
slap-1 has an association with ephas and nmdars have a connection with ephbs [ 44 , 45 ] .
ephb may excite the enlistment of ephas and promote the recruitment of slap-1 and nmdars . despite the analogous localization
, slap-1 does not affect the baseline activity of nmdars through a src - dependent stimulation of the receptors .
slap-1 may have a role in the negative feedback which regulates the number of the nmdars and prevent excitotoxicity .
this effect of slap-1 is based on the proteasomal degradation of redundant nmdars in neurons .
the described role of slap in neurons seems to be essential for the adequate neuronal functions .
rheumatoid arthritis ( ra ) is a common autoimmune disease that is associated with progressive disability and systemic complications .
symmetric synovial inflammation of multiple joints , especially the small joints of the hands and the feet , is characteristic for the disease .
the ongoing inflammation leads to cartilage destruction , bone erosions , and subsequent joint deformities .
although the current treatment strategy , especially the use of biologicals , improved largely the outcome of the disease , still only a small portion of the patients are in sustained remission and systemic complications ( including cardiovascular risk ) still represent a significant challenge .
tnf- plays an essential role in the pathogenesis of ra through promotion of angiogenesis , suppression of regulatory t - cell activation , and cytokine and chemokine expression . according to the recently published data of our research group , tnf- treatment downregulates the expression of -chain of cd4 t - lymphocytes reversibly and selectively in a dose dependent way .
tnf- induces the expression of slap which promotes the proteasomal but not the lysosomal degradation of the -chain .
tnf- treatment does not alter the slap mrna level , suggesting that tnf- controls slap activity through mirna mediated posttranscriptional silencing .
cd4 + t - lymphocytes isolated from ra patients expressed more than 2-fold higher slap levels than the t - cells of healthy donors .
tnf- treatment enhances the expression of slap in the cd4 + t - lymphocytes of healthy donors and dmard treated ra patients but does not alter the expression of cd4 + t - cells isolated from biological dmard ( etanercept , certolizumab pegol ) treated patients .
a spontaneous mutation in zap70 protein uncoupled proximal tcr signal transduction and led to severe symmetrical arthritis in skg mice upon exposure to zymosan .
according to recently published data , slap deficiency dramatically reduced both the incidence and severity of zymosan - induced chronic autoimmune arthritis in skg mice .
the protective role of slap deficiency was associated with the increased number of regulatory t - cells and decreased amount of th17 cells .
slap expression is increased in several cancers including chronic myeloid leukemia ( cml ) , chronic lymphocytic leukemia ( cll ) , and prostate cancer . by contrast , its expression is decreased in acute myeloid leukemia ( aml ) , myeloma , and colon cancer . furthermore , slap is associated with several oncogenic signaling pathways . it was recently reported that the transmembrane protooncogene c - kit is degraded through a slap dependent pathway .
slap binds to the wt c - kit and initiates its ubiquitylation and degradation in the proteasome .
by contrast , slap does not have a similar regulatory effect on the oncogenic c - kit variant ( c - kit d816v ) .
oncogenic c - kit associates with slap and phosphorylates it on three different tyrosines ( y120 , y258 , and y273 ) ; the phosphorylated forms of slap do not alter the downstream signaling of the mutant c - kit .
these data suggest that slap regulates wt c - kit signaling , but the oncogenic variant escapes from this negative feedback regulation .
the fms like tyrosine kinase ( flt3 ) is a receptor tyrosine kinase , which is predominantly expressed in hematopoietic progenitor cells .
after ligand stimulation , slap colocalizes with the flt3-itd and targets it for c - cbl dependent ubiquitylation and proteasomal degradation .
about 30% of patients with aml have mutation in the flt3 gene and gain of function mutations contribute to the initiation of aml .
the expression of slap is increased in patients with acute promyelocytic leukemia ( apl ) carrying flt3-itd mutation as compared to the flt3-wt .
fli-1 is a transcription factor , a member of the e26 transformation - specific ( ets ) protein family .
furthermore , the fli-1 locus is an integration site for the friend murine leukemia virus , which induces erythroleukaemia in responsive mice .
slap protein and mrna levels are overexpressed in fli-1 transformed erythroblasts . slap binds to both the phosphorylated and unphosphorylated forms of the erythropoietin receptor ( epor ) .
slap expression prevents the epo - induced differentiation , whose effect is associated with the inhibition of stat5 activation and bcl - x upregulation .
, slap is expressed in colon epithelium , but it is significantly downregulated in colorectal tumors .
interestingly , silencing of slap promotes tumor progression , while overexpression inhibits tumor growth and invasiveness .
slap promotes the destabilization of the src substrate ephrin type - a receptor 2 ( epha2 ) in the intestinal cells .
epha2 plays a critical role in the regulation of several intracellular signal pathways which mediate cell migration , invasion , and angiogenesis .
the inhibitory effect of slap appears to be independent from c - cbl but has an association with the ubiquitination factor ube4a and with the ptyr594-epha2 .
slap proteins are expressed in a variety of cell types which indicates a conserved function of these proteins .
both slap-1 and slap-2 have a prominent role in the negative regulation of several membrane bound receptors and receptor tyrosine kinases , thereafter slaps play a central role in the regulation of intracellular signal transduction and cell reactivity .
the proper function of slaps is necessary for immunoreceptor repertoire configuration and helps to avoid uncontrolled cell activation , proliferation , and migration .
slap molecules are involved in the ubiquitination of proteins , which may lead to proteasomal degradation .
selective regulation of slap molecules in different cell types may allow the fine control of cell activation and differentiation .
exploring the precise role of slap proteins will contribute to the understanding of many yet unknown physiological regulatory processes .
in addition , tissue specific targeting of slap may provide valuable therapeutic approaches in diverse diseases including ra , osteoporosis , immunodeficiency , and different cancers .
 although src - like adaptor proteins ( slap-1 and slap-2 ) were mainly studied in lymphocytes , where they act as negative regulators and provide fine control of receptor signaling , recently , several other functions of these proteins were discovered . 
 in addition to the well - characterized immunoregulatory functions , slap proteins appear to have an essential role in the pathogenesis of type i hypersensitivity , osteoporosis , and numerous malignant diseases . 
 both adaptor proteins are expressed in a wide variety of tissues , where they have mostly inhibitory effects on multiple intracellular signaling pathways . in this review , 
 we summarize the diverse effects of slap proteins . 