TI - Discussion . AB - Although much progress has been made on elucidating the PYK2 downstream signaling pathways , relatively little is known about the regulatory mechanisms of PYK2 . Our studies in this report demonstrated that SOCS3 might have a previously unidentified role in negatively regulating PYK2 function . The expression of SOCS3 is decreased due to aberrant methylation , and its transcriptional silencing is associated with malignant tumor behaviors [27] . Human SOCS3 gene is composed of the noncoding exon 1 , intron 1 and exon 2 . In our studies , the methylation - and non-methylation-specific primers were used to detect methylation status of SOCS3 in the exon 1 , intron 1 and exon 2 in A549 cells respectively . We found SOCS3 methylation in the exon 2 , which lies in a region of 92-250 bp downstream to the translation start site , although no detectable methylation was found in the promotor region containing the noncoding exon 1 and intron 1 , which was consistent with the previous findings [28] . This demonstrated that methylation in the exon 2 was associated with the decrease of SOCS3 expression in A549 cells . The demethylation agent 5-aza-2'-deoxycytidine reactivated SOCS3 , which further indicated that SOCS3 expression was attenuated by the associated methylation . Taken together , the exon 2 of SOCS3 appeared to be a crucial target for methylation in A549 cells . However , the significance of methylation in exons has not been elucidated . It has been shown that SOCS3 transfection inhibited proliferation of A549 cells [15] . We therefore examined the effect of SOCS3 up-regulation on migration of A549 cells . In our studies , we found that SOCS3 restoration inhibited cell migration . The negative regulation of SOCS3 was previously reported in PDGF -induced fibroblast [29] and HGF-promoted keratinocyte migration [30] , which was mediated by STAT3 . However , STAT3 pathway may not be involved in SOCS3 methylation-induced migration of A549 cells , because STAT3 binding sites [25] are not included in the exon 2 of SOCS3 . These data supported that SOCS3 methylation-associated inactivation might facilitate cell motility and SOCS3 reactivation by 5-aza-2'-deoxycytidine treatment inhibited migration of A549 cells . A growing number of reports have demonstrated that PYK2 plays a significant role in development of various tumors . In metastatic glioma cells and hepatocellular carcinoma , PYK2 expression is significantly elevated [31,32] . PYK2 becomes Tyr402 -PHOSphorylated in invasive breast cancer [33] and SCLC cells [34] and PYK2-associated ERK1/2 activation participates in up-regulating the adhesive ability of PCa cells [35] . Pyk2 becomes activated by a number of cytokines that are also known to regulate SOCS3 , such as SDF-1/CXCL12 [2,18] . As a negative regulator for PYK2 in T cells , SOCS3 showed migration suppression activity [36] . We also notice that SOCS3 binds to the PHOSphorylated Tyr397 of FAK , a close homolog of PYK2 , and inhibits its kinase activity or induces degradation by proteasome pathway [19] . Thus , similar mechanisms of interaction and inhibition could apply to PYK2 . We found that PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations were inhibited with the increasing expression of SOCS3 by the treatment of 5-aza-2'-deoxycytidine . PYK2 seemed to be down-regulated by proteasome-mediated degradation , because pretreatment with beta-lactacystin restored PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations to some extent and PYK2 mRNA levels were unchanged no matter whether treatment or not . As we concluded , inhibited migration of A549 cells was associated with the reduced PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations after SOCS3 restoration . We further examined the effects of SOCS3 mutants on cell motility , and the associated PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations . When SOCS3-SH2 mutant exogenously expressed in A549 cells , the PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations , as well as cell migration were unchanged regardless of whether transfection or not . Moreover , the interaction of exogenous SOCS3-SH2 mutant with PYK2 was not detected . These findings indicated that the SH2 region inactivated mutant deprived SOCS3 of the ability to down-regulate PYK2-associated signaling and function in A549 cells . Therefore , the SH2 domain is crucial for SOCS3 regulating PYK2 . Compared with control cells , A549 cells with SOCS3-KIR mutant showed decreased PYK2 expression . Tyr402 and ERK1/2 PHOSphorylations , as well as cell mobility . Pretreatment with beta-lactacystin restored PYK2 expression , Tyr402 and ERK1/2 PHOSphorylations and cell migration to some extent . We proposed that the binding of SOCS3 with PYK2 via SH2 domain and SOCS-box -mediated proteasome -dependent degradation of PYK2 led to the suppressed migration of transfected A549 cells . The roles of SOCS3-KIR mutant were observed definitely , however the potential interaction as we presumed between the exogenous SOCS3-KIR mutant and PYK2 was not detected . We supposed that SOCS3-KIR mutant interacted with PYK2 principally by the central SH2 domain , generating the inhibition effects , however the binding was a functional interaction , unstable and readily reversible , and the effects would be transient without the help of KIR domain , thus could not be detected by immunoprecipitation in vitro . Therefore we concluded that the KIR domain is possibly required for a more stable binding of SOCS3 with PYK2 based on the SH2 domain . Further investigation is required to demonstrate our point of view . The SOCS-box mutant had no influence on PYK2 expression , but Tyr402 and ERK1/2 PHOSphorylations were reduced in contrast to control cells . The migration of transfected A549 cells was also inhibited . These data denoted that the SOCS-box inactivated mutant down-regulated PYK2-associated signaling and function possibly by binding to PYK2 via the SH2 domain and inhibiting PYK2 activation by the KIR domain . PYK2 was detected in cell lysates immunoprecipitated with anti-myc tag antibody , which suggested a direct interaction between SOCS-box mutant and PYK2 . Taken together , the stable interaction between exogenous SOCS3 and PYK2 is dependent on the SH2 and KIR domains of SOCS3 , which results in the observed decrease in PYK2 expression , leading to the subsequent Tyr402 and ERK1/2 inactivations . Studies have demonstrated that Tyr402 PHOSphorylation of PYK2 leads to binding of the SH2 domain and activation of Src [37] or integration of the SH2 domain of CHK and inhibition of breast cancer cell migration [38] . Our results are suggestive of an important role of SOCS3 in blocking Tyr402 PHOSphorylation . However , it remains to be determined whether Tyr402 mediates the interaction between SOCS3 and PYK2 . Moreover , the precise role of SOCS3 in regulating migration-associated cytoskeleton proteins downstream of PYK2 has not been characterized in relation to lung cancer metastasis . Taken together , our results indicate that SOCS3 interacts with PYK2 and inhibits PYK2-associated migration of A549 cells . Further research is needed to clarify the pathological significance of PYK2 expression and interaction with SOCS3 in various signaling pathways . Studies of SOCS3 may foster new anti-chemotactic approaches to suppress lung cancer metastasis .