| IntroductionProline-rich tyrosine kinase 2(PYK2)is a ubiquitously expressed non-receptor protein tyrosine kinase that has emerged as a crucial molecule in integrating signals from receptor tyrosine kinases and intracellular signaling molecules in processes such as cell survival,proliferation and motility.PYK2 was hard to detect in early embryo stages,however macrophage cells derived from PYK2-knockout embryos demonstrate severe migration and function defects.Conversely,enhanced PYK2 signaling increases cell motility and promotes cell survival in an anchorage-independent manner.Along these lines,a number of groups have reported that PYK2 protein level and/or its activity are upregulated in invasive cancer cells.Ligand binding of receptor tyrosine kinases results in autophosphorylation at Tyr402,which serves as a binding site for several Src homology 2(SH2)domain-containing proteins,including Src kinases.The PYK2-Src dual kinase complex leads to further phosphorylation of PYK2,and also to phosphorylation and activation of a number of cytoskeleton-linked proteins,which transduce signals to downstream pathways,such as the mitogen-activated protein (MAP)kinase cascades,and to control of cell proliferation,survival and motility.At present,little is known about the molecular mechanisms that negatively regulate PYK2 function in vivo.Suppressors of cytokine signaling 3(SOCS3)is a member of SOCS family that acts in a feedback loop to inhibit cytokine responses and activation of the STAT3 pathway in various cells.Evidence indicates that SOCS3 protein regulates other signaling pathways,as well.The expression of SOCS3 protein is decreased due to aberrant methylation in the promoter region,which frequently occurs in various types of human tumors.The SOCS3 protein is structurally characterized by distinct domains that include a N-terminal kinase inhibitory region,a central SH2 domain and a C-terminal homology region termed SOCS-box.The SH2 and KIR domains of SOCS3 protein mediate interaction with phosphorylated tyrosine residues,and in many cases, negatively regulate activities of tyrosine kinases.The SOCS-box may act as a bridge between SOCS-SH2 interacting proteins and E3 ubiquitin ligases,and promote turnover by targeting proteins for polyubiquitination and proteasome-mediated degradation.There is direct evidence that SOCS3 can not only regulate the JAK/STAT3 signaling pathway and also the FAK tyrosine kinase.We report here that the demethylation agent 5-aza-2'-deoxycytidine treatment and SOCS3 mutants transfection result in up-regulation of SOCS3 protein and investigate the subsequent effects on PYK2 protein level,Tyr402 and ERK1/2 activity in human lung adenocarcinoma A549 cells.SOCS3 was found to interact with PYK2 and facilitate degradation of PYK2,and inhibit Tyr402 and ERK1/2 phosphorylation and PYK2-associated migration of A549 cells.These results identify SOCS3 as a potential novel regulator of PYK2 signaling.Materials and methodsCell lines culture and transfection:Human bronchial epithelial cell line(HBE) were grown in RPMI 1640,and human lung adenocarcinoma cell line(A549)were cultured in DMEM,both supplemented with 10%fetal bovine serum.The SOCS3-SH2, SOCS3-KIR and SOCS-box mutants were kind gifts from Dr.Kristiina Vuori (Burnham Institute,CA,USA).The SOCS3-SH2 mutant is a full length SOCS3 without the SH2 region,consisting of the KIR and SOCS-box domains.The KIR region is knocked out in the SOCS3-KIR mutant,with the SH2 and SOCS-box regions remained.And the SOCS-box mutant contains the SH2 and KIR domains,with the SOCS-box deleted.For the reactivation study,A549 cells(50-60%confuence)were treated with 10μM 5-aza-2'-deoxycytidine(Sigma)for 6 days.DNA and RNA as well as protein were extracted and analyzed as described below.To exogenously express SOCS3 for protein analysis,A549 cells(80-90%confluence)were transfected with either SOCS3 mutants or pcDNA3 vector for 48h using Lipofectamin2000(Invitrogen), according to the manufacturer's protocol.Where indicated,the proteasome inhibitor,β-lactacystin(Santa Cruz)at 25μM was added to cells and maintained throughout the experiment.Methylation-specific PCR:MSP was carried out as described.The Methl Primer Express software v1.0(ABI)was used to design the methylation-specific and unmethylation-specific primers.Sequences of methylation-specific primers in the exon 2 of SOCS3 gene were 5'-TTC GAG GTG TTC GAG TAG TC-3'(forward)and 5'-AAC GAT CTT CCG ACA AAA AT-3'(reverse);Sequences of unmethylation-specific primers were 5'-TTT TTT GAG GTG TTT GAG TAG TT-3' (forward)and 5'-AAC AAT CTT CCA ACA AAA ATA CT-3'(reverse),and the length of PCR products were both 159bp.The conditions for PCR amplification were at 40 cycles of 94℃for 40s,63℃(methylation)and 62℃(unmethylation)for 40s,then 72℃for 40s.Sequences of two other MSP primer sets for exon 1 and intron 1 of SOCS3 gene are available upon request.RT-PCR analysis:Total RNA was prepared using Trizol(Invitrogen).cDNA was generated by reverse transcription of 1μg total RNA using Reverse Transcriptase M-MLV(Takara).PCR for PYK2 was performed using 25μl of reverse transcribed reaction mix with Taq polymerase(Takara).The primer sequences of PYK2 were 5'-AGA TTC CCG ACG AAA CCC-3'(forward)and 5'-CAC GGC GAA CAT CCA GAC-3'(reverse),and that ofβ-actin were 5'-AAA TCG TGC GTG ACA TTA A-3'(forward)and 5'-CTCGTCATACTCCTGCTTG-3'(reverse).The lengths of PYK2 andβ-actin PCR products were 629bp and 455bp respectively.The condition for PYK2 amplification was at 35 cycles of 95℃for 40s,54℃for 40s and 72℃for 40s.The PCR forβ-actin was performed in the same manner as PYK2,except that 50℃for annealing was used at 30 cycles of PCR.Western blot and co-immunoprecipitation analysis:Cells were washed twice with ice-cold phosphate-buffered saline(PBS)and lysed in M-PER Reagent(PIERCE) containing 1 mM PMSF and phosphatase inhibitor mix.The following antibodies were used in this study:anti-myc tag(Applygen,China),anti-SOCS3,anti-PYK2, anti-Tyr40,anti-ERK1/2,and anti-β-actin(Santa Cruz).Cells were lysed at 6d after 5-aza-2'-deoxycytidine treatment or 48h post-transfection,and the lysates were subjected to SDS-PAGE followed by western blot analysis.When indicated,cells were pretreated withβ-lactacystin at a concentration of 25μM in DMEM 4h before 5-aza-2'-deoxycytidine treatment or transfection and maintained throughout the experiments.The co-immunoprecipitation study was performed using Profound mammalian c-myc tag IP/Co-IP kit(PIERCE),according to the manufacturer's protocol.Clarified cell extracts were preincubated with agarose coupled with anti-c-myc tag antibody at 4℃overnight followed by SDS-PAGE and immunoblotting with anti-PYK2 antibody coupled with DAB detection.Transwell cell migration assay:Cell migration assay was performed using a 24-well Transwell chamber(Costar).At 6 days following 5-aza-2'-deoxycytidine treatment,cells(1×10~4)were detached and seeded in 24-well plate of 8μm pore size inserts and kept cultured in DMEM for another 12h.48h after SOCS3 mutants transfection,cells were treated similarly as described above.The cells were allowed to migrate forward to the DMEM containing 10%FBS in bottom chamber.The non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were fixed with 4% paraformaldehyde and stained with hematoxylin.The number of migrated cells was counted in the randomly selected 5 high power fields under microscope.Data presented are representative of three individual wells.Statistical analysis:SPSS for windows 13.0 statistical analysis soft was applied to complete data processing.Independent-samples t-test was used to evaluate the differences of optical density between cells with various treatments indicated.Results were considered statistically significant when the P-value was less than 0.05.ResultsPYK2 expression,Tyr402 and ERK1/2 phosphorylation,as well as migratory ability were up-regulated in A549 cells:We compared the level of PYK2 protein and activity,as indicated by Tyr402 phosphorylation,as well as activated ERK1/2 in A549 and HBE cells.As a normal control for A549 cells,HBE cells expressed a low level of PYK2 protein with an undetectable level of phosphorylated Tyr402.A basal level of activated ERK1/2 was also observed in HBE cells.However,we found much higher level of PYK2 protein,Tyr402 and ERK1/2 phosphorylation in A549 cells. Additionally,more migratory A549 cells(16.9±3.6)at 12h were detected than HBE cells(7.9±1.9,P＜0.01)by Transwell assay.Therefore,we presumed that the migration of A549 cells was correlated with the activities of both PYK2 and ERK1/2.ERK1/2 may play a role in signaling transduction to regulate the migration of A549 cells.Methylation-associated down-regulation of SOCS3 expression in A549 cells:We compared the expression of SOCS3 in A549 and HBE cells by western blot assay,and found that SOCS3 protein level was much lower in A549 than HBE cells.Researches on human cancers have indicated that down-regulation of SOCS3 expression was correlated with aberrant methylation.Thus,in our studies,three methylation- and non-methylation-specific primer sets were used to detect methylation status of SOCS3 gene in the exon 1,intron 1 and exon 2 in A549 cells respectively.SOCS3 gene methylation in the exon 2,which lies in 92-250 bp downstream to the translation start site was found in A549 cells,although no detectable methylation was found in the noncoding exon 1 and the intron 1.Moreover,no methylation was observed in HBE cells.This demonstrates that methylation in the exon 2 may lead to the decrease of SOCS3 expression in A549 cells.The non-methylation-specific amplification instead of methylation was detected after the treatment of demethylation agent 5-aza-2'-deoxycytidine,which further demonstrated the methylated SOCS3 gene in A549 cells.SOCS3 protein reexpression inhibited migration of A549 cell and the associated PYK2 and ERK1/2 activities:To investigate whether the SOCS3 protein involves in regulating migration of A549 cells,we compared the number of migratory cells with the treatment of demethylation compound,5-aza-2'-deoxycytidine or not.SOCS3 expression was restored by 5-aza-2'-deoxycytidine,and cell migration was inhibited. The number of migratory cells with or without 5-aza-2'-deoxycytidine treatment were 16.9±3.6 and 9.2±3.4,respectively(P＜0.01).We further examined the activities of PYK2 and ERK1/2 in A549 cells treated.We found the phosphorylated PYK2 and ERK1/2 decreased upon 5-aza-2'-deoxycytidine treatment and the decreased level of Tyr402 phosphorylation may be due to the reduced PYK2 protein,which may be attributed to the process of proteolytic degradation,because pretreatment withβ-lactacystin elevated PYK2 protein level in treated A549 cells,and the mRNA level of PYK2 was unaffected.Thus,SOCS3 protein may mediate degradation of PYK2 and play a role in down-regulating PYK2-associated migration of A549 cells.Roles of SOCS3 mutants in regulating activities of PYK2 and ERK1/2 and migration of A549 cells:We utilized A549 cells to investigate the roles of SOCS3 mutants in regulating the activities of PYK2 and ERK1/2 as well as cell migration.The expression of exogenous SOCS3-SH2 mutant protein was detected in A549 cells,as indicated by the myc tag protein.Concurred with PYK2 protein,the activities of Tyr402 and ERK1/2 were identical to the vector alone transfected cells.PYK2 and ERK1/2 remained activated in those transfectants.The number of migratory cells with vector and SOCS3-SH2 mutant transfection were 16.1±3.8 and 15.7±3.5,respectively (P=0.725).It seems that exogenous expression of SOCS3-SH2 mutant had no effect on the migratory ability of A549 cells.In SOCS3-KIR mutant transfected A549 cells,decreased PYK2 and ERK1/2 activities directly correlated with inhibited cell migration.The number of migratory cells with vector as well as SOCS3-KIR mutant transfection were 16.1±3.8 and 10.3±2.9,respectively(P＜0.01).Thus,cells with KIR region inactivated SOCS3 showed reduced PYK2 protein and diminished cell migration.To further determine whether the down-regulation in cell migration was induced by SOCS-box-mediated proteasome-dependent degradation of PYK2,pretreatment withβ-lactacystin was performed in transfected A549 cells,and the mRNA level of PYK2 was also examined by RT-PCR analysis.We found thatβ-lactacystin restored PYK2 protein level,and the number of migratory A549 cells also increased.However,the mRNA level of PYK2 was the same.These results indicated that exogenous SOCS3 suppressed migration of A549 cells may be correlated with proteasome pathway-mediated degradation of PYK2.Next,A549 cells were transfected with SOCS-box mutant protein.Compared with the vector alone transfected cells,Tyr402 and ERK1/2 phosphorylation were inhibited, despite that these cells expressed similar level of PYK2 protein as control cells.The number of migratory cells with vector and SOCS-box mutant transfection were 16.1±3.8 and 13.8±4.5,respectively(P=0.227).The SOCS-box inactivated mutant failed to decrease the level of PYK2 or inhibit cell migration significantly.The suppressed cell migration may be due to the kinase inhibitory activity of SOCS3.Thus, SOCS-box is required for SOCS3-induced degradation of PYK2 and inhibition of cell migration.Interaction of exogenous SOCS3 mutants and PYK2 in A549 cells:We further investigated whether exogenously expressed SOCS3 mutants interacted with PYK2 in the transfected A549 cells.PYK2 was co-immunoprecipitated with exogenous myc tag protein,which is clearly seen in SOCS-box mutant-transfected A549 cells but not vector control cells.However,neither the SOCS3-SH2 nor -KIR mutant/PYK2 co-IP products was observed in transfected A549 cells.These results collectively suggested that the SH2 and KIR region are essential for SOCS3 protein interacting with PYK2, and the formation of SOCS3/PYK2 complex may result in inhibited migration of A549 cells.DiscussionAlthough significant progress has been made in elucidating the PYK2 downstream signaling pathways,relatively little is known about the regulatory mechanisms of PYK2 activity.Our studies in this report demonstrate that the SOCS3 protein may have a previously unidentified role in negatively regulating PYK2 function.The expression of SOCS3 protein is decreased due to aberrant methylation,and its transcriptional silencing is associated with malignant tumor behavior.Human SOCS3 gene is composed of 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 gene in the exon 1,intron 1 and exon 2 in A549 cells respectively.We found SOCS3 gene methylation in the exon 2,which lies in 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 the intron 1,which was consistent with the previous findings.This demonstrates that methylation in the exon 2 leads to the decrease of SOCS3 expression in A549 cells.The demethylation agent, 5-aza-2'-deoxycytidine reactivated SOCS3 expression,further indicating that SOCS3 expression is attenuated by the associated methylation.Taken together,the exon 2 of SOCS3 gene appears to be crucial target for methylation in A549 cells.However,the significance of methylation in gene exons has not been elucidated.It has been shown that SOCS3 gene transfection inhibited proliferation of A549 cells.We therefore examined the effect of SOCS3 reexpression on migration of A549 cells.In our studies,We found that SOCS3 restoration inhibited migration of A549 cells. The negative regulation of SOCS3 was previously reported in PDGF-induced fibroblast and HGF-promoted keratinocyte migration,which was mediated by STAY3.However, STAT3 pathway may not be involved in SOCS3 gene methylation-induced migration of A549 cells,because STAT3 binding sites are not included in the exon 2 of SOCS3 gene. These data support that SOCS3 methylation-associated inactivation correlates with motility of A549 cells,and SOCS3 reexpression by the 5-aza-2'-deoxycytidine treatment inhibits migration ofA549 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 cancer,PYK2 expression was significantly elevated.PYK2 becomes Tyr402-phosphorylated in invasive breast cancer cells and SCLC cells,and PYK2-associated ERK1/2 activity participates in up-regulating the adhesive ability of human PCa cells.Pyk2 becomes activated by a number of cytokines that are known to regulate SOCS3 protein,such as SDF-1/CXCL12.As one of the negative regulators for PYK2 in T cells,SOCS3 showed migration suppression activity.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.Thus,similar mechanisms of interaction and inhibition could apply to PYK2.We found that the level of PYK2 protein,Tyr402 and ERK1/2 phosphorylation decreased with the increasing expression of SOCS3 by the treatment of 5-aza-2'-deoxycytidine.PYK2 seems to be suppressed by proteasome pathway-mediated protein degradation,because pretreatment withβ-lactacystin restored PYK2 protein level,Tyr402 and ERK1/2 phosphorylation to some extent and PYK2 mRNA levels were identical no matter whether treatment or not. As we concluded,inhibited migration of A549 cells is associated with the reduced PYK2 protein,Tyr402 and ERK1/2 phosphorylation level after SOCS3 restoration.We further examined the effects of SOCS3 mutants transfection on motility of A549 cells,and the associated PYK2 protein level,Tyr402 and ERK1/2 phosphorylation.When SOCS3-SH2 mutant exogenously expressed in A549 cells,the PYK2 protein level,Tyr402 and ERK1/2 phosphorylation,and the mobility of transfected A549 cells were similar to control cells.These findings indicated that SH2 region inactivated mutant deprived SOCS3 of the ability to down-regulate PYK2-associated signaling and function in A549 cells.Therefore,the SH2 region is crucial for SOCS3 regulating PYK2.A549 cells with SOCS3-KIR mutant showed decrease in the level of PYK2 protein.Tyr402 and ERK1/2 phosphorylation as well as the mobility of transfected A549 cells was also inhibited compared with control cells. Pretreatment withβ-lactacystin restored PYK2 protein level,Tyr402 and ERK1/2 phosphorylation,and cell migration to some extent.We propose that binding of PYK2 via SH2 domain and SOCS-box-mediated proteasome-dependent degradation of PYK2 leads to decreased mobility of transfected A549 cells,because PYK2 mRNA levels were identical no matter whether transfection or not.The KIR domain is possibly required for a more stable binding based on SH2 domain,because the potential interaction as we presumed between exogenous SOCS3-SH2 or -KIR mutant and PYK2 was not detected.The SOCS-box mutant transfection had no influence on the level of PYK2 protein,but Tyr402 and ERK1/2 phosphorylation decreased in contrast to control cells.The migration of transfected A549 cells was reduced.These data denoted that SOCS-box inactivated mutant transfection down-regulated PYK2-associated signaling and function possibly by binding of PYK2 via SH2 domain and inhibiting PYK2 kinase activity by KIR domain.PYK2 protein 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 protein is dependent on the SH2 and KIR domains of SOCS3,which results in the observed decrease in PYK2 protein level, leading to subsequent Tyr402 and ERK1/2 inactivation.Studies have demonstrated that Tyr402 phosphorylation of PYK2 leads to binding of the SH2 domain and activation of Src,or integrating the SH2 domain of CHK and inhibition of breast cancer cell migration.Our results are suggestive of an important role of SOCS3 on 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.Additional future research is needed to clarify the pathological significance of PYK2 protein and its interactions with SOCS3 in various signaling pathways.Studies of SOCS3 may foster new anti-chemotactic approaches to suppress lung cancer metastasis.Conclusions1 Down-regulationof SOCS3 by methylation might promote lymph node metastases of NSCLCs.2 Overexpression of PYK2 might facilite lymph node metastases of NSCLCs.3 Exogenous SOCS3 interacted with PYK2,depending on the SH2 and KIR domains. SOCS3 might mediate PYK2 degradation by proteasome pathway,inhibiting phosphorylations of Tyr402 and ERK1/2,and negatively regulating cell migration.
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