Abstract
BackgroundSuppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor kinase and has been found crucial to cell motility. However, little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer.MethodsThe methylation status of SOCS3 was investigated in HBE and A549 cell lines by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine or transfected with three SOCS3 mutants with various functional domains deleted. Besides, cells were pretreated with a proteasome inhibitor β-lactacystin where indicated. The effects of SOCS3 up-regulation on PYK2 expression, PYK2 and ERK1/2 phosphorylations were assessed by western blot using indicated antibodies. RT-PCR was used to estimate PYK2 mRNA levels. Transwell experiments were performed to evaluate cell migration.ResultsSOCS3 expression was found impaired in A549 cells and higher PYK2 activity was correlated with enhanced cell migration. We identified that SOCS3 was aberrantly methylated in the exon 2, and 5-aza-2'-deoxycytidine restored SOCS3 expression. Reactivation of SOCS3 attenuated PYK2 expression and phosphorylation, cell migration was inhibited as well. Transfection studies indicated that exogenous SOCS3 interacted with PYK2, and both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of SOCS3 contributed to PYK2 binding. Furthermore, SOCS3 was found to inhibit PYK2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of PYK2 in a SOCS-box-dependent manner and interfered with PYK2-related signaling events, such as cell migration.ConclusionThese data indicate that SOCS3 negatively regulates cell motility and decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells. These results also suggest a negative role of SOCS3 in PYK2 signaling, and a previously unidentified regulatory mechanism for PYK2 function.
Highlights
Suppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells
Up-regulation of Proline-rich tyrosine kinase 2 (PYK2) expression, Tyr402 and ERK1/2 phosphorylations, as well as cell migration in A549 cells PYK2 expression and activation, as indicated by Tyr402 phosphorylation, and ERK1/2 phosphorylation were evaluated in HBE and A549 cells respectively
The non-methylation specific amplification instead of methylation was detected after the demethylation agent 5-aza-2'deoxycytidine treatment, and SOCS3 expression was restored (p < 0.05, Fig. 3b), which further demonstrated that SOCS3 was methylated in A549 cells
Summary
Suppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor kinase and has been found crucial to cell motility. Little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer. Proline-rich tyrosine kinase 2 (PYK2) is a ubiquitously expressed non-receptor protein tyrosine kinase that contributes to integrate signals from receptor tyrosine kinases and intracellular signaling molecules in processes such as cell survival [1], proliferation [2] and motility [3]. Inhibition of Pyk mostly results in decreased cell migration [9,10] Along these lines, report has been shown that PYK2 expression and/or its activity are up-regulated in invasive cancer cells [11]. Little is known about the molecular mechanisms that negatively regulate PYK2 function in lung cancer
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