TI - Roles of SOCS3 mutants in regulating PYK2 expression , Tyr402 and ERK1/2 activations and migration of A549 cells . AB - Concurred with PYK2 expression , cells with the SH2 domain deleted SOCS3 showed PHOSphorylations of Tyr402 and ERK1/2 were identical to that in the vector alone-transfected cells ( p gt 005 , data not shown ) . The numbers of migrated cells transfected with vector and SOCS3-SH2 mutant were 16.1 + - 3.8 , 15.7 + - 3.5 , respectively ( p gt 005 , data not shown ) . Cells with the KIR domain inactivated SOCS3 showed reduced PYK2 expression , as well as PYK2 and ERK1/2 PHOSphorylations ( p lt 005 , Fig 5a ) , which were in parallel with inhibited cell migration . The numbers of migrated cells with vector or SOCS3-KIR mutant transfection and beta-lactacystin pretreatment were 16.1 + - 3.8 , 10.3 + - 2.9 and 13.8 + - 3.6 , respectively ( p lt 005 , Fig 5c ) . To further determine whether the suppressed cell migration was induced by SOCS-box -mediated proteasome -dependent degradation of PYK2 , pretreatment with beta-lactacystin was performed in transfected cells and PYK2 mRNA levels were also examined . We found that beta-lactacystin elevated PYK2 expression , and the number of migrated cells also increased . However , PYK2 mRNA levels were unchanged ( p gt 005 , Fig 5b ) . These results indicated that the inhibited migration of A549 cells was possibly correlated with proteasome-mediated degradation of PYK2 . Next , A549 cells were transfected with SOCS-box mutant . Compared with the vector alone-transfected cells , Tyr402 and ERK1/2 PHOSphorylations were inhibited ( p lt 005 , Fig 6a ) , despite that these cells expressed similar level of PYK2 as control cells ( p gt 005 , Fig 6a ) . Pretreatment with beta-lactacystin was also performed in transfected cells and no effects were detected on PYK2 expression , Tyr402 and ERK1/2 activations ( p gt 005 , Fig 6a ) . PYK2 mRNA levels were unchanged as well ( p gt 005 , Fig 6b ) . The numbers of migrated cells with vector or SOCS-box mutant transfection and beta-lactacystin pretreatment were 16.1 + - 3.8 , 13.8 + - 4.5 and 14.2 + - 4.2 , respectively ( p gt 005 , Fig 6c ) . The SOCS-box inactivated mutant failed to decrease PYK2 expression and inhibit cell migration . The suppressed PHOSphorylations of Tyr402 and ERK1/2 were possibly due to the kinase inhibitory activity of SOCS3 .