FIG. 4.

Etk mediates the activation of STAT3 by v-Src. (A) The kinase-defective mutant of Etk (EtkKQ) functions as a dominant-negative mutant. The upper blot shows the expression levels of Etk and EtkKQ in parental WB cell and four stable cell lines as demonstrated by immunoblotting with anti-Etk antibody. The lower blot shows Etk kinase activities in WB and its stable lines. EtkKQ-M is a mixture of all four stable clones. Lysates of cells were immunoprecipitated with anti-Etk antibody, followed by in vitro kinase assays. The autophosphorylated Etk is shown. (B) Introducing v-Src into WB and its derivatives by recombinant retrovirus. WB and its derivatives were infected by retrovirus containing v-Src and the puromycin resistance gene. Lysates from equal numbers of puromycin resistant cells from each infection were used for Western blotting with antibody specific to v-Src. (C) EtkKQ blocks STAT3 DNA binding activity induced by v-Src. Nuclear extracts from WB and its derivatives as indicated were subjected to EMSA using a ³²P-labeled hSIE probe. A 133-fold molar excess of cold hSIE or a nonspecific oligonucleotide (NS) was used as the competitor, and supershifting (STAT3* indicates supershifted complex) was performed with anti-STAT3 antibody. The anti-STAT1 antibody was included as a control. (D) EtkKQ inhibits v-Src-induced tyrosine phosphorylation of STAT3. Lysates from cells as indicated were used for Western blotting with an antibody specific to STAT3 phosphorylated at position 705 (phospho-STAT) (upper blot) or the anti-STAT3 antibody (lower blot). (E) EtkKQ does not generally block tyrosine phosphorylation on v-Src targets. Lysates from cells as indicated were used for Western blotting with antiphosphotyrosine antibody (anti-pY) or antitubulin antibody.