Tyrosine phosphorylation and activation of Etk upon coexpression with v-Src in 293 cells. 293 cells were transiently transfected with wild-type (WT) or kinase-defective (KD), T7-tagged Etk and/or v-Src as indicated above each lane. Two days later, cells were lysed and the cell lysates were used for immunoprecipitations with anti-T7 antibody. (A) As shown on the left, the immunocomplexes were subjected to in vitro kinase assays in the presence of [γ-³²P]ATP and enolase as an exogenous substrate. Phosphorylated proteins were separated by SDS-PAGE and detected by autoradiography. Kinase assay products similar to those of lanes 2 and 3 of the blot shown on the left were separated on a longer gel and detected by autoradiography. The estimated migration of STAT3 protein is indicated by the asterisk. (B) Immunocomplexes (lanes are as described for panel A) were resolved by SDS-PAGE and subjected to Western blotting (WB) with antiphosphotyrosine antibody (anti-PY) or anti-Etk antibody.

Src phosphorylates Etk at Y566. (A) Mutation at Y566 decreases the Src-stimulated tyrosine phosphorylation of Etk. 293 cells were cotransfected with T7 tagged-wild type or mutant Etk and v-Src as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitations with anti-T7 antibody followed by Western blotting (WB) with antiphosphotyrosine antibody (anti-PY) or anti-Etk antibody. (B) Src phosphorylates Etk on Y566 in vitro. A constitutively active Src (c-Src 527F) was transfected into 293 cells. Cell lysates were subjected to immunoprecipitations (IP) with anti-c-Src antibody (anti-Src) or a control antibody (C), and the precipitated proteins were used for in vitro kinase assays in the presence of GST-Etk-K(K445Q), GST-Etk-K(Y566F), or GST-Etk-K(K445Q, Y566F) as the substrate. Phosphorylated proteins were separated by SDS-PAGE and detected by autoradiography, as shown at the top of the panel. The asterisk and arrow indicate the phosphorylated Src and GST fusion proteins, respectively. The amount of each GST fusion protein used in the kinase assay is shown at the bottom of the panel to demonstrate an equal input.

Etk associates with STAT3 in vivo via the PH domain of Etk. (A) Schematic representation of the Etk protein and its mutants. Various structural domains are also labeled. RP, the two direct repeats in the Etk sequence (48); PK, protein kinase domain. (B) Association of STAT3 with Etk or its deletion mutants. 293 cells were transfected with STAT3 and/or T7-tagged wild-type or mutant Etk as indicated. Cell lysates were immunoprecipitated with anti-T7 antibody, followed by Western blotting with anti-STAT3 antibody to detect Etk-bound STAT3 (top) or with anti-Etk antibody to demonstrate the expression of Etk and its mutants (middle). Similar cell lysates were subjected to Western blotting with anti-STAT3 antibody to detect the expression of STAT3 (bottom).

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.

Src induces tyrosine phosphorylation of JAK2. JAK2 was immunoprecipitated from lysates of WB and its derivatives. The immunoprecipitates (IP) were used for Western blotting (WB) with antiphosphotyrosine (α-pY) or anti-JAK2 antibody.

Dominant-negative inhibition of Etk reduces soft agar cloning efficiency of v-Src-transformed WB cells. Cells (5 × 10⁴) of each type as described for Fig. 3 were seeded in soft agar dishes (60-mm diameter). Colonies were stained and counted after 1 week.

Etk and c-Src 378G synergize in STAT3 activation and transformation of NIH3T3 cells. (A) Expression levels of Etk (upper blot) or c-Src mutant (lower blot) in stable cell lines. NIH3T3 cells stably expressing Etk and c-Src378G were generated by retrovirus-mediated gene transfer. Pools of drug-resistant cells were lysed, and the cell lysates were used for Western blotting with anti-Etk antibody or anti c-Src antibody. (B) Etk potentiates the transforming activity of c-Src 378G. Cells as described in for panel A were used for soft agar colony formation assay. Cells (5 × 10⁵) were seeded in each dish, and colonies were stained and counted after 4 weeks. (C) Etk activates STAT3 in synergy with c-Src378G. Parental NIH3T3 cells and stable lines as described for Fig. 6A were transiently transfected with 1.5 μg of pGASLuc STAT3 reporter and 0.5 μg of pRKβ-gal. Each experiment was carried out in triplicate, and the error bars represent standard deviations. For each experiment, the luciferase activity was normalized to β-galactosidase activity to account for transfection efficiency.

Expression of Etk enhances the soft agar cloning efficiency of Hep3B cells. (A) Overexpression of Etk (upper blot) or EtkKQ (lower blot) in Hep3B stable transfectants. Cell lysates from Hep3B or its stable transfectants were subjected to Western blotting with anti-Etk antibody. The positions of Etk and EtkKQ are indicated. (B) Soft agar colony formation assay for Hep3B and stable transfectants. Hep3B Etk and Hep3B EtkKQ are mixtures of the four stable lines shown in panel A. For each experiment, 10⁴ cells were seeded. Colonies with diameters larger than 1 mm and between 0.3 and 1 mm were separately counted after 4 weeks of incubation, and the numbers given are averages of at least two independent experiments.

Dominant-negative inhibition of STAT3 abrogates the effect of Etk on transformation of Hep3B cells. (A) Expression levels of Etk and STAT3D in Hep3B stable transfectants. Each stable transfectant was a mixture of several clones. Western blot analysis with anti-Etk (upper blot) or anti-STAT3 (lower blot) was performed with cell lysates containing equal amounts of proteins. The positions of Etk and STAT3 are indicated. (B) Soft agar colony formation assay. Cells described above were seeded as described for Fig. 7A. Pictures were taken after 4 weeks of incubation.

Model depicting participation of Etk in cell transformation (see text).