Overexpressed Csk Tyrosine Kinase Is Localized in Focal Adhesions, Causes Reorganization of (cid:97) v (cid:98) 5 Integrin, and Interferes with HeLa Cell Spreading

The C-terminal Src kinase p50 csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not signiﬁcantly suppressed, while the amount of p50 csk and Csk activity increased at least 10-fold during 3 days of induction. Immunoﬂuorescence for the induced p50 csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125 FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the (cid:97) v (cid:98) 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These synthesized wild-type fragment in pG ation, E. mutation csk was into the phagemid from which single- stranded was phage oligonucleotides so that their 5 (cid:57) halves (30 nucleotides) were complementary to the 30 nucleotides preceding the triplets encoding the ﬁrst amino acids of the SH3 or SH2 domain and their 3 (cid:57) halves (30 nucleotides) were complementary to the sequences downstream of the last triplets of these domains. Upon annealing of these oligonucleotides to the csk single-stranded DNA, in vitro DNA synthesis of the second strand, ligation, and propagation in E. coli mutS for assays. The activity of p50 csk was measured in immunoprecipitates from TKB lysates, using two different assays. In one assay, activity was measured as ﬂuorographic signals on ﬁlms; in the other, the incorporation of [ (cid:103) - 32 P]ATP into poly(Glu/Tyr) was measured in a liquid scintillation counter. For the ﬁrst assay, the last two washes of the immunoprecipitates were done in the assay buffer (10 mM HEPES [pH 7.4], 5 mM MnCl 2 ). As a substrate, 600 ng of baculovirus-derived, puriﬁed Src K295M (a generous gift David Morgan) was used per reaction. The reaction was initiated by the addition of ATP (to 3 (cid:109) M) and 10 (cid:109) Ci [ (cid:103) - 32 P]ATP (3,000 Ci/mmol), incubated for 5 min at 30 (cid:56) C, terminated by heating in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and analyzed by electrophoresis and autoradiography. The poly(Glu/Tyr) assays were performed with equal amounts of immunoprecipitates from TKB lysates for each time point essentially as described previously (13). After two washes in the assay buffer (10 mM HEPES [pH 7.4], 5 mM for Csk; 50 HEPES 7.4], , (cid:109) for c-Src or Src-family TKs), the reaction was started by addition of poly(Glu/Tyr) (4:1; Sigma) to 1 mg/ml, ATP to ﬁnal concentrations of 3 (cid:109) M for Csk and 1 (cid:109) M for c-Src and Src family TKs, and 3 (cid:109) Ci of [ (cid:103) - 32 P]ATP (3,000 Ci/mmol). After careful mixing, the samples were incubated for 10 min at 30 (cid:56) C. The reactions were stopped by bringing the mixture to ﬁnal concentrations of 3 mM ATP, 30 mM EDTA, and 1.5 mg of BSA per ml; 20- (cid:109) l samples were spotted in duplicate onto Whatman 3MM ﬁlter paper squares. The squares were 10% trichloroacetic pyrophosphate for 15 min, then three to four times for 15 min each in 5% trichloroacetic acid, and ﬁnally in ethanol and dried, and their radioactivity was measured in a liquid scintillation counter. As controls, immunoprecipitates