Cooperative activation of Src family kinases by SH3 and SH2 ligands

Abstract

Src family nonreceptor tyrosine kinases are kept in a repressed state by intramolecular interactions involving the SH3 and SH2 domains of the enzymes. Ligands for these domains can displace the intramolecular associations and activate the kinases. Here, we carried out in vitro activation experiments with purified, down-regulated hematopoietic cell kinase (Hck), a Src family kinase. We show that SH3 and SH2 ligands act cooperatively to activate Src family kinases: the presence of one ligand lowers the concentration of the second ligand necessary for activation. To confirm the findings in intact cells, we studied Cas, a Src substrate that possesses SH2 and SH3 ligands. In contrast to wild-type Cas, mutant forms of Cas lacking the SH3 or SH2 ligands were unable to stimulate Src autophosphorylation when expressed in Cas-deficient fibroblasts. Cells expressing the Cas mutants also showed decreased amounts of activated Src at focal adhesions. The results suggest that proteins containing ligands for both SH3 and SH2 domains can produce a synergistic activation of Src family kinases.

Figure 1

Cooperativity between the SH3 and SH2 domains of Hck. [A] Hck activity was measured using the continuous spectrophotometric assay. Assays were carried out in the presence of varying concentrations of SH3 ligand at fixed concentrations of SH2 ligand. [B] Hck activity was measured in the presence of varying concentrations of SH2 ligand at fixed concentrations of SH3 ligand. Activation constants were determined as described in the text.

Figure 2

(A) Domain arrangement of Cas showing the SH3 domain (SH3), proline-rich region (Pro), substrate region containing 15 repeats of YXXP, serine-rich region (Ser), C-terminal Src binding sequence (SBS), and helix-loop-helix region (HLH). Within the SBS, the sequences of the Src SH3 ligand (RPLPSPP) and SH2 ligand (YDYV) are shown. The PPX mutant carries mutations in the proline rich sequence (underlined) that impair interaction with the SH3 domains of SFKs. The Y668F mutation (underlined) blocks interaction with the SH2 domain of Src. The Y668F/PPX double mutant has both the binding sequences mutated. (B) Autophosphorylation of Src in Cas^−/− cells. Cells expressing wild-type or mutant forms of Cas (or vector control) were lysed and analyzed by SDS-PAGE with Western blotting. The membrane was probed with antibodies for autophosphorylated Src (pY416), then stripped and reprobed with antibodies against total Src, Cas, and tubulin as a loading control. (C) Co-immunoprecipitation of Src and Cas. Cas^−/− cells expressing wild-type or mutant forms of Cas (or vector control) were lysed and subjected to anti-Cas immunoprecipitation reactions. Proteins in the precipitates were transferred to membrane and detected by anti-FLAG Western blotting (for Src). The membrane was stripped and reprobed with anti-Cas antibody (the arrowhead indicates the position of Cas).

Figure 3

Decreased amounts of activated Src in cells expressing Cas mutants. Cas^−/− cells were co-transfected with Src and Cas (wild-type or mutants). Cas expressing cells were identified as YFP positive and analyzed for Src activation. Cells were immunostained 24 hours after transfection with anti-pY416 antibody to visualize activated Src. Nuclei were visualized with DAPI. [A]: YFP vector control. [B]: wild-type Cas. [C]: Y668F Cas mutant. [D]: PPX mutant. [E]: Y668F/PPX double mutant. [F]–[J]: Superimposed DIC and YFP images of Cas^−/− cells expressing YFP-tagged WT or mutant forms of Cas. A similar cytosolic distribution was observed for all forms of Cas.