The SH3 Domain of p56^lck Is Involved in Binding to Phosphatidylinositol 3′-Kinase from T Lymphocytes

Abstract

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56^lck fusion proteins and cell extracts to map regions on p56^lck that are involved in binding to phosphatidylinositol 3′-kinase (PI3K). Deletions within the SH3 domain of p56^lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56^lck sequences. The binding of PI3K from T-cell extracts to p56^lck was not blocked by antiphosphotyrosine antibodies, but p56^lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56^lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56^lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56^lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56^lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56^lck.