The SH3 domain of p56lck 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-p561k fusion proteins and cell extracts to map regions on p56kk that are involved in binding to phosphatidylinositol 3'-kinase (P13K). Deletions within the SH3 domain of pS6kk abolished binding of P13K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of P13K activity bound to p56kk sequences. The binding of P13K from T-cell extracts to p56kk was not blocked by antiphosphotyrosine antibodies, but p56-kkbound P13K activity was sensitive to phosphatase treatment. The SH3 domain of p56kk also bound the majority of P13K 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 P13K activity bound to the SH2 domain of p56k in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56kk, 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 p56kk. In the other mode, involving P13K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of pS6kk.The Ick gene is a member of the src family of genes, which encode a series of eight closely related protein tyrosine kinases (for a review, see reference 23?. The product of the ick gene is a 56-kDa protein (p561 ) that is expressed predominantly in cells of the lymphoid lineage, primarily T lymphocytes and lymphoid tumor cell lines (28, 30, 37).Expression of p56" has also been observed in human carcinomas of the lung and colon (50).Members of the Src family of tyrosine kinases are characterized by an NH2-terminal unique region followed by three regions that contain different degrees of homology among all members of the family (23, 42). In p561ck, Src homology region 3 (SH3) extends from amino acids 56 to 114. The SH2 domain includes amino acids 115 to 221 and the SH1, or catalytic, domain comprises most of the remainder of the C-terminal half of the protein. Recent evidence indicates that the SH2 region of Src-like kinases functions as a protein association domain that is important in the formation of multienzyme complexes that regulate signal transduction through tyrosine phosphorylation (5, 27).Several proteins have been reported to form complexes with pS6lc in T lymphocytes. The NH2-terminal unique region of p56"kk has been shown to associate with the C-terminal region of two T-cell transmembrane glycoproteins, CD4 and CD8 (3,39,40,45,48). The accessory role of CD4 and CD8 in T-cell signalling has led to the suggestion that p56"'" may participate in antigen-stimulated signal transduction events (18,36)....

The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3'-kinase.

“…However, several recent reports demonstrate that, upon TCR stimulation, p85 associates with the src family kinases Ick and fyn (48,49,67), and TCR stimulation results in serine phosphorylation of p85 and threonine phosphorylation of the pllO subunit of P13 kinase (50 …”

The Kinase, SH3, and SH2 Domains of Lck Play Critical Roles in T-Cell Activation after ZAP-70 Membrane Localization

“…The same mutation at this residue of the SH3 domain abrogated association of Src with various molecules (26). A number of molecules have been shown to bind the SH3 domain of Lck, such as HS1 (9,30,31,64,79), p120 Cbl (23,29,58), p120 Ras-GAP (2, 3, 11), Ras-GAP-associated molecules p62 and p190 (3), and phosphatidylinositol 3-kinase (53,71). It should VOL.…”

Phosphoinositide Kinases

“…The proline-rich motifs of p85α mediate binding to SH3 domains of src family kinases including src itself, lck, lyn, and fyn (63)(64)(65)(66)(67)(68)(69). The SH3 domain of the cytoplasmic tyrosine kinase abl also associates with p85α (63).…”