Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase

Abstract

Ligand binding of multi-chain antigen receptors and hematopoietin/cytokine receptors results in rapid activation of protein tyrosine kinase (PTK)-dependent signalling molecules such as phosphatidylinositol 3-kinase (PI 3-kinase). Co-precipitation studies have shown that Srcrelated PTK, such as Lyn, associates with the p85 regulatory subunit of PI 3-kinase via SH2 and SH3 domain binding with their cognate ligands. More recent studies have shown that the proto-oncogene product Cbl co-precipitates with p85 following engagement of cytokine and antigen receptors. As opposed to in vitro co-precipitation studies, the yeast two-hybrid screen reveals in vivo protein ± protein interactions. Using the yeast two-hybrid screen, we demonstrate an in vivo association of Lyn's SH3 and SH2 domains with the proline-rich domain of Cbl. Lyn's SH3 and SH2 domains do not interact with p85 in the yeast two-hybrid screen, as would be predicted from glutathione-S-transferase (GST) fusion protein pull-down or co-immunoprecipitation studies from whole cell lysates. However, the SH3 domain of p85 interacts with the proline-rich domain of Cbl. When yeast were transformed with catalytic Lyn, an interaction between p85's SH2 domain and Cbl occurred. From the data, we propose the following three step process of PI 3-kinase activation: (1) complexes of Lyn ± Cbl and Cbl ± p85 exist without ligand stimulation, (2) upon ligand binding, Lyn becomes active and phosphorylates Cbl, and (3) Cbl's tyrosine phosphorylated residue serves as a docking site for the SH2 domains of p85 ± thereby stabilizing the complex and activating PI 3-kinase. The yeast two-hybrid system can be used to dissect the precise mechanisms of in vivo protein ± protein interactions, including those between phosphotyrosine and SH2-containing proteins.

Involvement of Shc and Cbl-PI 3-kinase in Lyn-dependent proliferative signaling pathways for G-CSF

“…Similar results were seen with LY294002, which reversibly inhibits PI 3-kinase by competing for its substrate binding site (data not shown). Through coordination of SH3 and SH2 domains with proline-rich and phosphotyrosine motifs, Lyn can couple to PI 3-kinase via an adaptor molecule, such as Cbl (Dombrosky-Ferlan and Corey, 1997;Hunter et al, 1999). We observed that G-CSF-induced tyrosine phosphorylation of Cbl also correlated strictly with G-CSF-induced proliferation (Figure 5c).…”

Perlecan Proteolysis Induces an α2β1 Integrin- and Src Family Kinase-dependent Anti-apoptotic Pathway in Fibroblasts in the Absence of Focal Adhesion Kinase Activation

“…PI3K is a downstream target of integrin-FAK/SFK pathways and can be activated through various and nonmutually exclusive pathways (34 -36). Upon integrin ligation, activation of PI3K can result from the binding of the Src homology 3 domain of Src with a proline-rich region within the p85 subunit of PI3K or through interactions between p85 with tyrosine-phosphorylated docking proteins such as Cb1 (22,23,37). Integrin-dependent FAK autophosphorylation on Tyr 397 may lead to the recruitment of Src homology 2 domain containing signaling proteins such as PI3K (22).…”

Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis

“…As these assays do not measure the relative strength of association, we cannot exclude contribution of sequences outside the SH3 domain to efficient binding. The 516-535 proline-rich cluster of human GADD34 and the homologous 511-530 cluster of MyD116 are structurally the most likely sites responsible for the interaction with the SH3 domain of Lyn because they conform the consensus Lyn SH3 binding site (26,27). Interestingly, a deletion overlapping the proline-rich cluster of GADD34 has been reported to abrogate binding of HRX leukemic fusion proteins (6), implicating this region of GADD34 as a site of multiple protein interactions.…”

“…pcDNA3-Lyn and its K275R mutant have been described (17). Glutathione S-transferase (GST)-Lyn bacterial expression constructs are derivatives of pGEX-2T (Amersham Pharmacia) carrying Lyn cDNA fragments (26). All DNA constructs were verified by sequencing.…”