The phosphatidylinositol polyphosphate 5-phosphatase SHIP1 associates with the dok1 phosphoprotein in bcr-Abl transformed cells.

The initial phase of chronic myelogenous leukemia (CML) is triggered by constitutive protein tyrosine kinase activity of the chimeric kinase p210(bcr-abl) (Bcr-Abl). A major substrate of Bcr-Abl was recently identified as the RasGAP-associated 62 kDa docking protein Dok1. Here, we report complex formation between endogenous Dok1 and the SH2 domain-containing phosphatidylinositol polyphosphate 5-phosphatase SHIP1 in hematopoietic cells expressing Bcr-Abl. Expression of Bcr-Abl induced tyrosine phosphorylation of both Dok1 and SHIP1 and the formation of a Dok1/SHIP1 complex. Tyr(P) SHIP1 was also bound to Shc in Bcr-Abl expressing cells. A small amount of Shc/SHIP1/Dok1 trimolecular complex was detected and this was due to binding of Dok1 to SHIP1 that was bound to Shc. In contrast, association of Dok1 with SHIP1 or RasGAP was mutually exclusive. Both the SH2 domain of SHIP1 and the PTB domain of Dok1 were required for complex formation between the two proteins. Neither the specific activity of SHIP1 as an inositol phosphate 5-phosphatase nor the subcellular localization of SHIP1 appeared to be altered by tyrosine phosphorylation. However, the Dok1/SHIP1 complex was only detected in the cytosolic fraction of Bcr-Abl transformed hematopoietic cells. We propose that interaction between Dok1 and SHIP1 modulates the ability of these two proteins to interact with other cytosolic binding partners.

The N terminus of hamster polyomavirus middle T antigen carries a determinant for specific activation of p59c-Fyn.

Transformation by rodent polyomaviruses is mediated primarily by middle T antigen, a membrane-bound protein that does not carry an intrinsic enzymatic activity but interacts and subverts the activity of cellular regulators of proliferation. The multiple protein partners of murine polyomavirus (Py) middle T antigen include the tyrosine kinases c-Src and, to a lesser extent, c-Fyn and c-Yes. By contrast, the hamster polyomavirus (HaPV) middle T antigen selectively activates the c-Fyn gene product. This difference may account for the contrasting tumor patterns induced by the two viruses. The sequences of the respective N-terminal and C-terminal functional domains of murine Py and HaPV middle T antigens are highly conserved whereas the intervening stretches are clearly divergent, leading to the speculation that this divergence may direct the specificity for tyrosine kinase activation. We have addressed this issue by constructing a chimera middle T antigen molecule carrying the N-terminal domain from HaPV (exon 1) in phase with the other two domains from murine Py (exon 2). The biological properties of this chimera molecule are indistinguishable from those of HaPV middle T antigen; it specifically activates p59c-Fyn and carries the transforming phenotype of the HaPV middle T antigen on rat fibroblasts.

Functional interaction between the SH2 domain of Fyn and tyrosine 324 of hamster polyomavirus middle-T antigen.

Middle-T antigen of mouse polyomavirus (MomT) associates with the cellular tyrosine kinases c-Src, c-Yes, and Fyn, while middle-T antigen of hamster polyomavirus (HamT) exclusively binds Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. Here we show that the kinase domain of Fyn is sufficient for association with MomT but not for binding of HamT. We further demonstrate that a Fyn mutant lacking the SH2 domain is able to bind MomT but fails to associate with HamT, indicating that the SH2 domain of Fyn is essential for stable association with HamT. HamT, but not MomT, contains a tyrosine residue, Tyr-324, in the sequence context YEEI. Mutation of Tyr-324 to phenylalanine led to a drastic reduction of associated Fyn and abolished the oncogenicity of HamT. This suggests that Tyr-324 is the major phosphotyrosine residue mediating the binding of HamT to the SH2 domain of Fyn. These findings show that mouse and hamster polyomaviruses use different strategies to target Src-related tyrosine kinases.

Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src.