Identification of Syk -interacting proteins by a yeast two-hybrid system
Abstract
Syk is a 72 kDa protein tyrosine kinase that is expressed most prominently in hematopoietic cells. Syk couples the antigen receptor to multiple intracellular signaling pathways that are mediated, in part, by its interactions with downstream positive and negative effectors such as phospholipase Cγ, Vav, Fgr, and Cbl. While these interactions are fairly well understood, additional protein-protein interactions that modulate Syk function, such as its intracellular localization and translocation in response to receptor engagement, are poorly understood. To investigate these questions, we used a yeast two-hybrid screen to identify cDNA's for candidate Syk-interacting proteins from a bone marrow cDNA library. The p85 regulatory subunit of PI3K (phosphatidylinositol-3 kinase) and tensing were identified as Syk-interacting proteins. The interaction of p85 with Syk requires the (N+C)SH2 domain of p85 and phosphotyrosines of the Syk linker region. Phosphopeptide binding assays showed that the C-terminal SH2 domain of p85 preferentially interacts with Syk phosphotyrosine 317. Since the combined function of PI3K and Syk is important for FcγRIIA-induced phagocytosis, the effect of Syk-p85 interaction on phagocytosis was investigated in artificial system, FcγRIIA-expressing COS7 cells. Interestingly, the ability of COS7 cells in a phagocytosis assay was increased by Syk(WT) whereas it was completely abrogated by the inactive form of Syk (Syk(K396R)), Syk(Y317F), Syk(Y342F/346F), and Syk(Y317F/Y342F/Y346F). This result suggests that protein interactions with phosphotyrosines, Y317, Y342, and Y346 as well as catalytic activity are required for phagocytosis. Tens2 does not require phosphorylation for its interaction with Syk, which occurs in the linker B region. This interaction was confirmed by the co-expression of Syk and tensing in insect cells using baculoviral vectors to express epitope-tagged proteins. The localization of tensing using anti-tensing antibody in HUMEC1 and MCF 10A cells was investigated, which shows a punctate pattern similar to tensin.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Immunology|Molecular biology
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