PP60SRC BINDING PROTEIN AFAP-110

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SH2 and SH3 domains represent important cell signalling motifs that are conserved among a number of kinases known to mediate mitogenesis and transformation. SH2 domains function by interacting with phosphotyrosine containing substrates, while SH3 domains are hypothesized to link proteins to either cytoskeletal structures and/or ras signaling pathways. The integrity of the SH2 and SH3 domains of the nonreceptor tyrosine kinase pp60 (Src) are critical for mediating transformation. These domains are likely to be important to be important for mediating pp60 signaling processes, as well. Therefore, the identity of cellular proteins that interact with the SH2 and/or SH3 domains of pp60 will likely represent important components in signal transduction and transformation. In this proposal, the mechanism by which the novel pp60 binding protein, AFAP-110 (for Actin Filament Associated Protein, 110 kDa) forms a stable complex with pp60 will be examined. AFAP-110 can independently form a stable interaction with either the Src or Fyn SH2 and SH3 domains. AFAP-110 does encode peptide motifs that represent consensus Sr SH2 and SH3 binding motifs, thus AFAP-110 is distinctive in that it represents both an SH2 and SH3 binding protein. Identification of these motifs will be accomplished by introducing mutations that abrogate SH2 or SH3 mediated interactions. A hypothesis will be tested that indicates the mechanism of stable complex formation between AFAP-110 and pp60 occurs by a two-step binding process, governed by a primary SH43 mediated interaction, followed by tyrosine phosphorylation and SH2 mediated stable complex formation. The potential role of AFAP-110 in modulating transformation will be examined by competitively disrupting the pp60. AFAP-110 stable complex, in vivo. AFAP-110 will also serve as a model for "peptide mimetics", whereby the SH2 and/or SH3 binding motifs will be overexpressed in cells susceptible to transformation by Src. In this way, the SH2 and/or SH3 domains would be blocked from interactions with cellular substrates. Should this interruption effect the transformed phenotype, then this technique could be applied to human cancer cell lines that express activated tyrosine kinases, to determine the role of those kinases in effecting phenotype.