Mechanism of Signal Transduction by Nonreceptor Protein-tyrosine Kinases

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1. The Src homology 3 domain (SH3) is a protein domain involved in the assembly of proteins participating in the signal transduction by protein-tyrosine kinases. A cDNA clone encoding a new protein ligand to the SH3 domain of Fyn PTK was isolated. The clone was identified by screening mouse embryo cDNA library with Fyn・SH3/GST fusion protein as a probe. The new SH3 ligand protein is also expressed in adult brain. The protein contains two proline-rich sequences with a specificity in binding to Fyn and Src SH3 domains, a new SH3 domain, and potential ligand sequences for SH2 domains. Antibody specific to the newly identified protein immunoprecipitated and immunoblotted a protein from the teratocarcinoma cell lysate and from the lysate of COS-7 cells transfected with the cloned cDNA.The protein was tyrosine-phosphorylated by Fyn expressed from cotransfected fyn cDNA in COS-7 cells. 2. A 70-kDa protein with high affinity to Fyn SH3 was found in the Jurkat cell lysate by affinity binding to Fyn SH3/GST fusion protein. The protein was eluted from SDS-PAGE gels, digested with trypsin, and the peptides were separated. The results of gas-phase microsequencing of the peptides indicated that the protein is closely related to GAP-binding phosphoprotein, p62. The SH3 domains are known to bind to proline-rich sequences. The proline-rich sequences in p62 were expressed as GST fusion proteins and the binding of the GST fusion proteins to various SH3/GST fusion proteins were examined. The results indicate that each proline-rich sequence has a specific affinity to a subset of SH3 domains.