Ishino, Masaho

Sasaki, Hiroko

Sasaki, Terukatsu

Several proteins of Jurkat cells were identified on SDS-PAGE gels by Coomassie Blue staining that bound specifically to affinity matrices made of five different Src homology 3 (SH3) domains fused to glutathione S-transferase (GST). Purification of the major specific band of approximately 70kDa with affinity beads of the SH3 domain of Fyn tyrosine kinase resulted in an identification of a GAP-associated p62-related protein as a ligand to the Fyn and Src SH3 domains. Indeed, from a lysate of a Rous sarcoma virus-transformed rat fibroblast line, Src co-precipitated with the 70kDa and also bound to a puta-tive SH3 binding sequence of p62. Bacterially expressed GST fusion proteins containing sequences encompassing each of the proline-rich putative SH3 binding sites of p62 bound to a subset of SH3 domains with different affinities. Phos-pholipase Cgannma 2-SH3 also revealed strong binding to the bacterially expressed p62 fusion proteins in vitro but did not show primary binding to the cellular 70kDa. The multiple SH3 binding sequences with different affinities to various SH3 mole-cules together with their phosphorylation on tyrosine residue(s) suggest a role of p62 as a foothold on which signal transduction proteins, including Src-family kinases, link together

Multiple Proline-rich Regions of GAP-associated Phosphoprotein p62 Bind with Different Affinities to the Src Homology 3 Domains of Fyn and Src

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