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9 October 1997, Volume 15, Number 15, Pages 1823-1832
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Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor
Jiping Tang1, Gen-Sheng Feng2 and Wei Li1,a

1The Ben May Institute for Cancer Research and the Department of Pharmacological and Physiological Sciences, the University of Chicago, 5841 South Maryland Avenue, Chicago Illinois 60637 USA

2The Department of Biochemistry and Molecular Biology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202-5121, USA

aAuthor for correspondence


The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, among which p21cdc42/rac-activated kinase cascade, Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes, have been implicated. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor (Li et al., 1992 Mol. Cell. Biol. 12: 5824 - 2833). To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, pre-existing phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. We found that Nck does not directly bind to EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. We present evidence demonstrating that the Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62. (1) The Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS - PAGE. (2) SH2 domains from Nck and GAP compete for binding to p62 in vitro. (3) Purified GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCgamma, PI-3 kinase, SHC, and Grb2 did not bind p62. (4) Tryptic phosphopeptide maps of the Nck- and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation.


src-homology domains; adapters; receptor tyrosine kinases; signal transduction

Received 21 February 1999; revised 17 June 1999; accepted 17 June 1999
9 October 1997, Volume 15, Number 15, Pages 1823-1832
Table of contents    Previous  Abstract  Next   Article  PDF