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Molecular Cloning of p125Nap1, a Protein That Associates with an SH3 Domain of Nck

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Abstract

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125Nap1dose not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125Nap1was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125Nap1may clarify the protein-protein interaction in the downstream signaling of Nck.

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1

The first two authors contributed equally to this work.

2

Present address: Cardiovascular Research Center, Massachusetts General Hospital-East, and Department of Medicine, Harvard Medical School, Charlestown, MA 02129.

3

To whom correspondence may be addressed. M.K. fax: 81-78-382-2080; K.Y. fax: 617-726-5806.

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