Cloning, sequencing, and overexpression of SH2/SH3 adaptor protein Nck from mouse thymus.

Nck, an oncogenic protein containing one SH2 and three SH3 domains, is a common target for phosphorylation by a variety of cell surface receptors. The absence of a recognizable catalytic domain of Nck suggests that Nck serves as a linker molecule to couple cell surface receptors to downstream effector molecules that regulate cellular responses induced by receptor activation. In this study, we isolated Nck cDNA from mouse thymus by screening a phase expression library using monoclonal antibody (mAb) B16-5 which recognizes the SH3 domain of phospholipase C-gamma 1. Nck protein was purified from an extract of the Sf9 cell that had been infected with recombinant baculovirus containing mouse Nck cDNA. The purified protein exhibited an apparent molecular mass of 47 kDa on SDS-polyacrylamide gels. Nck antibody was generated in rabbits using purified protein as an antigen. The distribution of Nck protein in rat tissues examined by immunoblots showed that Nck is expressed widely, and that brain, spleen, and tests contain more Nck than other tissues examined.