WISKOTT-ALDRICH SYNDROME PROTEIN PHYSICALLY ASSOCIATES WITH NCK THROUGH SRC HOMOLOGY-3 DOMAINS

In the second of a series of experiments designed to identify p47(nck) -Src homology 3 (SH3)-binding molecules, we report the cloning of SAKA P II (Src A box Nck-associated protein II) from an HL60 cDNA expressio n library. This molecule has been identified as a cDNA encoding the pr otein product of WASP, which is mutated in Wiskott-Aldrich syndrome pa tients. Studies in vivo and in vitro demonstrated a highly specific in teraction between the SH3 domains of p47(nck) and Wiskott-Aldrich synd rome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antib odies recognized a protein of 66 kDa by Western blot (immunoblot) anal ysis. In vitro translation studies identified the 66-kDa protein as th e protein product of WASP, and subcellular fractionation experiments s howed that p66(WASP) i, mainly present in the cytosol fraction, althou gh significant amounts are also present in membrane and nuclear fracti ons. The main p47(nck) region implicated in the association with p66(W ASP) was found to be the carboxy-terminal SH3 domain.