RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120(GAP)

The Pas GTPase-activating protein p120(GAP) is a multidomain protein consis ting of a variety of noncatalytic domains that may be involved in its regul ation. RACK1 is a membrane-associated protein that binds the C2 domain of P KC and is related in sequence to the beta subunit of heterotrimeric G-prote ins which has been implicated in binding to PH domains. Because p120(GAP) c ontains both PH and C2/CaLB domains we determined whether it is also a RACK 1 binding protein. Coimmunoprecipitation experiments indicate that p120(GAP ) associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do n ot. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated wit h recombinant RACK1. Finally, serine/threonine phosphorylation appears to b e involved in regulating this association. These results suggest that p120( GAP) and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP. (C) 2001 Academic Press.