RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP.
The Ras GTPase-activating protein p120GAP is a multidomain protein consisting of a variety of noncatalytic domains that may be involved in its regulation. RACK1 is a membrane-associated protein that binds the C2 domain of PKC and is related in sequence to the beta subunit of heterotrimeric G-proteins which has been implicated in binding to PH domains. Because p120GAP contains both PH and C2/CaLB domains we determined whether it is also a RACK1 binding protein. Coimmunoprecipitation experiments indicate that p120GAP associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do not. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated with recombinant RACK1. Finally, serine/threonine phosphorylation appears to be involved in regulating this association. These results suggest that p120GAP and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP.
Koehler
J A
JA
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, M5G 1X5, Canada.
Moran
M F
MF
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D000075370:Receptors for Activated C Kinase; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020729:p120 GTPase Activating Protein / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D010455:Peptides; D000075370:Receptors for Activated C Kinase; D011993:Recombinant Fusion Proteins; D020729:p120 GTPase Activating Protein; C070301:peptide I; D010749:Phosphoprotein Phosphatases