IDENTIFICATION OF THE BINDING SURFACE ON CDC42HS FOR P21-ACTIVATED KINASE

The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesi s, intracellular trafficking, and cytoskeletal organization. The Rho s ubfamily, of which Cdc42Hs is a member, is involved in cell morphogene sis through a GTPase cascade which regulates cytoskeletal changes. Cdc 42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-ac tivated serine/ threonine kinases (PAKs). We have determined previousl y the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistr y 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 4 6 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a K-D of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of N-15-Cd c42Hs bound to PBD46. A H-1,N-15-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (be ta 2) and a portion of the loop between the first alpha-helix (alpha 1 ) and beta 2 (switch I). A complex of PBD46 bound to N-15-Cdc42Hs.GMPP CP exhibited extensive chemical shift changes in the H-1,N-15-HSQC spe ctrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding doma in on Cdc42Hs is similar to, but more extensive than, the c-Raf-bindin g domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].