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, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. 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 domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].
Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, and Department of Chemistry, Leicester University, Leicester, LE1 7RK, UK.
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D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein; D054462:p21-Activated Kinases
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; C515911:PAK3 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein