Residues in Cdc42 that specify binding to individual CRIB effector proteins

Biochemistry. 2000 Feb 15;39(6):1243-50. doi: 10.1021/bi991567z.

Abstract

Cdc42 is a member of the Rho family of small G proteins. Signal transduction events emanating from Cdc42 lead to cytoskeletal rearrangements, cell proliferation, and cell differentiation. Many effector proteins have been identified for Cdc42; however, it is not clear how certain effectors specifically recognize and bind to Cdc42, as opposed to Rac or Rho, or in many cases, which effector controls what cellular events. Mutations were introduced into Cdc42 at residues: Met1, Val8, Phe28, Tyr32, Val33, Thr35, Val36, Phe37, Asp38, Tyr40, Val42, Met45, Ile46, Glu127, Ala130, Asn132, Gln134, Lys135, and Leu174. Measurements were made of their equilibrium binding constants to the Cdc42 binding domains of the CRIB effectors ACK, PAK, and WASP and to the GTPase-activating protein Rho GAP. Generally, mutations in the effector loop have an equally deleterious effect on binding to all CRIB proteins tested, though the F37A mutation resulted in significant selectivity. Residues outside the effector loop were found to be important for binding of Cdc42 to CRIB containing proteins and also to contribute to selectivity. Mutations such as V42A and L174A resulted in large, selective changes in binding to specific CRIB effectors. Neither mutation resulted in alteration in PAK binding, whereas both severely disrupt binding to ACK and only L174A disrupted binding to WASP. These mutations are interpreted using the structures of the Cdc42/ACK and Cdc42/WASP complexes to give insight into how effectors can specifically recognize Cdc42. Those mutations in Cdc42 that inhibit certain interactions, while retaining others, should aid investigations of the role of specific effectors in Cdc42 signaling in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Binding Sites / genetics
  • Consensus Sequence / genetics
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding / genetics
  • Protein Structure, Secondary / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Scintillation Counting
  • Spectrometry, Fluorescence
  • cdc42 GTP-Binding Protein / chemistry
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*
  • rac GTP-Binding Proteins / metabolism*

Substances

  • Peptide Fragments
  • Recombinant Fusion Proteins
  • cdc42 GTP-Binding Protein
  • rac GTP-Binding Proteins