Residues in Cdc42 that specify binding to individual CRIB effector proteins

被引:62
作者
Owen, D
Mott, HR
Laue, ED
Lowe, PN
机构
[1] Univ Cambridge, Cambridge Ctr Mol Recognit, Dept Biochem, Cambridge CB2 1GA, England
[2] Glaxo Wellcome Med Res Ctr, Stevenage SG1 2NY, Herts, England
关键词
D O I
10.1021/bi991567z
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 effecters 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, Va18, Phe28, Tyr32, Va133, Thr35, Va136, Phe37, Asp38, Tyr40, Va142, 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 effecters 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 effecters. 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 effecters can specifically recognize Cdc42. Those mutations in Cdc42 that inhibit certain interactions, while retaining others, should aid investigations of the role of specific effecters in Cdc42 signaling in vivo.
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页码:1243 / 1250
页数:8
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