Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Research Abstract Details 

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Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Abstract Text:

G Thompson,D Owen,P A Chalk,P N Lowe,

p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac. They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif. The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding. Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques. The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac. A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM. Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132. A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75. PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type. The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42. mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar. Calorimetric measurements revealed that binding was associated with a relatively slow heat change. It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac. In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient.

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Publishing Authors By Initials

G Thompson,D Owen,PA Chalk,PN Lowe,

For similar enzymes and coenzymes: enzymes: hydrolases: acid anhydride hydrolases: gtp phosphohydrolases: gtp-binding proteins: monomeric gtp-binding proteins: rho gtp-binding proteins: rac gtp-binding proteins research abstracts see: enzymes and coenzymes: enzymes: hydrolases: acid anhydride hydrolases: gtp phosphohydrolases: gtp-binding proteins: monomeric gtp-binding proteins: rho gtp-binding proteins: rac gtp-binding proteins research

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Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Biochemistry

VOLUME: 37

Page Numbers: 7885-91

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 26

MONTH: May

YEAR: 1998

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370623

Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Keywords Mesh Terms:

KEYWORDS: rac GTP-Binding Proteins

MESH TERMS: chemistry

Chemical & Substance for Abstract: Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. Information

Substance Name: rac GTP-Binding Proteins

Registry Number: EC 3.6.5.2

Grant and Affiliation Information for Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.

AFFILIATION: Exploratory Chemistry Unit, Glaxo Wellcome Medicines Research Centre, Hertfordshire, United Kingdom.

Country: UNITED STATES

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MEDLINETA: Biochemistry

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