Identification of a Putative Effector for Cdc42Hs with High Sequence Similarity to the RasGAP-related Protein IQGAP1 and a Cdc42Hs Binding Partner with Similarity to IQGAP2

doi:10.1074/jbc.271.36.21732

Identification of a Putative Effector for Cdc42Hs with High Sequence Similarity to the RasGAP-related Protein IQGAP1 and a Cdc42Hs Binding Partner with Similarity to IQGAP2*

From the Departments of Biochemistry,
^‡ Molecular and Cell Biology, and Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853

^¶ To whom correspondence should be addressed:
Dept. of Pharmacology, Cornell University, Ithaca, NY 14853.
Tel.: 607-253-3888; Fax: 607-253-3659; E-mail: rac1{at}cornell.edu.

Abstract

Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass ∼180 kDa from rabbit liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5′-3-O-(thio)triphosphate-bound forms of Cdc42. Binding of p180 to GTP-bound Cdc42 maintains it in the GTP-bound state. Another cytosolic protein, with an apparent molecular mass of 175 kDa (p175), was also found to interact with Cdc42, but this association showed less dependence on guanine nucleotides. Both p180 and p175 were capable of binding to Rac1 but not to RhoA or Ha-Ras. The limit functional domain of the Cdc42-GAP protein did not compete with p180 or p175 for binding to Cdc42. However, the Cdc42-binding domain from mPAK-3, a member of the PAK (p21 activated kinase) family of serine/threonine kinases, competed with both proteins. The binding of p180 or p175 was inhibited by mutations of the putative effector loop of Cdc42. p180 and p175 also bound less effectively to a Cdc42/Ras chimera in which loop 8 from Ras was substituted for the predicted loop 8 in Cdc42 that includes a 13-amino acid insert present in all Rho family members but absent in Ras. Microsequencing of a p180 peptide revealed 92% identity with the human IQGAP1 protein, while two peptides derived from p175 were 89 and 100% identical to human IQGAP2. These findings identify IQGAP1 and IQGAP2 as a new class of target/effectors that utilize both regions of the switch I domain and an insert region distinct to Rho proteins for binding to Cdc42.

Footnotes

↵^§ Supported by National Institutes of Health Grant GM08210.
↵* This work was supported by National Institutes of Health Grant 47458. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PAK

p21-activated serine/threonine kinase

GST

glutathione S-transferase

GTPγS

guanosine 5′-3-O-(thio)triphosphate

PBD

p21-binding domain

PAGE

polyacrylamide gel electrophoresis

Mant-GMP-PNP

N-methylanthraniloyl adenosine 5′-(β,γ-imino)triphosphate

GAP

GTPase-activating protein.
²D. Leonard and R. Cerione, unpublished data.
³D. A. Leonard, R. Satoskar, W. J. Wu, S. Bagrodia, R. A. Cerione, and D. Manor, manuscript in preparation.
↵⁴W. J. Wu and R. A. Cerione, unpublished data.
↵⁵T. Nomanbhoy, unpublished data.
- Received May 7, 1996.
- Revision received June 19, 1996.