Characterization of Rac and Cdc42 Activation in Chemoattractant-stimulated Human Neutrophils Using a Novel Assay for Active GTPases*
- From the Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Abstract
A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versusRhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.
Footnotes
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↵* This work was supported in part by U. S. Public Health Service Grants GM39434 and GM44428 (to G. M. B.). This is manuscript number 12082-IMM from the Scripps Research Institute.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.
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↵‡ Supported by a fellowship from the Association Pour la Researche Sur Le Cancer during the tenure of this work and is currently a recipient of a National Arthritis Foundation Postdoctoral Fellowship.
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↵§ To whom correspondence should be addressed: Depts. of Immunology and Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-8217; Fax: 619-784-8218; E-mail:bokoch{at}scripps.edu.
- Abbreviations:
- GEF
-
guanine nucleotide exchange factors
- PAK
-
p21-activated kinase
- PBD
-
p21-binding domain
- GST
-
glutathione S-transferase
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate
- PI 3-kinase
-
phosphatidylinositol 3-kinase
- GAP
-
GTPase-activating protein(s)
- PMA
-
phorbol myristate acetate
- fMLP
-
fMet-Leu-Phe
- DTT
-
dithiothreitol
- BHK
-
baby hamster kidney
- PAGE
-
polyacrylamide gel electrophoresis
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- Received November 20, 1998.
- Revision received February 12, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
