Phosphoinositide 3-Kinase-dependent and -independent Activation of the Small GTPase Rac2 in Human Neutrophils*
- From the Department of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Abstract
The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonistsN-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.
Footnotes
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↵* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, from Kato Memorial Bioscience Foundation, and from Fukuoka Cancer Society.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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↵‡ To whom correspondence should be addressed. Tel.: 81-92-642-6213; Fax: 81-92-642-6215; E-mail:hsumi{at}mailserver.med.kyushuu.ac.jp.
- Abbreviations:
- PtdIns(3,4,5)P3
-
phosphatidylinositol 3,4,5-trisphosphate
- PtdIns(4,5)P2
-
phosphatidylinositol 4,5-bisphosphate
- PI3K
-
phosphoinositide 3-kinase
- fMLP
-
N-formyl-methionyl-leucyl-phenylalanine
- LTB4
-
leukotriene B4
- Gi
-
the Gi class of heterotrimeric GTP-binding protein
- PMA
-
phorbol 12-myristate 13-acetate
- PKC
-
protein kinase C
- GST
-
glutathioneS-transferase
- RBD
-
Rac-binding domain
- PTX
-
pertussis toxin
- GTPγS
-
guanosine 5′-3-O-(thio)-triphosphate
- PAGE
-
polyacrylamide gel electrophoresis
- Giα
-
the a subunit of Gi
- GEF
-
guanine nucleotide exchange factor
- PH
-
pleckstrin homology
-
- Received February 23, 1999.
- Revision received April 13, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
