Distinct Mechanisms Mediate the Initial and Sustained Phases of Integrin-mediated Activation of the Raf/MEK/Mitogen-activated Protein Kinase Cascade*
- From the Department of Pharmacology and The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365
Abstract
Integrin-mediated adhesion to the extracellular matrix activates the canonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activation of Raf-1, an upstream regulator of mitogen-activated protein kinase, occurs in two phases. Efficient early activation of Raf required Raf-Ras interaction but was not affected by protein kinase C (PKC) inhibitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PKC inhibitors. The combination of PKC inhibition and lack of Ras binding completely blocked integrin-mediated Raf activity. The activity of a membrane-bound Raf mutant that is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by adhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapidly stimulated to wild-type levels by cell adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation of wild-type and mutant Raf proteins was ablated by cytochalasin D, demonstrating that cytoskeletal organization is required for activation of Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respectively, and that integrin-mediated adhesion may regulate a cytoskeleton-associated factor(s) responsible for Raf activation.
Footnotes
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↵* This work was supported in part by National Institutes of Health (NIH) Grant GM26165 (to R. L. J.).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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↵‡ A Lineberger Comprehensive Cancer Center Postdoctoral Fellow (National Cancer Institute, NIH, Training Grant CA09156-23).
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↵§ To whom correspondence should be addressed. Dept. of Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Mary Ellen Jones Bldg. #920, CB# 7365, Chapel Hill, NC 27599-7365. Tel.: 919-966-4383; Fax: 919-966-5640; E-mail: arjay{at}med.unc.edu.
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↵2 A. Howe and R. Juliano, unpublished observations.
- Abbreviations:
- ECM
-
extracellular matrix
- MAPK
-
mitogen-activated protein kinase
- RTK
-
receptor tyrosine kinase
- MEK
-
mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
- PKC
-
protein kinase C
- DMEM
-
Dulbecco’s modified Eagle’s medium
- Fn
-
fibronectin
- PBS
-
phosphate-buffered saline
- TPA
-
12-O-tetradecanoylphorbol-13-acetate
- BIM
-
bisindolylmaleimide
- PAGE
-
polyacrylamide gel electrophoresis
- BSA
-
bovine serum albumin
- cytoD
-
cytochalasin D
- WT
-
wild type.
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- Received April 9, 1998.
- Revision received July 24, 1998.
