Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc*
- Adriana A. Paulucci-Holthauzen‡,
- Leoncio A. Vergara§,
- Larry J. Bellot‡,
- David Canton¶,
- John D. Scott¶1 and
- Kathleen L. O'Connor‡∥2
- ‡Sealy Center for Cancer Cell Biology and Departments of ∥Surgery, and of §Neurosciences and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555 and the ¶Howard Hughes Medical Institute, Department of Pharmacology, University of Washington, Seattle, Washington 98195
- 2 To whom correspondence should be addressed: Dept. of Surgery, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0525. Tel.: 409-772-1852; Fax: 409-747-1938; E-mail: kloconno{at}utmb.edu.
Abstract
Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.
Footnotes
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↵3 The abbreviations used are: PKA, protein kinase A; AKAP, A-kinase anchoring proteins; AKAR1, A-kinase activity reporter; FRET, fluorescence resonance energy transfer; Fc, sensitized acceptor emission or corrected FRET; PDE, phosphodiesterase; PKI, protein kinase inhibitor; RII, type II regulatory; NT, non-targeting; LPA, lysophosphatidic acid; PIPES, 1,4-piperazinediethane-sulfonic acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; siRNA, small interfering RNA; IBMX, isobutylmethylxanthine.
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↵4 A. A. Paulucci-Holthauzen, M. Chen, and K. L. O'Connor, unpublished observation.
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants R21-GM071928 and R01-CA109136. The costs of publication of this article were defrayed in part by the payment of page charges. This 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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↵1 Supported by National Institutes of Health Grant HL088366 and the Fondation Leducq Transatlantic Network.
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- Received July 22, 2008.
- Revision received December 19, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
