title: Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.
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.
authors.affiliation: Sealy Center for Cancer Cell Biology and Departments of Surgery, and of Neurosciences and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Adriana A
authors.initials: AA
authors.lastname: Paulucci-Holthauzen
authors.forename: Leoncio A
authors.initials: LA
authors.lastname: Vergara
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Bellot
authors.forename: David
authors.initials: D
authors.lastname: Canton
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: O'Connor
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007124:Immunoenzyme Techniques; D007546:Isoquinolines / Q000494:pharmacology; D010766:Phosphorylation; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D054758:A Kinase Anchor Proteins; D007546:Isoquinolines; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide