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.
Highlights
Cell motility is controlled by a complex network of signals that are initiated by binding to the extracellular matrix
We and others (6 –9) have suggested that Protein kinase A (PKA) activity gradients exist in migrating cells to facilitate polarization of cells and proper spatial signaling for the organization of distinct actin cytoskeletal structures
We find that these gradients are found in close proximity to where integrins interact with the extracellular matrix
Summary
Cell motility is controlled by a complex network of signals that are initiated by binding to the extracellular matrix. To determine the spatial distribution of cAMP/PKA activity, Clone A cells transfected with either AKAR1, a non-phosphorylatable mutant (S475A) of AKAR1, CFP-only, or YFP-only constructs were allowed to migrate on laminin-1 and fixed.
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