Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII and a GFP-RII fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII-associated microspikes are highly dynamic and that the coupling of RII to actin is tight, as t... More
Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII and a GFP-RII fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII-associated microspikes are highly dynamic and that the coupling of RII to actin is tight, as the movement of both actin and RII are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RII and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RII is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RII fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc.
