Anchored Protein Kinase A in Signal Transduction

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DESCRIPTION (provided by applicant): A-kinase anchor proteins (AKAPs) mediate targeting of signals carried by cAMP. AKAPs bind protein kinase All (PKAII) and have unique domains that target the tethered kinase to docking sites in organelles. Colocalization of anchored PKA with substrate/effector proteins enables efficient reception and precisely focused transmission of cAMP signals. Critical aspects of cell physiology are controlled by encounters between activated PKA and effectors that are embedded in or juxtaposed to cortical actin cytoskeleton. Little is known about properties of AKAPs that directly bind with specialized regions of actin cytoskeleton. AKAPKL isoforms are adapted for coupling PKAII to properties and proteins of the cortical F-actin network. AKAPKL proteins have an RII tethering site, anchoring domains that bivalently ligate and cross-link F-actin and a domain that binds NSF. However, AKAP-KL4 routes PKAII to the lateral surface of polarized cells, whereas AKAP-KL2 is enriched at the apical surface. Thus the anchor proteins deliver PKAII to different microenvironments and constellations of effectors. An AKAP paradigm predicts that unique combinations of intrinsic targeting domains and cognate docking molecules account for specific locations and functions of AKAP-PKAII complexes. The principal investigator and his group will characterize a) targeting domains that control asymmetric distribution of AKAP-KL2 and AKAP-KL4 in polarized MDCK cells and b) docking proteins that guide different AKAP-PKAII complexes to structurally and functionally distinct destinations in actin cytoskeleton. A molecular basis for specific docking interactions will be determined and tools developed to selectively disrupt anchoring of PKAII by AKAP-KL2 or AKAP-KL4. A central aim is to elucidate a physiological role for the epically-oriented AKAPKL2-PKAII complex. The proposition that this anchored PKAII complex simultaneously controls a) phosphorylation and translocation of a channel protein and b) organization of the local F-actin network, will be systematically investigated. Downstream targets that mediate cytoskeleton remodeling will be characterized. The principal investigator and his group will investigate the structural basis and physiological functions for anchored PKAI-Iike PKA in C. elegans in vivo. They have discovered unique structural features in the AKAPce and Rce that confer isoform-selective high affinity binding activity. Wild type and mutant transgenes will be introduced into AKAPce or Rce null C. elegans. Biochemical and physiological consequences will be assayed in vivo.