• AKAP350 Targets to the Golgi Apparatus Where it Interacts with CLIC5B and CIP4/S

      Shanks, Ryan; Department of Biochemistry and Molecular Biology (2002-03)
      A-kinase anchoring proteins (AKAPs) are defined by their ability to scaffold PKA, but their function depends upon their targeting of PKA and other scaffolded signaling proteins to specific subcellular compartments. We have investigated one AKAP, AKAP350, which can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The AKAP350 gene is multiply spliced to create three carboxyl terminal splice variants which we have designated AKAP350A, AKAP350B and AKAP350C. Immunocytochemistry in HCA-7 cells demonstrated that AKAP350A was localized specifically to the Golgi apparatus. GFP-fusion proteins representing the carboxyl terminus of AKAP350A identified a carboxyl terminal region responsible for the Golgi apparatus targeting of AKAP350A. Yeast two-hybrid analysis was utilized to screen a rabbit gastric parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813) which is weakly homologous to pericentrin. This screen yielded two positive clones representing rabbit chloride intracellular channel 1 (CLIC1) and rabbit Cdc42 interacting protein 5 (CIP5). Further yeast-two hybrid binary analysis determined that CLIC1 and CIP5 bound to AKAP350 through adjacent domains located with in the PHR. CLIC1 belongs to a family of proteins which all contain a high degree of homology in their carboxyl termini, and this conserved domain is responsible for several CLIC family member’s ability to bind AKAP350. We isolated the human homologue of bovine p64, CLIC5B, from an HCA-7 colonic adenocarcinoma cell cDNA. A splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the molecular weight of a major CLIC immunoreactive protein in HCA-7 cells. Immunocytochemistry determined that CLIC5B colocalized with AKAP350 at the Golgi apparatus. Yeast-two hybrid binary analysis determined that the final 120 amino acids of CLIC5B interacted with AKAP350. Furthermore, expression of a GFP-fusion protein containing the final 120 amino acids targeted to the Golgi apparatus in HCA-7 cells. CIP5 contains high homology to the human protein Cdc42 interacting protein 4 (CIP4). Yeast-two hybrid binary analysis determined that the first 117 amino acids of both human CIP4 and CIP5 interacted with AKAP350. Immunocytochemistry in HCA-7 cells determined with an antibody recognizing CIP4 and CIP5 localized to the Golgi apparatus. These results suggest that AKAP350 associates with CLIC proteins and CIP4/5, and these proteins interact with the AKAP35A splice variant at the Golgi apparatus.