Abstract
AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611-6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178-410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.
Highlights
AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus
The results suggest that AKAP350 associates with chloride intracellular channel (CLIC) proteins and that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells
We have identified the interaction of AKAP350 with CLIC family members, the colocalization of CLIC5B and AKAP350 at the Golgi apparatus of HCA-7 cells
Summary
Vol 277, No 43, Issue of October 25, pp. 40973–40980, 2002 Printed in U.S.A. II. ASSOCIATION OF AKAP350 WITH A NOVEL CHLORIDE INTRACELLULAR CHANNEL (CLIC) FAMILY MEMBER*. AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The results suggest that AKAP350 associates with CLIC proteins and that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells. The study of signaling molecules scaffolded in specific locations within the cell has led to an expanded understanding of the cellular functions that they control. Scaffolding proteins are utilized in the cell to localize signaling proteins to specific areas facilitating precise cellular function. Often an AKAP will scaffold both kinases and phosphatases, thereby coordinating specific signaling events [6]. Using yeast two-hybrid screening we show that the chloride intracellular channel (CLIC) family of proteins interacts with a region of AKAP350 previously denoted as the peri-. The results suggest that AKAP350 may scaffold CLIC proteins at discrete sites within the cell
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