AKAP350 at the Golgi apparatus. 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. 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.
Mesh Terms:
A Kinase Anchor Proteins, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Base Sequence, Blotting, Northern, Carrier Proteins, Chloride Channels, Cytoskeletal Proteins, DNA, Complementary, Golgi Apparatus, Humans, Microfilament Proteins, Molecular Sequence Data, Precipitin Tests, Protein Binding, Sequence Homology, Amino Acid, Two-Hybrid System Techniques
A Kinase Anchor Proteins, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Base Sequence, Blotting, Northern, Carrier Proteins, Chloride Channels, Cytoskeletal Proteins, DNA, Complementary, Golgi Apparatus, Humans, Microfilament Proteins, Molecular Sequence Data, Precipitin Tests, Protein Binding, Sequence Homology, Amino Acid, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Oct. 25, 2002
PubMed ID: 12163479
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