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.
Shanks
Ryan A
RA
Department of Medicine, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA.
Larocca
M Cecilia
MC
Berryman
Mark
M
Edwards
John C
JC
Urushidani
Tetsuro
T
Navarre
Jennifer
J
Goldenring
James R
JR
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D054758:A Kinase Anchor Proteins; C119719:AKAP9 protein, human; D048868:Adaptor Proteins, Signal Transducing; C411466:CLIC5 protein, human; D002352:Carrier Proteins; D018118:Chloride Channels; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins