Browsing Department of Biochemistry and Molecular Biology Theses and Dissertations by Subjects
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AKAP350: A Centrosome Associated Scaffold ProteinA-kinase anchoring proteins (AKAPs) are recognized as key components of compartmentalization and transduction in intracellular cAMP signaling. They allow localization of the Type II c AMP-dependent protein kinase to specific subcellular domains, effectively positioning the enzyme near its substrate to await activation by cAMP. The role of AKAPs as protein scaffolds allows binding of multiple enzymes, regulatory molecules, and structural elements, functioning as a virtual platform for modulation of specific cellular events (i.e. membrane channel activity, receptor clustering). We have cloned a novel 350 kDa AKAP (AKAP350) from human gastric cDNA, and identified partial clones in human lung and rabbit parietal cells. The genomic region containing AKAP350, found on chromosome 7q21, is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, an NMDA receptorassociated protein. We identified three unique AKAP350 C-termini (AKAP350A, -B, and -C) resulting from alternative splicing of the 3' end of the gene. AKAP350 is associated with centrosomes, as well as with the cleavage furrow during anaphase and telophase by immunocytochemistry. Polyclonal antibodies to individual AKAP350 Cterminal splice variants demonstrate tissue dependent combinations of centrosomal and non-centrosomal distribution. In the polarized HCA-7 colon cell line AKAP350A is purely non-centrosomal while AKAP350B and -C are centrosomal. Anti-AKAP350C is limited to mitotic cells, suggesting that this isoform may be expressed only at entry into M phase. A yeast two-hybrid screen of a rabbit parietal cell library identified a novel TACC (Transforming Acidic Coiled coil Containing) protein family member as a ligand of the final pair of arginine residues in the AKAP350A splice variant. A GFP fusion with the novel AKAP interacting protein verified co-localization with AKAP350 at the centrosome exclusively during mitosis. Microinjection of dividing sea urchin embryos with GST fused to the AKAP interacting protein arrested cell division. Therefore, the AKAP350 protein scaffold may function as a large docking station, providing kinase / phosphatase signals for coordination of cytoskeletal dynamics as well as cell division.