• AKAP350: A Centrosome Associated Scaffold Protein

      Schmidt, Hank; Department of Biochemistry and Molecular Biology (2000-06)
      A-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.
    • The regulation of steroid 11 [beta]-hydroxylase gene expression in cultured bovine adrenocortical cells

      Naseeruddin, Syed Ahmed; Department of Cell and Molecular Biology (1989-11)
      Second messenger systems play an important role in regulating gene expression. Adrenocorticotropin (ACTH) acting through cAMP dependent protein kinase is known to induce the adrenal cytochrome P450 steroidogenic enzymes, 11~-hydroxylase and 17a-hydroxylase by increasing transcription of the genes for these enzymes. In this thesis, activators of Ckinase and IGF-I were studied to examine their effects on enzyme activity and gene expression. IGF-I has an important function in llP-hydroxylase expression. An absence of IGF-1 results in a reduced level of both cAMP-stimulated 11~hydroxylase enzyme activity and mRNA. This effect was specific for 11~- · hydroxylase, as 17a-hydroxylase mRNA levels and enzyme activity were not affected in the absence of IG F-1. Since these enzymes were regulated differently by IGF-1, it was possible they were. regulated differently by A-kinase activating agents. Although the agents stimulated both 17a-hydroxylase and llP-hydroxylase, llP-hydroxylase activity was more sensitive to cAMP levels, and declined at cAMP concentrations_ above 100 μM, suggesting that A-kinase may downregulate in the presence of excess cAMP. _Although some steroids serve as negative regulators of 11~hydroxylase activity; it was unknown whether these effects were transcriptional. In the _presence of androstenedione, enzyme activity was inhibited, bt1st mRNA levels remained unchanged, suggesting a posttranscriptional 8:Ction. In the absence of ascorbate, which prevents oxidative damage. to. llf3-hydroxylase, the enzyme activity was decreased; mRNA levels- were unaffected, suggesting that ascorbate also acts posttranscriptionally. Effects of protein kinase C activation were analyzed using the phorbol ester, TPA. Although TPA alone had no effect, it decreased the levels of cAMP induced mRNA and enzyme activity of llf3-hydroxylase. Angiotensin II had similar effects.