AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes

Abstract

Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. We have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants were also identified. Both of the major splice variants showed tissue-and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that AKAP350 was associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not alter its localization. During the cell cycle, AKAP350 was associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrated noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types.Transduction of signals from extracellular stimuli is most commonly accomplished via ligand-receptor binding and generation of a second messenger response. While increases in intracellular second messengers have traditionally been viewed as global cellular events, second messenger effects are often limited to particular regions or organelles within cells. Investigations over the past decade have led to a greater understanding of the mechanisms responsible for the compartmentalization of second messenger effects. These studies have identified a diverse group of scaffolding proteins that sequester both protein kinases and protein phosphatases within specific cellular domains (1, 2). In the case of cAMP-dependent protein kinases, protein kinase A-anchoring proteins (AKAPs) 1 tether the protein kinase A holoenzyme through binding to the regulatory subunit dimer. A growing group of AKAPs that bind the regulatory subunit of type II protein kinase A (R II ) have been reported over the past several years. The first R II -binding protein was identified over 15 years ago when microtubule-associated protein 2 (MAP-2) was described (3, 4). Since that time, several AKAPs have been identified, localizing the type...

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

“…These AKAPs possess a conserved centrosomal targeting sequence called the PACT domain [40,54]. AKAP450 acts as a centrosomal scaffold for a variety protein kinases and phosphatases, including a subpopulation of PP2A [29,40,[78][79][80]. Although the exact subunit composition of centrosomal PP2A, or its associated proteins and targets are not known, cyclin G2 could associate with PP2A via AKAP450.…”

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer

“…The fusion partner of BRAF, the AKAP9 gene, belongs to the group of A-kinase anchor proteins that have the common function of binding to the regulatory subunit of PKA and targeting it to discrete locations within the cell (29). AKAP9, in particular, has predominantly centrosomal and Golgi compartmentalization (19)(20)(21). The fusion protein lacks the C-terminal centrosomal domain and, as expected, loses its centrosomal localization in cancer cells.…”

“…Further confirmation was obtained by doublecolor FISH with probes corresponding to the BRAF and AKAP9 genes ( Figure 2B). Since the BRAF gene is located on chromosome 7q34 (18) and AKAP9 on 7q21-q22 (19)(20)(21), the AKAP9-BRAF fusion results from inv(7)(q21-22q34) ( Figure 2C). Using primers located within the 5′ and 3′ untranslated region of AKAP9 and BRAF, respectively, the AKAP9-BRAF coding cDNA sequence was amplified from the tumor, cloned into the pCR-XL-TOPO vector, and sequenced (GenBank accession number AY803272).…”

“…The wild-type AKAP9 gene encodes a 453-kDa protein capable of binding the type II regulatory subunit (RII) of protein kinase A (PKA) as well as other signaling proteins, anchoring them to the centrosome and Golgi apparatus (19)(20)(21). Several isoforms arising through alternative splicing are selectively expressed in most human tissues at low abundance (19,20).…”

cDNA cloning of a novel human gene NAKAP95, neighbor of A-kinase anchoring protein 95 (AKAP95) on chromosome 19p13.11–p13.12 region

“…PKA type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs) (Scott and Macartney, 1994;Rubin 1994;Hausken and Scotte, 1996;Faux and Scott 1996). A number of different AKAPs which direct different compartmentalizations have been found: AKAP79/75 direct the RII to postsynaptic densities and cortical actin (Carr et al 1992;Li et al 1996), AKAP250/ Gravin to filopodia (Nauert et al 1997), AKAP350 to centrosomes (Schmidt et al 1999), AKAP100 to sarcoplasmic reticulum (McCartney et al 1995), AKAP220 to peroxisome (Lester et al 1996), AKAP85 to Golgi apparatus (Keryer et al 1993), and AKAP84/149 to mitochondria (Chen et al 1997).…”