The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.
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cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus. (+info)
Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.
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A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene. (+info)
Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220.
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The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme. (+info)
NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum.
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Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1-30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1-63 fused to GFP are necessary and sufficient for ER targeting. Residues 14-33 of N1 are especially important for targeting to ER; however, residues 1-33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1-30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network. (+info)
Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.
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A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates. (+info)
Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex.
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Regulation of N-methyl-D-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor-associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity. (+info)
Conservation and function of a bovine sperm A-kinase anchor protein homologous to mouse AKAP82.
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Protein kinase A regulates sperm motility through the cAMP-dependent phosphorylation of proteins. One mechanism to direct the activity of the kinase is to localize it near its protein substrates through the use of anchoring proteins. A-Kinase anchoring proteins (AKAPs) act by binding the type II regulatory subunit of protein kinase A and tethering it to a cellular organelle or cytoskeletal element. We showed previously that mAKAP82, the major protein of the fibrous sheath of the mouse sperm flagellum, is an AKAP. The available evidence indicates that protein kinase A is compartmentalized to the fibrous sheath by binding mAKAP82. To characterize AKAP82 in bovine sperm, a testicular cDNA library was constructed and used to isolate a clone encoding bAKAP82, the bovine homologue. Sequence analysis showed that the primary structure of bAKAP82 was highly conserved. In particular, the amino acid sequence corresponding to the region of mAKAP82 responsible for binding the regulatory subunit of protein kinase A was identical in the bull. Bovine AKAP82 was present in both epididymal and ejaculated sperm and was localized to the entire principal piece of the flagellum, the region in which the fibrous sheath is located. Finally, bAKAP82 bound the regulatory subunit of protein kinase A. These data support the idea that bAKAP82 functions as an anchoring protein for the subcellular localization of protein kinase A in the flagellum. (+info)
mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes.
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The compartmentalization of second messenger-activated protein kinases contributes to the fidelity of hormone-mediated signal transduction events. For example, the cAMP-dependent protein kinase is tethered at specific intracellular locations through association with A-kinase anchoring proteins (AKAPs). We now report the cloning of mAKAP, an anchoring protein found predominantly in heart, skeletal muscle and brain, and whose expression is induced in neonatal ventriculocytes by treatment with hypertrophic stimuli. mAKAP is targeted to the nuclear membrane of differentiated myocytes. Analysis of mAKAP-green fluorescent protein (GFP) fusion constructs revealed that nuclear membrane targeting is conferred by two regions of the protein, between residues 772-915 and 915-1065, which contain spectrin-like repeat sequences. Heterologous expression of the mAKAP targeting sequences displaced the endogenous anchoring protein from the nuclear membrane, demonstrating that mAKAP targeting is saturable. Collectively, these data suggest that a domain containing spectrin-like repeats mediates targeting of the anchoring protein mAKAP and the cAMP-dependent protein kinase holoenzyme to the nuclear membrane in response to differentiation signals. (+info)