AKAP signaling complexes at the cytoskeleton.
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Targeting of protein kinases and phosphatases to the cytoskeleton enhances the regulation of signal transduction events. The assembly of cytoskeletal signaling complexes facilitates the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. These signals influence fundamental cell properties, such as shape, movement and division. Targeting of the cAMP-dependent kinase (PKA) to the cytoskeleton is achieved through interaction with A-kinase anchoring proteins (AKAPs). AKAPs maintain multivalent signaling complexes by binding additional enzymes, including kinases and phosphatases. (+info)
Targeting of an A kinase-anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1.
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Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoring proteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathione S-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites. (+info)
Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIalpha regulatory subunit of cAMP-dependent protein kinase.
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In skeletal muscle, transcription of the gene encoding the mouse type Ialpha (RIalpha) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIalpha protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIalpha or RIIalpha fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIalpha subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RIalpha-green fluorescent protein template abolishes localization, indicating that dimerization of RIalpha is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIalpha at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Ialpha homologue R(CE) with AKAP(CE) and for in vitro binding of RIalpha to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIalpha tethering at this site. (+info)
AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex.
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In cardiac myocytes, the slow component of the delayed rectifier K(+) current (I(Ks)) is regulated by cAMP. Elevated cAMP increases I(Ks) amplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, I(Ks) channels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the I(Ks) amplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the I(Ks) amplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of I(Ks) requires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying I(Ks). (+info)
The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.
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The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor. (+info)
Expression of a down-regulated target, SSeCKS, reverses v-Jun-induced transformation of 10T1/2 murine fibroblasts.
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Line 10T1/2 mouse fibroblast overexpressing the v-Jun oncoprotein were morphologically altered, grew into multilayered foci in culture and formed colonies when suspended in agar. The growth rate of the v-Jun-transformed 10T1/2 cells was not changed significantly from that of the untransformed parental cells, but the saturation density of the transformed cultures exceeded that of normal controls by a factor of 2. mRNA extracted from v-Jun-transformed 10T1/2 cells was analysed for differential gene expression with DNA micro-array technology. One of the targets downregulated by v-Jun was identified as SSeCKS (Src-suppressed C kinase substrate). Re-expression of SSeCKS in v-Jun-transformed fibroblasts reversed the transformed phenotype of the cells. Their ability to form foci was reduced to background levels, the number and size of agar colonies was lowered by a factor of 10 and the saturation density was significantly diminished. However, expression of SSeCKS had little effect on the morphology of v-Jun-transformed 10T1/2 cells. These data suggest that the SSeCKS protein has growth-attenuating properties. Down-regulation of SSeCKS may be essential for Jun-induced transformation. (+info)
Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression.
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We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C. (+info)
Gene trap insertion reveals two open reading frames in the mouse SSeCKS gene: the form predominantly detected in the nervous system is suppressed by the insertion while the other, specific of the testis, remains expressed.
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Scaffold proteins play an important role in regulating signal transduction by targeting kinases and phosphatases in close proximity to their relevant substrates. SSeCKS protein has been described as a protein kinase C and A (PKC/PKA) anchoring protein as well as a PKC substrate with a tumor suppressor activity. In this study, we report the generation, via gene trapping in embryonic stem cells of mice carrying an insertion in the mouse SSeCKS gene. Through the molecular analysis of the insertion site, we show that SSeCKS contains two alternative promoters directing the synthesis of mRNAs (P1- and P2-mRNA), encoding two different proteins, one of which would be a truncated form of the other. Interestingly, these RNAs are differentially expressed, P2 being found exclusively in the male germ line, while P1 exhibits a dynamic and wider pattern of expression during embryonic development and in the adult; its expression is predominant in the nervous system. Finally, we show that P1- but not P2-mRNA expression is abolished by the insertion and furthermore that mice homozygous for the mutation lack SSeCKS in all tissues except the male germ cells. Nevertheless and surprisingly, these mice do not exhibit any obvious phenotype. The functional implications of these observations are discussed. (+info)