Compensatory stabilization of RIIbeta protein, cell cycle deregulation, and growth arrest in colon and prostate carcinoma cells by antisense-directed down-regulation of protein kinase A RIalpha protein.
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The cyclic AMP-dependent protein kinase (PKA) exists in two isoforms, PKA-I (type I) and PKA-II (type II), that contain an identical catalytic (C) subunit but distinct regulatory (R) subunits, RI and RII, respectively. Increased expression of RIalpha/PKA-I has been shown in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. We have shown previously that a single-injection RI, antisense treatment results in a reduction in RIalpha and PKA-I expression and sustained inhibition of human colon carcinoma growth in athymic mice (M. Nesterova and Y. S. Cho-Chung, Nat. Med., 1: 528-533, 1995). Growth inhibition accompanied reduction in RIalpha/PKA-I expression and compensatory increases in RIIbeta protein and PKA-IIbeta, the RIIbeta-containing holoenzyme. Here, we report that these in vivo findings are consistent with observations made in cancer cells in culture. We demonstrate that the antisense depletion of RIalpha in cancer cells results in increased RIIbeta protein without increasing the rate of RIIbeta synthesis or RIIbeta mRNA levels. Pulse-chase experiments revealed a 3-6-fold increase in the half-life of RIIbeta protein in antisense-treated colon and prostate carcinoma cells with little or no change in the half-lives of RIalpha, RIIalpha, and Calpha proteins. Compensation by RIIbeta stabilization may represent a novel biochemical adaptation mechanism of the cell in response to sequence-specific loss of RIalpha expression, which leads to sustained down-regulation of PKA-I activity and inhibition of tumor growth. (+info)
mRNA mutations of type I protein kinase A regulatory subunit alpha in T lymphocytes of a subject with systemic lupus erythematosus.
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Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by multiple T lymphocyte immune effector dysfunctions. Protein kinase A (PKA) isozymes contribute to the regulation of T cell immune effector functions. In SLE T cells, there is a profound deficiency of PKA-I isozyme activity characterized by both reduced RI alpha transcript and RI alpha protein levels. To identify a molecular mechanism(s) for this isozyme deficiency, we utilized single-strand conformation polymorphism (SSCP) analysis to detect structural changes in the cDNA. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band. Sequence analyses demonstrated that a shifted SSCP band from SLE T cells carried heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats-regions that are susceptible to transcript editing and/or molecular misreading. By contrast, no genomic mutations were identified. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in a subject with SLE. Mutant RI alpha transcripts are pathophysiolgically significant, for they can encode diverse, aberrant RI alpha isoforms, including truncated, dominant-negative subunits, resulting in deficient PKA-I activity. We propose that deficient PKA-I isozyme activity contributes to the pathogenesis of SLE by hindering effective signal transduction and impairing T cell effector functions. (+info)
Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the carney complex.
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Carney complex (CNC) is an autosomal dominant multiple neoplasia syndrome, which has been linked to loci on 2p16 and 17q22-24. We recently reported that PRKAR1A, which codes for the type 1A regulatory subunit of protein kinase A (PKA), is a tumor suppressor gene on chromosome 17 that is mutated in some CNC families. To evaluate the spectrum of PRKAR1A mutations, we identified its genomic structure and screened for mutations in 54 CNC kindreds (34 families and 20 patients with sporadic disease). Fourteen families were informative for linkage analysis: four of four families that mapped to 17q had PRKAR1A mutations, whereas there were no mutations found in seven families exhibiting at least one recombination with 17q. In six of the latter, CNC mapped to 2p16. PRKAR1A mutations were also found in 12 of 20 non-informative families and 7 of 20 sporadic cases. Altogether, 15 distinct PRKAR1A mutations were identified in 22 of 54 kindreds (40.7%). In 14 mutations, the sequence change was predicted to lead to a premature stop codon; one altered the initiator ATG codon. Mutant mRNAs containing a premature stop codon were unstable, as a result of nonsense-mediated mRNA decay. Accordingly, the predicted truncated PRKAR1A protein products were absent in these cells. We conclude that (i) genetic heterogeneity exists in CNC; and (ii) all of the CNC alleles on 17q are functionally null mutations of PRKAR1A. CNC is the first human disease recognized to be caused by mutations of the PKA holoenzyme, a critical component of cellular signaling. (+info)
Resonant mirror biosensor analysis of type Ialpha cAMP-dependent protein kinase B domain--cyclic nucleotide interactions.
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A resonant mirror biosensor was used to study cyclic nucleotide-receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide-binding properties of the wild-type isolated B domain of the cAMP-dependent protein kinase type Ialpha regulatory subunit and its Ala-334-Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP-saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X-100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild-type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor-determined kinetic constants. Biosensor-determined relative inhibition constants (Ki' = Ki(cAMP)/Ki(analog)) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published Ki' values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The Ki' values for the wild-type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide-receptor interactions. (+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)
Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme.
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Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIalpha mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Calpha catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy. (+info)
Binding of PKA-RIIalpha to the Adenovirus E1A12S oncoprotein correlates with its nuclear translocation and an increase in PKA-dependent promoter activity.
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The adenovirus type 12 (Ad12) E1A12S oncoprotein utilizes the cAMP/protein kinase A (PKA) signal transduction pathway to activate expression of the viral E2 gene, the products of which are essential for viral replication. A central unsolved question is, however, whether E1A12S interacts directly with PKA in the process of promoter activation. We show here that E1A12S binds to the regulatory subunits (R) of PKA in vitro and in vivo. Interaction depends on the N-terminus and the conserved region 1 (CR1) of E1A12S. Both domains are also essential for the activation of viral E2 gene expression. Infection of cells with Ad12 leads to the cellular redistribution of RIIalpha from the cytoplasm into the nucleus. Furthermore, RIIalpha is also located in the nucleus of cells transformed by E1 of Ad12 and transient expression of E1A12S leads to the redistribution of RIIalpha into the nucleus in a N-terminus- and CR1-dependent manner. Cotransfection of E1A12S with RIIalpha results in strong activation of the E2 promoter. Based on these results we conclude that E1A12S functions as a viral A-kinase anchoring protein redistributing RIIalpha from the cytoplasm into the nucleus where it is involved in E1A12S-mediated activation of the E2 promoter. (+info)
Nuclear translocation of the catalytic subunit of protein kinase A induced by an antisense oligonucleotide directed against the RIalpha regulatory subunit.
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The regulatory (R) subunits of cAMP-dependent protein kinase (PKA) are implicated in the regulation of cell proliferation and differentiation. There are two isoforms of PKA that are distinguished by two types of R subunit, RI and RII. Evidence suggests that RI is associated with proliferation and RII is associated with cell differentiation. Previous work in this laboratory has demonstrated that depletion of the RIalpha subunit by treatment with an antisense oligonucleotide (ODN) induces differentiation in leukemia cells and growth arrest and apoptosis in epithelial cancer cells. Using the prostate cancer cell line PC3M as a model system, we have developed a cell line that overexpresses a retroviral vector construct containing the RIalpha antisense gene. This cell line has been characterized and the effectiveness of the construct determined. In the work presented here, we demonstrate by immunocytochemistry that treatment with RIalpha antisense ODN induces translocation of the Calpha subunit of PKA to the nucleus of PC3M prostate cancer cells. The translocation of Calpha triggered by exogenous antisense ODN treatment mirrors that observed in cells endogenously overexpressing the antisense gene. Triggering the nuclear translocation of the Calpha subunit of PKA in the cell may be an important mechanism of action of RIalpha antisense that regulates cell growth independent of adenylate cyclase and cellular cAMP levels. The nuclear localization of the Calpha subunit of PKA may be an essential step in revealing the mechanism whereby this critical kinase regulates cell growth. (+info)