Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates.

Identification and characterization of novel mutations in the human gene encoding the catalytic subunit Calpha of protein kinase A (PKA).

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

Background: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility.

Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing.

Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus.

Identification and characterization of novel PKA holoenzymes in human T lymphocytes.

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes.

Induction of Cbeta splice variants and formation of novel forms of protein kinase A type II holoenzymes during retinoic acid-induced differentiation of human NT2 cells.

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha.

Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication.

Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin.

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c.

HA95 is a protein of the chromatin and nuclear matrix regulating nuclear envelope dynamics.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane.

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform.

Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids.

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95.

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95.

Identification and quantitation of cAMP-dependent protein kinase R subunit isoforms in subcellular fractions of failing human myocardium.

Isoforms of regulatory (R) subunits of cAMP-dependent protein kinase were identified immunochemically and quantified in soluble and washed particulate fractions of failing human left ventricular myocardium. The predominant isoforms in both fractions were RI alpha and RII alpha. Both isoforms were present in comparable amounts in these fractions, although RII alpha subunits were somewhat more prevalent than RI alpha subunits in washed particulate fractions.

Differential localization of protein kinase A type II isozymes in the Golgi-centrosomal area.

Selectivity in the action of cAMP may be mediated by compartmentalized pools of cyclic AMP-dependent protein kinase (PKA). PKA type II is directed to different subcellular loci by interaction of the type II regulatory subunits (RIIalpha, RIIbeta) with A-kinase anchoring proteins. In order to separately investigate the subcellular localization of PKA type II isozymes, monospecific antibodies to human RIIalpha and RIIbeta subunits of PKA were developed.

Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.

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.

Mapping of the gene encoding the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome region 3p21.3-p21.2.

We have determined the chromosomal localization of the gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase (locus PRKAR2A) to human chromosome 3 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. Furthermore, PCR analysis of a chromosome 3 mapping panel revealed the presence of a human RII alpha-specific amplification product only in cell lines containing the region 3p21.3-p21.

The gene encoding the C gamma catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon.

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence.

Characterization of the gene encoding the human type II cGMP-dependent protein kinase.

The type II cGMP-dependent protein kinase (cGK) plays a pivotal role in the regulation of intestinal fluid balance in man. Furthermore, mice carrying a null mutation for the gene encoding the type II cGK develop as dwarfs indicating that this enzyme has other less characterized roles. The present report describes the isolation and characterization of bacterial artificial chromosome (BAC)- and P1-derived artificial chromosome (PAC)-clones containing the gene encoding the human type II cGK.

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis.

Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI2C2) and type II (RII2C2) PKA.

Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95.

The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M.

Characterization of the human gene encoding the type I alpha and type I beta cGMP-dependent protein kinase (PRKG1).

The type I cGMP-dependent protein kinase (cGK) has been shown to play a crucial role in the relaxation of vascular smooth muscle by lowering the intracellular level of calcium. Two isoforms of type I cGK have been described, type I alpha and type I beta, differing only in their N-terminal parts. This report describes the cloning of the gene PRKG1 encoding both human type I cGK isoforms.

Characterization of the 5'-flanking region of the gene for the cAMP-inducible protein kinase A subunit, RIIbeta, in Sertoli cells.

Activation of cyclic AMP-dependent protein kinases (protein kinase A, PKA) by gonadotropins and cyclic AMP (cAMP) plays an important role in the regulation of testicular functions. A regulatory subunit, RIIbeta, of PKA is transcriptionally induced in rat Sertoli cells in response to treatment with cAMP. The present study addresses regulatory mechanisms leading to increased transcription of the rat RIIbeta gene.

Molecular cloning, upstream sequence and promoter studies of the human gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase.

The gene for the regulatory subunit RII alpha of cAMP-dependent protein kinase is highly regulated during spermatogenesis and a strong signal from a distinct short mRNA form is observed postmeiotically during spermatid elongation. This report presents the isolation and characterization of the 5'-flanking region (1.2 kb) and exon 1 of the human RII alpha gene.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity.