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 Cß, 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 Cß arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cß kinase family is exceptionally high, a small set of signature residues defining Cα and Cß subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cß clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

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 Cß, 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. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser(109) significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly(186) completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly(186) is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg(2+), which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser(109) or Gly(186) may be faced with abnormal development and possibly severe disease.

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. In the present report, we show that both Calpha and Cbeta are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

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. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.

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. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function.

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. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.