Anne-Katrine Kvissel

Splicing factor arginine/serine-rich 17A (SFRS17A) is an A-kinase anchoring protein that targets protein kinase A to splicing factor compartments.

Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kinase A anchoring proteins (AKAPs). AKAPs are divided into subclasses based on their ability to bind type I or type II PKA or both. Dual-specificity AKAPs were recently reported to have an additional PKA binding determinant called the RI specifier region. A bioinformatic search with the consensus RI specifier region identified a novel AKAP, the splicing factor arginine/serine-rich 17A (SFRS17A). Here, we show by a variety of protein interaction assays that SFRS17A binds both type I and type II PKA in vitro and inside cells, demonstrating that SFRS17A is a dual-specific AKAP. Moreover, immunofluorescence experiments show that SFRS17A colocalizes with the catalytic subunit of PKA as well as the splicing factor SC35 in splicing factor compartments. Using the E1A minigene splicing assay, we found that expression of wild type SFRS17A conferred regulation of E1A alternative splicing, whereas the mutant SFRS17A, which is unable to bind PKA, did not. Our data suggest that SFRS17A is an AKAP involved in regulation of pre-mRNA splicing possibly by docking a pool of PKA in splicing factor compartments.

G-patch domain and KOW motifs-containing protein, GPKOW; a nuclear RNA-binding protein regulated by protein kinase A

Background Post-transcriptional processing of pre-mRNA takes place in several steps and requires involvement of a number of RNA-binding proteins. How pre-mRNA processing is regulated is in large enigmatic. The catalytic (C) subunit of protein kinase A (PKA) is a serine/threonine kinase, which regulates numerous cellular processes including pre-mRNA splicing. Despite that a significant fraction of the C subunit is found in splicing factor compartments in the nucleus, there are no indications of a direct interaction between RNA and PKA. Based on this we speculate if the specificity of the C subunit in regulating pre-mRNA splicing may be mediated indirectly through other nuclear proteins. Results Using yeast two-hybrid screening with the PKA C subunit Cbeta2 as bait, we identified the G-patch domain and KOW motifs-containing protein (GPKOW), also known as the T54 protein or MOS2 homolog, as an interaction partner for Cbeta2. We demonstrate that GPKOW, which contains one G-patch domain and two KOW motifs, is a nuclear RNA-binding protein conserved between species. GPKOW contains two sites that are phosphorylated by PKA in vitro. By RNA immunoprecipitation and site directed mutagenesis of the PKA phosphorylation sites we revealed that GPKOW binds RNA in vivo in a PKA sensitive fashion. Conclusion GPKOW is a RNA-binding protein that binds RNA in a PKA regulated fashion. Together with our previous results demonstrating that PKA regulates pre-mRNA splicing, our results suggest that PKA phosphorylation is involved in regulating RNA processing at several steps.

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing.

Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates

It is well documented that the β‐gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N‐terminal end encoded by alternative use of several exons located 5′ to exon 2 in the human, bovine and mouse Cβ gene. In the present study, we demonstrate the expression of six novel human Cβ mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CβΔ4, were identified in low amounts at the mRNA level in NTera2‐N cells. We developed a method to detect CβΔ4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CβΔ4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP‐responsive element binding protein. Furthermore, co‐expression of CβΔ4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CβΔ4 variants associate with both RI and RII in a cAMP‐independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CβΔ4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

Protein Kinase A–Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein

Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the proteins capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.

Effect of acidosis on IL-8 and MCP-1 during hypoxia and reoxygenation in human NT2-N neurons.

Inflammation probably plays a significant role in perinatal brain injury. To study the contribution of locally produced cytokines, the effect on cell death of addition of IL-8 and MCP-1 or antibodies to these, and the impact of acidosis, human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and reoxygenated for 21 h. After 3 h of hypoxia with neutral medium, IL-8 was significantly increased compared to controls (150 (100-250)% vs. 100 (85-115)%, p=0.023). After 21 h of neutral reoxygenation, both IL-8 (380 (110-710)% vs. 150 (85-260)%, p=0.041) and monocyte chemoattractant protein-1 (MCP-1) (650 (440-2000)% vs. 310 (230-340)%, p=0.007) were significantly increased compared to controls. After 3 h of hypoxia, both IL-8 (p=0.002) and MCP-1 (p=0.008) were significantly lower in cells with acidotic compared with cells with neutral medium. Acidosis during reoxygenation, however, significantly increased IL-8 release, whereas MCP-1 release was diminished. Similar effects of acidosis were seen in normoxic controls. The cells also secreted RANTES and IP-10, but not 8 other cytokines tested. We found no effect on cell death, measured by MTT assay, of addition of IL-8, MCP-1 or antibodies to these. We conclude that human NT2-N neurons release IL-8 and MCP-1 during 21 h of reoxygenation after 3 h of hypoxia. Acidosis led to a differential effect on IL-8 and MCP-1, with increased IL-8 and decreased MCP-1, both during reoxygenation and in normoxic controls. IL-8 and MCP-1 had no effect on cell death.

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

BackgroundEpidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits.ResultsWe have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level.ConclusionOur results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

Protein kinase A type I activates a CRE-element more efficiently than protein kinase A type II regardless of C subunit isoform

BackgroundProtein kinase A type I (PKAI) and PKAII are expressed in most of the eukaryotic cells examined. PKA is a major receptor for cAMP and specificity is achieved partly through tissue-dependent expression and subcellular localization of subunits with different biochemical properties. In addition posttranslational modifications help fine tune PKA activity, distribution and interaction in the cell. In spite of this the functional significance of two forms of PKA in one cell has not been fully determined. Here we have tested the ability of PKAI and PKAII formed by expression of the regulatory (R) subunits RIα or RIIα in conjunction with Cα1 or Cβ2 to activate a co-transfected luciferace reporter gene, controlled by the cyclic AMP responsive element-binding protein (CREB) in vivo.ResultsWe show that PKAI when expressed at equal levels as PKAII was significantly (p < 0.01) more efficient in inducing Cre-luciferace activity at saturating concentrations of cAMP. This result was obtained regardless of catalytic subunit identity.ConclusionWe suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.