Search

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 protein’s 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.

Alkylphenols are common aquatic pollutants originating from industrial use of the compounds themselves or as biodegradation products of alkylphenol polyethoxylates. The cytotoxicity of a range of alkylphenols and alkylated non-phenolics were assessed in a primary culture of rainbow trout (Onchorhynchus mykiss) hepatocytes to construct a structure-toxicity relationship for this group of ubiquitous aquatic pollutants. Metabolic inhibition and loss of membrane integrity were used as cytotoxic endpoints through use of the cellular markers Alamar blue and 5-carboxyfluorescein diacetate acetoxymethyl ester, respectively. The results show that cytotoxicity increased with the hydrophobicity of the alkylphenols for compounds with logK(OW)4.9. Normal chained alkylphenols, branched alkylphenols and multi-substituted alkylphenols with logK(OW)4.9 deviated clearly from this relationship. The alkylphenols displayed greater cytotoxicity than alkylated non-phenolics and it is proposed that most alkylated non-phenolic caused non-polar narcosis (baseline toxicity) whereas the alkylphenols caused polar narcosis. Observations that metabolic inhibition occurred at lower concentrations than loss of membrane integrity for most chemicals indicated that interference with cellular metabolic functions was the main cause of cytotoxicity. Metabolic inhibition corresponded better than loss of membrane integrity to reported acute toxicity to fish, although the in vivo acute toxicity of hydrophobic compounds (logK(OW)2-3) was clearly underestimated by both endpoints.

It is well documented that the b-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 Cb gene. In the present study, we demonstrate the expression of six novel human Cb mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbD4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbD4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbD4 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 CbD4 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 CbD4 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 CbD4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

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.

In several cases of immunodeficiency and autoimmunity, the dysfunctional immune system is associated with either hypo- or hyperactive T and B cells. In autoimmune conditions such as systemic lupus erythematosus (SLE) and immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), it has been demonstrated that the regulatory effect of the signaling pathway of cyclic 3', 5' adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) is abrogated. PKA is well-known as a key regulator of immune responses in that it inhibits both early and late phases of antigen induced T and B cell activation. Here we will discuss a potential useful strategy for therapeutic interventions of dysfunctional T cells associated with SLE and HIV by modulation of the cAMP-PKA pathway. Therefore, we will describe the components and architecture of the cAMP-PKA signaling pathway in T cells in order to point out one or several steps which potentially may serve as targets for therapeutic intervention.

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.

Background Protein 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. Results We 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. Conclusion We suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.

It is well documented that the beta-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 Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 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 CbetaDelta4 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 CbetaDelta4 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 CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.

Alkylphenols act as estrogen mimics by binding to and transactivating estrogen receptors (ERs) in fish. In the present study, activation of ER-mediated production of the estrogenic biomarker vitellogenin (vtg) in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes was used to construct a structure–activity relationship for this ubiquitous group of aquatic pollutants. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure was assessed. The results showed that most alkylphenols were estrogenic, although with 3–300 thousand times lower affinity than the endogenous estrogen 17β-estradiol. Mono-substituted tertiary alkylphenols with moderate (C4–C5) and long alkyl chain length (C8–C9) in the para position exhibited the highest estrogenic potency. Substitution with multiple alkyl groups, presence of substituents in the ortho- and meta-position and lack of a hydroxyl group on the benzene ring reduced the estrogenic activity, although several estrogenic alkylated non-phenolics were identified. Co-exposures with the natural estrogen 17β-estradiol led to identification of additional estrogenic compounds as well as some anti-estrogens. A combination of low affinity for the ER and cytotoxicity was identified as factors rendering some of the alkylphenols non-estrogenic in the bioassay when tested alone.

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.