Elevated Protein Kinase A Activity Research Articles

Cyclic AMP-Dependent Protein Kinase Exhibits Antagonistic Effects on the Replication Efficiency of Different Human Papillomavirus Types.

Several types of widespread human papillomaviruses (HPVs) may induce the transformation of infected cells, provoking the development of neoplasms. Two main genera of HPVs are classified as mucosatropic alphapapillomaviruses and cutaneotropic betapapillomaviruses (α- and β-HPVs, respectively), and they both include high-risk cancer-associated species. The absence of antiviral drugs has driven investigations into the details of the molecular mechanisms of the HPV life cycle. HPV replication depends on the viral helicase E1 and the transcription factor E2. Their biological activities are controlled by numerous cellular proteins, including protein kinases. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of α-HPV11, α-HPV18, and β-HPV5. PKA stimulates the replication of both α-HPVs studied but has a more profound effect on the replication of high-risk α-HPV18. However, the replication of β-HPV5 is inhibited by activated PKA in human primary keratinocytes and U2OS cells. We show that the activation of PKA signaling by different pharmacological agents induces the rapid proteasomal degradation of the HPV5 E2 protein, which in turn leads to the downregulation of E2-dependent transcription. In contrast, PKA-stimulated induction of HPV18 replication is the result of the downregulation of the E8^E2 transcript encoding a potent viral transcriptional inhibitor together with the rapid upregulation of E1 and E2 protein levels. IMPORTANCE Several types of human papillomaviruses (HPVs) are causative agents of various types of epithelial cancers. Here, we report that ubiquitously expressed cyclic AMP-dependent protein kinase A (PKA) differentially regulates the replication of various types of HPVs during the initial amplification and maintenance phases of the viral life cycle. The replication of the skin cancer-related pathogen HPV5 is suppressed, whereas the replication of the cervical cancer-associated pathogen HPV18 is activated, in response to elevated PKA activity. To inhibit HPV5 replication, PKA targets the viral transcriptional activator E2, inducing its rapid proteasomal degradation. PKA-dependent stimulation of HPV18 replication relies on the downregulation of another E2 gene product, E8^E2, which encodes a potent transcriptional repressor. Our findings highlight, for the first time, protein kinase-related mechanistic differences in the regulation of the replication of mucosal and cutaneous HPV types.

Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via cAMP-PKA signal pathway.

To test our hypothesis that activation of protein kinase A (PKA) signal pathway by β-adrenergic agonist plays an important role in the protecting of cultured retinal pigment epithelial (RPE) cells against the hydroxychloroquine (HCQ) toxicity. Cultured human RPE cells were treated with 1) HCQ, 2) HCQ with salbutamol (a β2-adrenergic receptor agonist), and 3) HCQ with salbutamol and a PKA inhibitor, and compared these to 4) untreated cells (controls). After treated for 24h, cell vacuolation, cells viability, PKA and PKA kinase activity levels were determined by the measurement of the size of vacuoles using Image J software, the cell counting with a dye-exclusion testing, Western blot and PKA kinase detection, respectively. Cell vacuolation and cell death of cultured RPE cells were significantly increased by the treatment of HCQ. Salbutamol significantly elevated PKA and PKA activity levels and this was associated with the inhibition of the vacuolation and cell death. The PKA inhibitor significantly decreased the PKA levels and eliminated the protective effects of salbutamol on HCQ-treated RPE cells. The PKA pathway plays an important role in the protective effects of β2-adrenergic agonist on the RPE cells against HCQ toxicity. These findings reveal a novel potential strategy against HCQ retinopathy by treatment with PKA activating medications.

T-Darpp stimulates protein kinase A activity by forming a complex with its RI regulatory subunit

t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDpT39A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDpT39A cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp–mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.

Abstract 4093: t-Darpp stimulates protein kinase A activity by interacting with its RI regulatory subunit

Abstract t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway. t-Darpp over-expression results in enhanced activity of protein kinase A (PKA), but the mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines that over-express t-Darpp (SK.tDrp) or a T75A phosphorylation mutant (SK.tDrp.T75A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. Whereas SK.tDrp cells had elevated PKA activity, SK.tDrp.T75A cells did not. In agreement with the effects on activity, a proximity ligation assay showed that SK.tDrp cells, but not SK.tDrp.T75A cells, had diminished association of RI with PKAc. Moreover, t-Darpp was seen to bind directly to RI in co-immunoprecipitation studies. Cells that express both Darpp-32 and t-Darpp had PKA activity levels and RI-PKAc association that were similar to control SK.empty cells. This is consistent with our published findings that Darpp-32 reverses t-Darpp’s effects on trastuzumab resistance and CREB DNA binding activity (downstream of PKA). Taken together, our data suggest that T75 phosphorylation is crucial for t-Darpp-mediated PKA activation and this activation appears to occur through direct binding to RI and release of RI from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer. Citation Format: Dirk Theile, Shuhui Geng, Erin Denny, Jamil Momand, Susan E. Kane. t-Darpp stimulates protein kinase A activity by interacting with its RI regulatory subunit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2017-4093

Central Role of Protein Kinase A in Promoting Trigeminal Nociception in an In Vivo Model of Temporomandibular Disorders.

To investigate cellular changes in the spinal trigeminal nucleus (STN) and trigeminal ganglion (TG) associated with trigeminal nociception mediated by inflammation in the temporomandibular joint (TMJ). Male Sprague-Dawley rats (n = 86) were utilized to investigate cellular and behavioral responses to prolonged TMJ inflammation caused by bilateral injection of Complete Freund's Adjuvant (CFA) in the TMJ capsules. To investigate the cellular effects of protein kinase A (PKA) in the STN, rats were injected intrathecally with the selective PKA inhibitor KT5720 prior to injection of CFA into both TMJ capsules. Levels of calcitonin gene-related peptide (CGRP), active PKA, and ionized calcium-binding adapter molecule 1 (Iba1) in the STN and expression of phosphorylated extracellular regulated kinases (p-ERK) in the TG were determined with immunohistochemistry (n ≥ 3 experiments per test condition). Nocifensive head withdrawal responses to mechanical stimulation of the cutaneous tissue over the TMJ were monitored following CFA injection in the absence or presence of KT5720 (n = 7). Statistical analysis was performed using parametric analysis of variance (ANOVA) tests. Intrathecal injection of KT5720 significantly inhibited the stimulatory effect of CFA on levels of CGRP, PKA, and Iba1 in the STN. In addition, administration of KT5720 decreased the average number of CFA-induced nocifensive withdrawal responses to mechanical stimulation and the CFA-mediated increase in p-ERK expression in the ganglion. These findings provide evidence that elevated PKA activity in the STN promotes cellular events temporally associated with trigeminal nociception caused by prolonged TMJ inflammation.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation.

Enhanced cAMP-stimulated protein kinase A activity in human fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.

The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that has an antioxidative protective effect on various tissues. Here, we determined whether GLP-1 has a role in the pathogenesis of diabetic nephropathy using nephropathy-resistant C57BL/6-Akita and nephropathy-prone KK/Ta-Akita mice. By in situ hybridization, we found the GLP-1 receptor (GLP-1R) expressed in glomerular capillary and vascular walls, but not in tubuli, in the mouse kidney. Next, we generated C57BL/6-Akita Glp1r knockout mice. These mice exhibited higher urinary albumin levels and more advanced mesangial expansion than wild-type C57BL/6-Akita mice, despite comparable levels of hyperglycemia. Increased glomerular superoxide, upregulated renal NAD(P)H oxidase, and reduced renal cAMP and protein kinase A (PKA) activity were noted in the Glp1r knockout C57BL/6-Akita mice. Treatment with the GLP-1R agonist liraglutide suppressed the progression of nephropathy in KK/Ta-Akita mice, as demonstrated by reduced albuminuria and mesangial expansion, decreased levels of glomerular superoxide and renal NAD(P)H oxidase, and elevated renal cAMP and PKA activity. These effects were abolished by an adenylate cyclase inhibitor SQ22536 and a selective PKA inhibitor H-89. Thus, GLP-1 has a crucial role in protection against increased renal oxidative stress under chronic hyperglycemia, by inhibition of NAD(P)H oxidase, a major source of superoxide, and by cAMP-PKA pathway activation.

The Protein Kinase A Regulatory Subunit R1A (Prkar1a) Plays Critical Roles in Peripheral Nerve Development

Signaling through cAMP has been implicated in Schwann cell (SC) proliferation and myelination, but the signaling pathway components downstream of cAMP required for SC function remain unknown. Protein kinase A (PKA) is a potential downstream effector of cAMP. Here, we induced loss of Prkar1a, the gene encoding the type 1A regulatory subunit of PKA, in SC to study its role in nerve development; loss of Prkar1a is predicted to elevate PKA activity. Conditional Prkar1a knock-out in mouse SC (Prkar1a-SCKO) resulted in a dramatic and persistent axonal sorting defect, and unexpectedly decreased SC proliferation in Prkar1a-SCKO nerves in vivo. Effects were cell autonomous as they were recapitulated in vitro in Prkar1a-SCKO SC, which showed elevated PKA activity. In the few SCs sorted into 1:1 relationships with axons in vivo, SC myelination was premature in Prkar1a-SCKO nerves, correlating with global increase in the cAMP-regulated transcription factor Oct-6 and expression of myelin basic protein. These data reveal a previously unknown role of PKA in axon sorting, an unexpected inhibitory role of PKA on SC cell proliferation in vivo and define the importance of Prkar1a in peripheral nerve development.

ABCA1 Protein Enhances Toll-like Receptor 4 (TLR4)-stimulated Interleukin-10 (IL-10) Secretion through Protein Kinase A (PKA) Activation

ABCA1 is known to suppress proinflammatory cytokines. ABCA1 activates PKA and up-regulates anti-inflammatory cytokine IL-10. Elevated PKA transforms macrophages to M2-like phenotype. Disrupting lipid rafts by statins MCD, and filipin recuperates ABCA1 phenotype and likely functions downstream of ABCA1. By modulating cholesterol, ABCA1 activates PKA. This generates M2-like macrophages. ABCA1 does not simply suppress inflammatory response. It promotes M2-like activation and facilitates resolution. Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1(+/+) bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1(-/-) bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1(+/+) bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-β-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.

Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.

Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.