Search

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ., Competing Interests: Declaration of competing interest All authors declare that they don’t have any conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe . We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1-308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1-241) both resulted in more severe phenotypes than Mal3 (1-308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446 , was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, meiosis, and stress responses in Schizosaccharomyces pombe. We demonstrated that Pka1 is responsible for calcium tolerance. Loss of functional components of the PKA pathway such as Git3, Gpa2, Cyr1, and Pka1 yields a CaCl 2 -sensitive phenotype, while loss of Cgs1, a regulatory subunit of PKA, results in CaCl 2 tolerance. Cytoplasmic distribution of Cgs1 and Pka1 is increased by the addition of CaCl 2 , suggesting that CaCl 2 induces dissociation of Cgs1 and Pka1. The expression of Prz1, a transcriptional regulator in calcium homeostasis, is elevated in a pka1∆ strain and in a wild type strain under glucose-limited conditions. Accordingly, higher expression of Prz1 in the wild type strain results in a CaCl 2 -sensitive phenotype. These findings suggest that Pka1 is essential for tolerance to exogenous CaCl 2 , probably because the expression level of Prz1 needs to be properly regulated by Pka1.

The Hedgehog (Hh) signaling pathway has numerous roles in the control of cell proliferation, tissue patterning and stem cell maintenance. In spite of intensive study, the mechanisms of Hh signal transduction are not completely understood. Here I review published data and present a novel model of vertebrate Hh signaling suggesting that Smoothened (Smo) functions as a G-protein-coupled receptor in cilia. This is the first model to propose molecular mechanisms for the major steps of Hh signaling, including inhibition of Smo by Patched, Smo activation, and signal transduction from active Smo to Gli transcription factors. It also suggests a novel role for the negative pathway regulators Sufu and PKA in these processes.

A reduced sinoatrial node (SAN) functional reserve underlies the age-associated decline in heart rate acceleration in response to stress. SAN cell function involves an oscillatory coupled-clock system: the sarcoplasmic reticulum (SR), a Ca(2+) clock, and the electrogenic-sarcolemmal membrane clock. Ca(2+)-activated-calmodulin-adenylyl cyclase/CaMKII-cAMP/PKA-Ca(2+) signaling regulated by phosphodiesterase activity drives SAN cells automaticity. SR-generated local calcium releases (LCRs) activate Na(+)/Ca(2+) exchanger in the membrane clock, which initiates the action potential (AP). We hypothesize that SAN cell dysfunctions accumulate with age. We found a reduction in single SAN cell AP firing in aged (20-24 mo) vs. adult (3-4 mo) mice. The sensitivity of the SAN beating rate responses to both muscarinic and adrenergic receptor activation becomes decreased in advanced age. Additionally, age-associated coincident dysfunctions occur stemming from compromised clock functions, including a reduced SR Ca(2+) load and a reduced size, number, and duration of spontaneous LCRs. Moreover, the sensitivity of SAN beating rate to a cAMP stress induced by phosphodiesterase inhibitor is reduced, as are the LCR size, amplitude, and number in SAN cells from aged vs. adult mice. These functional changes coincide with decreased expression of crucial SR Ca(2+)-cycling proteins, including SR Ca(2+)-ATPase pump, ryanodine receptors, and Na(+)/Ca(2+) exchanger. Thus a deterioration in intrinsic Ca(2+) clock kinetics in aged SAN cells, due to deficits in intrinsic SR Ca(2+) cycling and its response to a cAMP-dependent pathway activation, is involved in the age-associated reduction in intrinsic resting AP firing rate, and in the reduction in the acceleration of heart rate during exercise.

Background: The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4., Results: We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA., Conclusions: In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in combination, in which PKA is often involved. Redundant signalling pathways confer a greater robustness to the response to glucose, while cooperative pathways provide a greater flexibility.

Cyclic-AMP stimulation of GTP cyclohydrolase I (GCH1) gene transcription was investigated in PC12 cells, the protein kinase A-deficient PC12 cell line 126-1B2 and C6 cells using transient transfection assays of proximal promoter reporter constructs and wild type or dominant negative proteins, chromatin immunoprecipitation and real-time quantitative PCR. These studies show that protein kinase A is necessary and sufficient for cAMP-dependent transcription conferred by both the cAMP regulatory element and the adjacent CCAAT-box. In intact cells these cis-elements were shown to bind cAMP response element binding protein, CCAAT-enhancer binding protein beta and nuclear factor-Y, with each protein controlling a different aspect of the cAMP response. Cyclic-AMP acting through protein kinase A stimulated promoter recruitment of CCAAT-enhancer binding protein beta, nuclear factor-Y and RNA polymerase II while depleting the promoter of cyclic-AMP response element binding protein. Stimulation of transcription by cAMP was not associated with increased acetylation of histones H3 and H4 at proximal promoter nucleosomes, indicating that histone acetyltransferases are not involved in this response. Nonetheless, pharmacological inhibition of histone deacetylase activity did increase histone H4 acetylation and the recruitment of RNA polymerase II, indicating that histone acetyltransferases are normally associated with the proximal promoter. Only in C6 cells, however, did inhibition of histone deacetylases stimulate transcription and synergize with cAMP. These experiments provide the first glimpse of the GCH1 gene promoter functioning within intact cells and supply evidence for the involvement of histone acetyltransferase-containing complexes in GCH1 gene transcription.

The human PRKAR1A gene encodes the regulatory subunit 1-alpha (RIalpha) of the cAMP-dependent protein kinase A (PKA) holoenzyme. Regulation of the catalytic activity of PKA is the only well-studied function of RIalpha. Inactivating PRKAR1A mutations cause primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC), an inherited syndrome associated with abnormal skin pigmentation and multiple neoplasias, including PPNAD. Histochemistry of tissues from CNC patients is indicative of autophagic deficiency and this led us to investigate the relationship between RIalpha and mammalian autophagy. We found that fluorescently tagged RIalpha associates with late endosomes and autophagosomes in cultured cells. The number of autophagosomes in prkar1a-/- mouse embryonic fibroblasts (MEFs) was reduced compared with wild-type MEFs. RIalpha co-immunoprecipitated with mTOR kinase, a major regulator of autophagy. Phosphorylated-mTOR levels and mTOR activity were dramatically increased in prkar1a-/- mouse cells, and in HEK 293 cells with RIalpha levels reduced by siRNA. Finally, phosphorylated-mTOR levels and mTOR activity were increased in CNC cells and in PPNAD tissues. These data suggest that RIalpha deficiency decreases autophagy by the activation of mTOR, providing a molecular basis to autophagic deficiency in PPNAD.

The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks.

Previous studies have shown that inhibition of L-type Ca(2+) current (I(Ca)) by cytosolic free Mg(2+) concentration ([Mg(2+)](i)) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I(Ca), rat cardiac myocytes and tsA201 cells expressing L-type Ca(2+) channels were whole cell voltage-clamped with patch pipettes in which [Mg(2+)] ([Mg(2+)](p)) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca(2+) channels (alpha(1C)/beta(2A)/alpha(2)delta), increasing [Mg(2+)](p) from 0.2 mM to 1.8 mM decreased peak I(Ca) by 76 +/- 4.5% (n = 7). Mg(2+)-dependent modulation of I(Ca) was also observed in cells loaded with ATP-gamma-S. With 0.2 mM [Mg(2+)](p), manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP(2A)) produced large changes in I(Ca) amplitude; however, with 1.8 mM [Mg(2+)](p), these same manipulations had no significant effect on I(Ca). With mutant channels lacking principal PKA phosphorylation sites (alpha(1C/S1928A)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) had only small effects on I(Ca). However, when channel open probability was increased by alpha(1C)-subunit truncation (alpha(1CDelta1905)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) greatly reduced peak I(Ca). Correspondingly, in myocytes voltage-clamped with pipette PP(2A) to minimize channel phosphorylation, increasing [Mg(2+)](p) produced a much larger reduction in I(Ca) when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg(2+) modulates the extent to which channel phosphorylation regulates I(Ca). This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.

Rationale: Mutant mice lacking the RIIbeta subunit of protein kinase A (regulatory subunit II beta(-/-)) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIbeta(-/-) and littermate wild-type (RIIbeta(+/+)) mice on multiple genetic backgrounds., Methods: Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEvxC57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3-4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment., Results: Relative to RIIbeta(+/+) mice, RIIbeta(-/-) mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIbeta(-/-) mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIbeta(-/-) mice was specific to ethanol injections, and was not related to altered blood ethanol levels., Conclusions: These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIbeta(-/-) mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.

This Teaching Resource provides an introduction to the use of cultured cells as an experimental approach in undergraduate laboratory research and the study of neuronal differentiation in PC12 cells. In addition, a thought experiment with answers is provided that can be used to assess student understanding of (i) the scientific method, (ii) signaling processes involved in cellular differentiation, and (iii) the use of pharmacological agents to manipulate a cell culture system.

Background: Accumulating evidence indicates that adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is involved in the neurobiological responses to ethanol. Previous reports indicate that mice lacking the RIIbeta subunit of PKA (RIIbeta(-/-)) voluntarily consume more ethanol than wild-type controls (RIIbeta(+/+)) using 2-bottle testing procedures. Although such procedures primarily measure consummatory behavior, operant self-administration procedures allow analysis of consummatory as well as appetitive or "ethanol-seeking" behavior (i.e., lever pressing is required to gain access to the ethanol solution). Therefore, we determined whether the high ethanol consumption characteristic of RIIbeta(-/-) mice would be complemented by increased appetitive ethanol-seeking behavior in an operant paradigm., Methods: RIIbeta(-/-) (n=8) and RIIbeta(+/+) (n=8) mice were initially sucrose-faded until they were lever responding for nonsweetened ethanol (10, 14, and 18%). Following the self-administration testing, RIIbeta(+/+) and RIIbeta(-/-) mice were given access to 2 bottles, one containing water and the other ethanol to replicate the voluntary ethanol drinking data previously from our laboratory. Finally, immediately after voluntary consumption all mice were again tested for self-administration of 10% ethanol. Alterations in the reinforcement schedule were also explored as RIIbeta(+/+) and RIIbeta(-/-) mice were tested for self-administration of 10% ethanol at FR-3 and FR-5 schedules., Results: The RIIbeta(-/-) mice displayed lower operant responding for ethanol and food reinforcement compared with RIIbeta(+/+) controls. However, this effect was driven by a significant increase in lever responses made by female RIIbeta(+/+) mice. When the excessive lever responses of the female RIIbeta(+/+) mice are accounted for, the RIIbeta(-/-) mice show ethanol lever responses comparable to controls. Following operant self-administration testing, RIIbeta(-/-) mice of both sexes consumed more ethanol solution compared with RIIbeta(+/+) mice during 2-bottle testing., Conclusions: Increased ingestion of ethanol by RIIbeta(-/-) mice is likely the result of altered PKA activity within neuronal pathways that control ethanol-consummatory behaviors. Conversely, the RIIbeta subunit of PKA appears not to play a critical role in neuronal pathways that regulate appetitive behaviors directed at obtaining ethanol. Finally, increased operant self-administration of food and ethanol by female wild-type mice was absent in female RIIbeta(-/-) mice, suggesting that normal PKA signaling may be part of a general, and sex-dependent, mechanism involved with reinforcement-seeking behavior.

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.

Select members of the Wnt family of secreted glycoproteins have been implicated in inducing the myogenic determinant genes Pax3, MyoD and Myf5 during mammalian embryogenesis, but the mechanism of induction has not been defined. We describe an unexpected role for protein kinase A (PKA) signalling via CREB in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer.

Background: Genetic and pharmacological evidence suggests that the cyclic adenosine monophosphate-dependent protein kinase A pathway modulates neurobiological responses to ethanol. Mutant mice lacking the RIIbeta subunit of protein kinase A (RIIbeta(-/-)) are resistant to ethanol-induced sedation and drink significantly more ethanol than littermate wild-type mice (RIIbeta(+/+)). We determined whether high ethanol intake by the RIIbeta(-/-) mice on alternate genetic backgrounds is reliably predicted by high basal levels of anxiety or resistance to the sedative effects of ethanol., Methods: Two-bottle choice procedures and a battery of behavioral tests (elevated plus maze, open-field activity, and zero maze) were used to assess voluntary ethanol consumption and basal levels of anxiety in RIIbeta(-/-) and RIIbeta(+/+) mice on either a C57BL/6J or a 129/SvEv x C57BL/6J genetic background. Additionally, ethanol-induced sedation and blood ethanol levels were determined in RIIbeta(-/-) and RIIbeta(+/+) mice after intraperitoneal injection of ethanol (3.8 g/kg)., Results: RIIbeta(-/-) mice on both genetic backgrounds consumed more ethanol and had a greater preference for ethanol relative to RIIbeta(+/+) mice. However, RIIbeta(-/-) mice showed reduced basal levels of anxiety when maintained on the C57BL/6J background but showed increased anxiety when maintained on the 129/SvEv x C57BL/6J background. Consistent with prior research, RIIbeta(-/-) mice were resistant to the sedative effects of ethanol, regardless of the genetic background. Finally, RIIbeta(-/-) and RIIbeta(+/+) mice showed similar blood ethanol levels., Conclusions: These results indicate that high ethanol consumption is associated with resistance to the sedative effects of ethanol but that basal levels of anxiety, as well as ethanol metabolism, do not reliably predict high ethanol drinking by RIIbeta(-/-) mice.

An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed Calpha(2) or C(s). We generated mice that lack Calpha(2) to determine its importance in the events downstream of cAMP production. Male Calpha(2) null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, Calpha(2) has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that Calpha(2) is required for bicarbonate to speed the flagellar beat and facilitate Ca(2+) entry channels. In addition, Calpha(2) is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, Calpha(2) null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility.

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.

Msn2 and Msn4 are two related transcriptional activators that mediate a general response to stress in yeast Saccharomyces cerevisiae by eliciting the expression of specific sets of genes. In response to stress or nutritional limitation, Msn2 and Msn4 migrate from the cytoplasm to the nucleus. Using GFP-tagged constructs and high-resolution time-lapse video microscopy on single cells, we show that light emitted by the microscope also triggers this migration. Unexpectedly, the population of Msn2 or Msn4 molecules shuttles repetitively into and out of the nucleus with a periodicity of a few minutes. A large heterogeneity in the oscillatory response to stress is observed between individual cells. This periodic behavior, which can be induced by various types of stress, at intermediate stress levels, is not dependent upon protein synthesis and persists when the DNA-binding domain of Msn2 is removed. The cAMP-PKA pathway controls the sensitivity of the oscillatory nucleocytoplasmic shuttling. In the absence of PKA, Msn4 continues to oscillate while Msn2 is maintained in the nucleus. We show that a computational model based on the possibility that Msn2 and Msn4 participate in autoregulatory loops controlling their subcellular localization can account for the oscillatory behavior of the two transcription factors.

The protein kinase A-deficient PC12 cell line PC12A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 perpendicular RegA perpendicular [cAMP] --> PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.

Stimulation of the beta(2)-adrenergic receptor (beta(2)AR) in human embryonic kidney (HEK) 293 cells causes a transient activation of Extracellular Signal-Regulated Kinase (ERK) 1/2. One of the mechanisms proposed for this activation is a PKA-mediated phosphorylation of the beta(2)AR that switches receptor coupling from G(s) to G(i) and triggers internalization of the receptor. To examine these phenomena, we characterized agonist activation of ERK1/2 in HEK293 cells by the endogenous beta(2)AR and in HEK293 cells stably overexpressing either the wild-type beta(2)AR or a substitution mutant beta(2)AR (PKA(-)) that lacks the cyclic AMP-dependent protein kinase (PKA) consensus phosphorylation sites (S261A, S262A and S345A, S346A). As the baseline, we established that epinephrine stimulation of the endogenous beta(2)AR in HEK293 cells (20-30 fmol/mg) caused a rapid and transient activation of ERK1/2 with an EC(50) of 5 to 6 nM. In contrast, the potency of epinephrine stimulation of ERK1/2 in cells stably overexpressing WTbeta(2)AR and PKA(-) (2-4 pmol of beta(2)AR/mg) was increased by over 100-fold relative to HEK293 cells, the EC(50) values being 20 to 60 pM. The nearly identical 100-fold shift in EC(50) for ERK1/2 activation in the PKA(-) and WTbeta(2)AR relative to that in the HEK293 showed that the PKA(-) are fully capable of activating ERK1/2. We also found maximal activation of ERK1/2 in the overexpressing cell lines at concentrations of epinephrine that cause no internalization (i.e., the EC(50) for internalization was 75 nM). Pertussis toxin pretreatment caused only a weak inhibition of epinephrine activation of ERK1/2 in the HEK293 (7-16%) and no inhibition in the PKA(-) cells. Finally we found that the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 microM) caused a >90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of beta(2)AR activation of ERK1/2 does not require PKA phosphorylation of the beta(2)AR, receptor internalization or switching from activation of G(s) to G(i) but clearly requires activation of a Src family member that may be downstream of PKA.

Conflicting reports have attributed 8-chloro-cAMP (Cl-cAMP)-mediated inhibition of tumor cell growth to either a toxic 8-chloro-adenosine (Cl-AdR) breakdown product or a Cl-cAMP-mediated decrease in ratio of Type I to Type II regulatory (R) subunits of protein kinase A (PKA). Using the MCF-7 human breast cancer and S49 mouse lymphoma cell lines as models, we show that the effects of Cl-cAMP and other cAMP analogs on growth and R subunit expression are unrelated. MCF-7 cell growth was insensitive to most analogs and inducers of cAMP, but was potently inhibited by Cl-cAMP acting through uptake and phosphorylation of its Cl-AdR breakdown product. Possible roles of adenosine receptors or P(2) purinoceptors in these Cl-cAMP-mediated growth effects were ruled out by studies with agonists and antagonists. Cholera toxin markedly decreased the ratio of Type I to Type II R subunits in MCF-7 cells without affecting growth, while growth inhibitory concentrations of Cl-cAMP or Cl-AdR had insignificant effects on this ratio. In S49 cells, where PKA activation is known to inhibit cell growth, PKA-deficient mutants retained sensitivity to both Cl-cAMP and the related 8-bromo-cAMP. Adenosine kinase (AK)-deficient S49 cells were inhibited only by higher concentrations of these 8-halogenated cAMP analogs. Of the commonly used cAMP analogs, only 8-(4-chlorophenylthio)-cAMP acted purely as a cyclic nucleotide-having no effect on PKA-deficient cells, but strongly inhibiting both wild-type and AK-deficient cells. Where growth inhibitory concentrations of most cAMP analogs reduced RI expression in the AK-deficient mutant, a functionally equivalent concentration of (N(6), O(2'))dibutyryl-cAMP maintained or increased this expression., (Copyright 2002 Wiley-Liss, Inc.)

Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence "cluster" analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this "fast linear" subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more "fast linear" sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of "fast linear sperm" induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.

Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disease characterized by impaired T lymphocyte immune effector functions. We have identified a disorder of signal transduction in SLE T cells involving the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Cyclic AMP-stimulated PKA-catalyzed protein phosphorylation is markedly diminished owing to profound deficiencies of both type I (PKA-I) and type II (PKA-II) isozyme activities. Deficient PKA-I isozyme is characterized by a significant reduction in the amount of type I regulatory beta subunit (RI beta) steady state mRNA by competitive polymerase chain reaction. This is associated with a 30% decrease in RI alpha protein and a 65% reduction in RI beta protein. Indeed, T cells from approximately 25% of SLE subjects have no detectable RI beta protein. Transient transfection of T cells not expressing RI beta protein with autologous SLE RI beta cDNA bypassed the block in translation, reconstituting PKA activity and augmenting IL-2 production. Of importance was the initial identification of novel RI alpha mRNA mutations characterized by heterogeneous transcript mutations, including deletions, transitions, and transversions. Most mutations are clustered adjacent to GAGAG motifs and CT repeats. By contrast, deficient PKA-II activity is the result of spontaneous dissociation of the cytosolic RII beta(2)C(2) holoenzyme, aberrant RII beta translocation to the nucleus from the cytosol, and retention of RII beta in the nucleus. In conclusion, distinct mechanisms account for deficient PKA-I and PKA-II isozyme activities in SLE T cells.

We have studied the function of protein kinase A (PKA) during embryonic development using a PKA-deficient mouse that retains only one functional catalytic subunit allele, either Calpha or Cbeta, of PKA. The reduced PKA activity results in neural tube defects that are specifically localized posterior to the forelimb buds and lead to spina bifida. The affected neural tube has closed appropriately but exhibits an enlarged lumen and abnormal neuroepithelium. Decreased PKA activity causes dorsal expansion of Sonic hedgehog signal response in the thoracic to sacral regions correlating with the regions of morphological abnormalities. Other regions of the neural tube appear normal. The regional sensitivity to changes in PKA activity indicates that downstream signaling pathways differ along the anterior-posterior axis and suggests a functional role for PKA activation in neural tube development.

The intracellular second messenger cAMP affects cell physiology by directly interacting with effector molecules that include cyclic nucleotide-gated ion channels, cAMP-regulated G protein exchange factors, and cAMP-dependent protein kinases (PKA). Two catalytic subunits, Calpha and Cbeta, are expressed in the mouse and mediate the effects of PKA. We generated a null mutation in the major catalytic subunit of PKA, Calpha, and observed early postnatal lethality in the majority of Calpha knockout mice. Surprisingly, a small percentage of Calpha knockout mice, although runted, survived to adulthood. This growth retardation was not due to decreased GH production but did correlate with a reduction in IGF-I mRNA in the liver and diminished production of the major urinary proteins in kidney. The survival of Calpha knockout mice after birth is dependent on the genetic background as well as environmental factors, but sufficient adult animals were obtained to characterize the mutants. In these animals, compensatory increases in Cbeta levels occurred in brain whereas many tissues, including skeletal muscle, heart, and sperm, contained less than 10% of the normal PKA activity. Analysis of sperm in Calpha knockout males revealed that spermatogenesis progressed normally but that mature sperm had defective forward motility.

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the melatonin response suggesting that melatonin may be acting through one of its Gi coupled cell surface receptors. This is confirmed by Western blots demonstrating the presence of MT1 receptors in PC12 cells. Coupling of the Gi protein to these receptors is demonstrated by failure of melatonin to suppress cell growth in PKA deficient A126-1B2-1 mutant PC12 cells. Similarly, melatonin failed to prevent cell proliferation when cells were incubated in the presence of the PKA inhibitor, Rp-cAMP. Retinoic acid and dexamethasone, agents known to effect PC12 cell growth and/or differentiation, displayed differential effects on the actions of melatonin. In the presence of melatonin and low concentrations of retinoic acid (100 nM), PC12 cell proliferation was stimulated compared to that seen with either agent alone, whereas no increase in cell proliferation was observed when higher concentrations of retinoic acid (100 microM) were used. The effects of dexamethasone on suppression of PC12 cell growth were additive with that of melatonin whereas, 1,25-dihydroxyvitamin D(3) (IC(50)=10 nM), which by itself had no effect on PC12 cell growth, was found to inhibit the melatonin response. This study demonstrates that inhibition of PC12 cell growth, at physiological concentrations of melatonin, is mediated by cAMP-dependent cell surface receptors and this response is altered by other growth factors known to effect PC12 cell proliferation and differentiation.

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.

A profound deficiency of type I protein kinase A (PKA-I or RIalpha/beta2C2) phosphotransferase activity occurs in the T lymphocytes of 80% of subjects with systemic lupus erythematosus (SLE), an autoimmune disorder of unknown etiology. This isozyme deficiency is predominantly the product of reduced or absent beta isoform of the type I regulatory subunit (RIbeta). Transient transfection of RIbeta cDNAs from SLE subjects into autologous T cells that do not synthesize the RIbeta subunit bypassed the block, resulting in RIbeta subunit synthesis and restoration of the PKA-Ibeta (RIbeta2C2) holoenzyme. Transfected T cells activated via the T cell surface receptor complex revealed a significant increase of cAMP-activatable PKA activity that was associated with a significant increase in IL-2 production. These data demonstrate that a disorder of RIbeta translation exists, and that correction of the PKA-I deficiency may enhance T lymphocyte effector functions in SLE.

cAMP-dependent protein kinase (PKA) is believed to play a critical role in the expression of long-lasting forms of hippocampal long-term potentiation (LTP). Can distinct patterns of synaptic activity induce forms of LTP that require different isoforms of PKA? To address this question, we used transgenic mice that have genetically reduced hippocampal PKA activity, and a specific pharmacological inhibitor of PKA, Rp-cAMPS. Transgenic mice [R(AB) mice] that express an inhibitory form of a particular type of regulatory subunit of PKA (type-Ialpha) showed significantly reduced LTP in area CA1 of hippocampal slices as compared with slices from wild-type mice. This impairment of LTP expression was evident when LTP was induced by applying repeated, temporally spaced stimulation (4 1-s bursts of 100-Hz applied once every 5 min). In contrast, LTP induced by applying just 60 pulses in a theta-burst pattern was normal in slices from R(AB) mice as compared with slices from wild-type mice. We found that Rp-cAMPS blocked the expression of LTP induced by both spaced tetra-burst and compressed theta-burst stimulation in hippocampal slices of wild-type and R(AB) mice, respectively. Since Rp-cAMPS is a PKA inhibitor that is not selective for any particular isoform of PKA and these R(AB) mice show reduced hippocampal PKA activity resulting from genetic manipulation of a single isoform of PKA regulatory subunit, our data support the idea that distinct patterns of synaptic activity can produce different forms of LTP that significantly engage different isoforms of PKA. In particular, theta-burst LTP significantly recruits isoforms of PKA containing regulatory subunits other than the mutant RIalpha subunit, whereas tetra-burst LTP requires PKA isoforms containing the mutant RIalpha subunit. Thus, altering both the total amount of imposed synaptic activity and the temporal spacing between bursts of imposed activity may subtly modulate the PKA dependence of hippocampal LTP by engaging distinct isoforms of PKA. In a broader context, our findings suggest that synaptic plasticity in the mammalian brain might be importantly regulated by activity-dependent recruitment of different isoforms of key signal transduction molecules.

To further elucidate the links among synaptic plasticity, hippocampal place cells, and spatial memory, place cells were recorded from wild-type mice and transgenic "R(AB)" mice with reduced forebrain protein kinase A (PKA) activity after introduction into a novel environment. Place cells in both strains were similar during the first exposure and were equally stable for recording sessions separated by 1 hr. Place cell stability in wild-type mice was unchanged for sessions separated by 24 hr but was reduced in R(AB) mice over the longer interval. This stability pattern parallels both the reduced late-phase long-term potentiation in hippocampal slices from R(AB) mice and the amnesia for context fear conditioning seen in R(AB) mice 24 but not 1 hr after training. The similar time courses of synaptic, network, and behavioral instability suggest that the genetic reduction of PKA activity is responsible for the defects at each level and support the idea that hippocampal synaptic plasticity is important in spatial memory.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by abnormal T cell signal transduction and altered T cell effector functions. We have previously observed a profound deficiency of total protein kinase A (PKA) phosphotransferase activity in SLE T cells. Here we examined whether reduced total PKA activity in SLE T cells is in part the result of deficient type II PKA (PKA-II) isozyme activity. The mean PKA-II activity in SLE T cells was 61% of normal control T cells. The prevalence of deficient PKA-II activity in 35 SLE subjects was 37%. Deficient isozyme activity was persistent over time and was unrelated to SLE disease activity. Reduced PKA-II activity was associated with spontaneous dissociation of the cytosolic RIIbeta2C2 holoenzyme and translocation of the regulatory (RIIbeta) subunit from the cytosol to the nucleus. Confocal immunofluorescence microscopy revealed that the RIIbeta subunit was present in approximately 60% of SLE T cell nuclei compared with only 2-3% of normal and disease controls. Quantification of nuclear RIIbeta subunit protein content by immunoprecipitation and immunoblotting demonstrated a 54% increase over normal T cell nuclei. Moreover, the RIIbeta subunit was retained in SLE T cell nuclei, failed to relocate to the cytosol, and was associated with a persistent deficiency of PKA-II activity. In conclusion, we describe a novel mechanism of deficient PKA-II isozyme activity due to aberrant nuclear translocation of the RIIbeta subunit and its retention in the nucleus in SLE T cells. Deficient PKA-II activity may contribute to impaired signaling in SLE T cells.

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.

To investigate the role of the cAMP-dependent protein kinase (PKA) in the desensitization and down-regulation of the D(1) dopamine receptor, we stably expressed the rat cDNA for this receptor in mutant Chinese hamster ovary (CHO) cell lines deficient in PKA activity. The 10260 mutant CHO cell line has been characterized as expressing less than 10% of type I and type II PKA activities relative to the parental 10001 CHO cell line. The 10248 mutant CHO line lacks type II PKA activity and expresses a defective type I PKA. The transfected parental and mutant cell lines were found to express approximately 1 pmol/mg D(1) receptor binding activity (B(max)) as determined using [(3)H]SCH-23390 binding assays. All three cell lines demonstrated similar levels of dopamine-stimulated adenylyl cyclase activity. Pretreatment of all three CHO cells with dopamine resulted in desensitization of the adenylyl cyclase response, although the maximum desensitization was attenuated by 20 and 40% in the 10260 and 10248 cell lines, respectively. Dopamine also promoted, in a time- and dose-dependent fashion, a >90% down-regulation of D(1) receptors in the parental cell line but only a 50 and 30% decrease in the 10260 and 10248 cells, respectively. Similarly, treatment of the cells with the membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP induced functional desensitization and down-regulation of the D(1) receptor, although it was not as great as that observed with agonist pretreatment. As with the agonist pretreatments, the 8-(4-chlorophenylthio)-induced responses were attenuated in the mutant cells with the 10248 line exhibiting the least desensitization/down-regulation. Our results suggest that PKA significantly contributes to the desensitization and down-regulation of D(1) receptors in CHO cells and that type II PKA may be the more relevant isoform with respect to regulating D(1) receptor function.

We characterized a regulatory element located in the -76 to -62 region of the human ferredoxin gene. This region bound to Sp1-like proteins with low affinity, as shown using electrophoretic mobility shift, competition, antibody binding, and Southwestern experiments. The similarity of the regulatory element to Sp1 extends beyond its DNA-binding domain, as cloned Sp1 functioned equally well when fused to a peptide that bound to an irrelevant site. The function of these Sp1-binding sites is mediated through the cAMP-dependent protein kinase (PKA) signaling pathway, because reporter genes downstream of the Sp1-binding sites were not activated in a PKA-deficient cell line. Transfection of the catalytic subunit of PKA restored activated transcription. Similar Sp1-binding sites identified in the CYP11A1 and CYP21 genes also controlled cAMP-dependent transcription of the reporter gene. Our finding of the function of Sp1-like proteins in steroidogenic gene transcription adds one more role Sp1 plays in controlling physiological events.

Objective: To estimate the prevalence of protein kinase A type I isozyme (PKA-I) deficiency in a cohort of systemic lupus erythematosus (SLE) patients, and to establish whether the isozyme deficiency is associated with SLE disease activity., Methods: Thirty-five SLE patients and 35 age-, sex-, and race-matched normal controls were studied. Fifteen subjects were restudied on at least 3 occasions over a 4-year interval. Clinical disease activity was estimated by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and the T cell activation markers CD25+ and HLA-DR+ were quantified by flow cytometry. PKA-I isozyme activities were quantified in enriched T cells. Statistical analyses were performed by Student's t-test, Mann Whitney U test, and Pearson product moment test., Results: The mean PKA-I activity in SLE T cells (540 pmoles/minute/mg of protein) was significantly lower than that in control T cells (1,578 pmoles/ minute/mg of protein) (P<0.001). The prevalence of isozyme deficiency in this cohort was 80%. During a 4-year interval, PKA-I activities remained significantly reduced, whereas SLEDAI scores significantly improved. There was no relationship between deficient PKA-I activity and either SLEDAI scores or the proportion of T cells bearing CD25+ or HLA-DR+ activation markers., Conclusion: There is a high prevalence of deficient T cell PKA-I isozyme activity in SLE that persists over time and is independent of SLE disease activity.

The vesicular acetylcholine transporter (VAChT) gene and the choline acetyltransferase (ChAT) gene comprise the cholinergic gene locus. We have studied the coordinate regulation of these genes by cyclic AMP-dependent protein kinase (PKA) in the rat pheochromocytoma cell line PC12 and PC12 PKA-deficient mutants. Both ChAT and VAChT mRNA increased approximately fourfold after treatment of PC12 cells with dibutyryl cyclic AMP (dbcAMP). ChAT and PKA activity were also increased by dbcAMP. The basal levels of ChAT and VAChT mRNAs in the PKA-deficient cell lines were both about six times lower than in wild-type PC12 cells, and were induced less than twofold by addition of dbcAMP. H-89 and H-9, specific inhibitors for PKA, reduced ChAT and VAChT mRNA levels to approximately one-third that of untreated cells and ChAT activity to approximately one-fourth that of untreated PC12 cells. Activation of PKA type II, but not PKA type I, increased ChAT activity approximately threefold. Analysis of reporter gene constructs indicates that PKA affects gene transcription at an upstream site in the cholinergic gene locus. These results demonstrate that the expression of the ChAT and VAChT genes is regulated coordinately at the transcriptional level, and a signaling pathway specifically involving PKA II plays an important role in this process.

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP-dependent kinase creates the more acidic species.

To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway.

Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsalpha- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsalpha is responsible for transducing the beta-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules.

Developing sensory systems are sculpted by an activity-dependent strengthening and weakening of connections. Long-term potentiation (LTP) and depression (LTD) in vitro have been proposed to model this experience-dependent circuit refinement. We directly compared LTP and LTD induction in vitro with plasticity in vivo in the developing visual cortex of a mouse mutant of protein kinase A (PKA), a key enzyme implicated in the plasticity of a diverse array of systems. In mice lacking the RIbeta regulatory subunit of PKA, we observed three abnormalities of synaptic plasticity in layer II/III of visual cortex in vitro. These included an absence of (1) extracellularly recorded LTP, (2) depotentiation or LTD, and (3) paired-pulse facilitation. Potentiation was induced, however, by pairing low-frequency stimulation with direct depolarization of individual mutant pyramidal cells. Together these findings suggest that the LTP defect in slices lacking PKA RIbeta lies in the transmission of sufficient net excitation through the cortical circuit. Nonetheless, functional development and plasticity of visual cortical responses in vivo after monocular deprivation did not differ from normal. Moreover, the loss of all responsiveness to stimulation of the originally deprived eye in most cortical cells could be restored by reverse suture of eyelids during the critical period in both wild-type and mutant mice. Such an activity-dependent increase in response would seem to require a mechanism like potentiation in vivo. Thus, the RIbeta isoform of PKA is not essential for ocular dominance plasticity, which can proceed despite defects in several common in vitro models of neural plasticity.

The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-fos mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIbeta subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIbeta mutant mice fail to induce either c-fos or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.

Systemic lupus erythematosus (SLE) T cells exhibit deficient type I protein kinase A (PKA-1) isozyme phosphotransferase activity, resulting in impaired phosphorylation of plasma membrane-associated proteins. To determine the mechanism of this isozyme deficiency, we studied 16 SLE subjects with a mean (+/-1 SD) SLE disease activity index of 16.7 +/- 8.8 and 16 normal controls. Immunoblotting of type I regulatory (RI) subunit protein in SLE and control T cells demonstrated no significant differences in the amount of protein. Analysis of isozyme kinetics in SLE T cells demonstrated a 2.2-fold increase in the Michaelis-Menten constant, a 2.5-fold increase in the apparent association constant for cAMP, a 3.8-fold decrease in the maximal velocity, and a reduction in the mean maximal binding of cAMP to the RI subunit compared with control T cells. Reduction of the Hill coefficient from 1.2 in normal T cells to 0.7 in SLE T cells indicated a loss of positive cooperativity between cAMP binding sites A and B. An increase in the apparent association constant for cAMP signifies relative resistance to cAMP, indicating that higher intracellular concentrations of cAMP are necessary to activate the isozyme. Because the R subunit of PKA is the only intracellular receptor for cAMP, the abnormal isozyme kinetics may account for the deficiency of PKA-I phosphotransferase activity and impaired PKA-I-catalyzed protein phosphorylation observed in SLE T cells. This disordered isozyme function may contribute to the altered signal transduction and observed cellular immune dysfunctions in SLE. Moreover, these altered isozyme kinetics raise the possibility of a structural defect(s) in the RI subunit.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.