Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates. Copyright (C) 2002 John Wiley Sons, Ltd.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Double-Stranded RNA-Activated Protein Kinase Is a Key Modulator of Insulin Sensitivity in Physiological Conditions and in Obesity in Mice

The molecular integration of nutrient-and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of kappa B kinase beta. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of kappa B kinase beta phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity. (Endocrinology 153:5261-5274, 2012)

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Maximal acceleration of calcium release refractoriness by β-adrenergic stimulation requires dual activation of protein kinase A and CaMKII in mouse ventricular myocytes

Time-dependent refractoriness of calcium (Ca2+) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca2+ spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca2+ spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca2+ sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark- to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca2+/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen) without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca2+ release refractoriness in mouse myocytes.

PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.

The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIα subunit of protein kinase A after oral administration

Overexpression of the RIα subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of 35S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIα subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Crosstalk between the Ras2p-controlled Mitogen-activated Protein Kinase and cAMP Pathways during Invasive Growth of Saccharomyces cerevisiae

The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the “afterdischarge.” In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5′-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19–36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.