Peptide Inhibitor Of Protein Kinase Research Articles

TNF−α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1-ERK-VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.

The Kinase Specificity of Protein Kinase Inhibitor Peptide.

G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in in vitro assays. We found that in addition to inhibiting PKA activity, both PKI (6–22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.

Protein Kinase Inhibitor Peptide as a Tool to Specifically Inhibit Protein Kinase A.

The protein kinase enzyme family plays a pivotal role in almost every aspect of cellular function, including cellular metabolism, division, proliferation, transcription, movement, and survival. Protein kinase A (PKA), whose activation is triggered by cyclic adenosine monophosphate (cAMP), is widely distributed in various systems and tissues throughout the body and highly related to pathogenesis and progression of various kinds of diseases. The inhibition of PKA activation is essential for the study of PKA functions. Protein kinase inhibitor peptide (PKI) is a potent, heat-stable, and specific PKA inhibitor. It has been demonstrated that PKI can block PKA-mediated phosphorylase activation. Since then, researchers have a lot of knowledge about PKI. PKI is considered to be the most effective and specific method to inhibit PKA and is widely used in related research. In this review, we will first introduce the knowledge on the activation of PKA and mechanisms related on the inhibitory effects of PKI on PKA. Then, we will compare PKI-mediated PKA inhibition vs. several popular methods of PKA inhibition.

Abstract 122: Pkialpha Protects Hearts From Beta-adrenergic Overstimulation

Introduction: Protein kinase inhibitor peptide α (PKIα) isoforms are abundantly expressed endogenous inhibitors of PKA in the heart. The physiological and pathophysiological relevance of PKIα has not been studied. Hypothesis: PKIα inhibits PKA activity in normal and stressed (post-myocardial infarction (MI)) hearts. Methods: PKIα knockout (KO) and c57bl/6 control (CTR) mice were used to test if PKIα affected cardiac β-adrenergic response and if played a role in post-MI hearts. Results: Protein abundance of PKIα was significantly increased in failing human and post-MI mouse ventricular tissue. The knockout of PKIα in mice did not affect the expression of the other two PKI isoforms. Nor did it change the expression of PKA regulatory and catalytic subunits. PKIα KO mice did not show alterations in basal cardiac morphology, function and life span of mice. Intra-LV hemodynamic measurements in response to different doses of isoproterenol showed that the loss of PKIα sensitizes the heart in response to isoproterenol (ISO) with a lower EC50 and the maximum response was increased more in KO mice than in control mice. ISO (10nM) increased myocyte contraction and Ca2+ transient amplitudes more in KO myocytes than control myocytes though 100nM ISO increased myocyte contraction and Ca2+ transients amplitude to the same. At the ISO concentration of 10nM, ISO increased more phosphorylation of the Ser16 site (PKA site) on phospholamban. In post-MI animals, the loss of PKIα enhances cardiac function at 1 week after MI but caused more myocardial infarction and functional depression at 8 weeks after MI. Conclusions: PKIα is an important regulator of cardiac β-adrenergic responses by antagonizing PKA. This mechanism could be protective to avoid β-adrenergic overstimulation, especially in post-MI patients and animal models.

Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

Abstract 19461: Endogenous Protein Kinase Inhibitor Peptide (pki) Regulates β-Adrenergic Responses of the Heart

Introduction: Protein kinase inhibitor peptide (PKI) isoforms are endogenous inhibitors of PKA and existing in the heart. The physiological relevance of PKI has not been studied. Aims: We will determine the role of PKIα (one of the major PKI isoforms in the heart) in β-adrenergic regulation of the heart with PKIα knockout (KO) versus c57bl/6 control (CTR) mice. Methods: In vivo cardiac function was evaluated by conventional echocardiography (ECHO) in PKIα KO mice. In vivo cardiac β-adrenergic response was determined by intra-LV hemodynamic measurements in response to different doses of isoproterenol (10-16g/g BW∼10-5g/g BW). Isolated ventricular myocytes were used to determine cellular responses to β-adrenergic agonist, isoproterenol (ISO, 1nM, 10nM and 100nM). Western blotting was used to determine phosphorylation levels of PKA-targets in the heart. Results: (1) The loss of PKI did not change basal cardiac morphology, function and life span of mice; (2) ISO (2μ g/g BW) enhanced cardiac function more in KO mice than in CTR mice when evaluated by ECHO and intra-LV hemodynamic measurements; (3) The loss of PKIα sensitizes the heart in response to ISO (Log[EC50] shifted from -9.2 to -10.8) and the maximum dp/dt increases more (by 25%) in KO mice than in CTR mcie; (4) PKIa KO myocytes were more sensitive to ISO in that 10nM ISO increased myocyte contraction and Ca2+ transient amplitudes more in KO myocytes; (5). At the ISO concentration of 10-9M, ISO increased more phosphorylation of the Ser16 site (PKA site) on phospholamban. Conclusion: PKI plays an important role in regulating cardiac β-adrenergic responses by antagonizing PKA.

Phosphodiesterase 3A (PDE3A) Deletion Suppresses Proliferation of Cultured Murine Vascular Smooth Muscle Cells (VSMCs) via Inhibition of Mitogen-activated Protein Kinase (MAPK) Signaling and Alterations in Critical Cell Cycle Regulatory Proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

Global Consequences of Activation Loop Phosphorylation on Protein Kinase A

Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr197 in the activation loop when expressed in Escherichia coli. Although mutation of Arg194 to Ala prevents autophosphorylation, phosphorylation of Thr197 can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr197 decreased the Km by ∼15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the αH-αI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu208 and Arg280.

Regulation of Protein Kinase A Activity by p90 Ribosomal S6 Kinase 1

Previously, we reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) binds to the active p90 ribosomal S6 kinase 1 (RSK1) (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell. Biol. 26, 4586-4600). Herein, by overexpressing hemagglutinin-tagged RSK1 fragments in HeLa cells we have identified the region of RSK1 that is responsible for the interaction with PKAc. PKAc bound to the last 13 amino acids of RSK1, which overlaps the Erk1/2 docking site. This interaction between PKAc and RSK1 required the phosphorylation of Ser-732 in the C terminus of RSK1. Depending upon its phosphorylation status, RSK1 switched interactions between Erk1/2 and PKAc. In addition, a peptide corresponding to the last 13 amino acids of RSK1 with substitution of Ser-732 with Glu (peptide E), but not Ala (peptide A), decreased interactions between endogenous active RSK1 and PKAc. RSK1 attenuated the ability of cAMP to activate PKA in vitro and this modulation was abrogated by peptide E, but not by peptide A. Similarly, in intact cells, cAMP-mediated phosphorylation of Bcl-xL/Bcl-2-associated death promoter on Ser-115, the PKA site, was reduced when RSK1 was activated by epidermal growth factor, and this effect was blocked by peptide E, but not by peptide A. These findings demonstrate that interactions between endogenous RSK1 and PKAc in intact cells regulate the ability of cAMP to activate PKA and identify a novel mechanism by which PKA activity is regulated by the Erk1/2 pathway.

Different role of cAMP dependent protein kinase and CaMKII in H3 receptor regulation of histamine synthesis and release

Histamine H3 autoreceptors induce a negative feedback on histamine synthesis and release. While it is known that cAMP/cAMP dependent protein kinase (PKA) and Ca2+/CaMKII transduction pathways mediate H3 effects on histamine synthesis, the pathways regulating neuronal histamine release are poorly known. Given the potential use of H3 ligands in cognitive diseases, we have developed a technique for the determination of H3 effects on histamine synthesis and release in brain cortical miniprisms. Potassium-induced depolarization effects were impaired by blockade of calcium entry through N and P/Q channels, as well as of CaMKII, but release was not affected by activators or inhibitors of the cAMP/PKA pathway (1-methyl-3-isobutylxanthine (IBMX), N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (db–cAMP) or myristoyl PKA inhibitor peptide 14-22 (PKI14-22). In contrast, forskolin stimulated histamine release, although independently of PKA. Stimulation of histamine H3 receptors with the agonist imetit markedly reduced the depolarization increase of histamine release, apparently through P/Q calcium channel inhibition. The H3 antagonist/inverse agonist thioperamide modestly stimulated histamine release. Thioperamide effect on release was not modified by the PKA inhibitor PKI14-22, but it was blocked by the CaMKII inhibitor KN-62. These results indicate that H3 autoreceptors regulate neuronal histamine release (1) independently of the cAMP/PKA cascade, and (2) through modulation of calcium entry and CaMKII activation during depolarization.

Inhibition of angiogenesis by extracellular protein kinase A

The cyclic-AMP dependent protein kinase (PKA) signaling pathway regulates cell growth, development, metabolism, and gene expression. Peripheral blood of cancer patients but not normal individuals, shows increased catalytic subunit levels of PKA (PKAc). We showed here that this extracellular form of PKAc (ECPKA) from conditioned media of cultured cancer cells as well as purified PKAc inhibit angiogenesis, using the in utero chicken embryo chorioallantoic membrane assay. Inhibition of angiogenesis is partially reversed by PKI, a peptide inhibitor of PKA, thus suggesting an anti-angiogenic role for ECPKA. The significance of ECPKA in cancer is discussed.

Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.

Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

A Genetically Encoded Pore for the Stochastic Detection of a Protein Kinase

Stochastic sensing is an emerging approach for the detection of a wide variety of analytes at the level of individual molecules. Detection is accomplished by observing the modulation of the current that flows through a single protein pore that has been engineered to bind an analyte of interest. Previously, protein analytes have been detected by using pores to which ligands have been appended at specific sites by targeted chemical modification. Here, we report the first genetically encoded stochastic sensor element for detecting a protein. A protein kinase inhibitor peptide sequence was incorporated into the alpha-hemolysin polypeptide, which was used to form a heteroheptameric pore containing a single copy of the inhibitor sequence. With this pore, the successful detection of the catalytic subunit of protein kinase A was demonstrated. This development should greatly facilitate the detection of active kinase subunits by stochastic sensing and the rapid screening of kinase inhibitors by an approach that yields kinetic information.

622 POSTER Discovery and characterization of a novel multi-targeted tyrosine kinase inhibitor with activity against c-ret, pdgfr, c-kit and c-src

Protein kinases are now the second largest group of drug targets, and most protein kinase inhibitors in clinical development are directed towards the ATP-binding site. However, these inhibitors must compete with high intracellular ATP concentrations and they must discriminate between the ATP-binding sites of all protein kinases as well the other proteins that also utilise ATP. It would therefore be beneficial to target sites on protein kinases other than the ATP-binding site. This review describes the discovery, characterisation and use of peptide inhibitors of protein kinases. In many cases, the development of these peptides has resulted from an understanding of the specific protein-binding partners for a particular protein kinase. In addition, novel peptide sequences have been discovered in library screening approaches and have provided new leads in the discovery and/or design of peptide inhibitors of protein kinases. These approaches are therefore providing exciting new opportunities in the development of ATP non-competitive inhibitors of protein kinases.

C5b-9-induced Endothelial Cell Proliferation and Migration Are Dependent on Akt Inactivation of Forkhead Transcription Factor FOXO1

Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

Oculomotor Plasticity During Vestibular Compensation Does Not Depend on Cerebellar LTD

Vestibular paradigms are widely used for investigating mechanisms underlying cerebellar motor learning. These include adaptation of the vestibuloocular reflex (VOR) after visual-vestibular mismatch training and vestibular compensation after unilateral damage to the vestibular apparatus. To date, various studies have shown that VOR adaptation may be supported by long-term depression (LTD) at the parallel fiber to Purkinje cell synapse. Yet it is unknown to what extent vestibular compensation may depend on this cellular process. Here we investigated adaptive gain changes in the VOR and optokinetic reflex during vestibular compensation in transgenic mice in which LTD is specifically blocked in Purkinje cells via expression of a peptide inhibitor of protein kinase C (L7-PKCi mutants). The results demonstrate that neither the strength nor the time course of vestibular compensation are affected by the absence of LTD. In contrast, analysis of vestibular compensation in spontaneous mutants that lack a functional olivo-cerebellar circuit (lurchers) shows that this form of motor learning is severely impaired. We conclude that oculomotor plasticity during vestibular compensation depends critically on intact cerebellar circuitry but not on the occurrence of cerebellar LTD.

Protein kinase inhibitor peptide (PKI): A family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.

Modulation of voltage-dependent Ba2+ currents in the guinea-pig gastric antrum by cyclic nucleotide-dependent pathways.

We have investigated whether the activation of cAMP- and cGMP-dependent pathways modifies the properties of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes using patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Both dibutyryl cAMP (db-cAMP, 0.1-1 mM), a membrane-permeable ester of cAMP, and isoproterenol, a selective beta-stimulant, inhibited I(Ba) in a concentration-dependent manner. Forskolin, but not dideoxy-forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of I(Ba). In the presence of either Rp-cAMP or the PKA (cAMP-dependent protein kinase) inhibitor peptide 5-24 (PKA-IP), neither forskolin nor db-cAMP inhibited I(Ba). After establishing a conventional whole-cell recording, the peak amplitude of I(Ba) gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp-cAMP reversibly enhanced I(Ba). Sodium nitroprusside (0.1-1 mM) and 8-Br-cGMP (0.1-1 mM) also inhibited I(Ba) in a concentration-dependent manner. The inhibitory effects of forskolin or db-cAMP on I(Ba) were not significantly changed by pretreatment with a cGMP-dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8-Br-cGMP on I(Ba) were not modified by PKA-IP. The membrane-permeable cyclic nucleotides db-cAMP and 8-Br-cGMP caused little shift of the voltage dependence of the steady-state inactivation and reactivation curves. Neither of the membrane-permeable cyclic nucleotides db-cAMP or 8-Br-cGMP had additive inhibitory effects on I(Ba). These results indicate that two distinct cyclic nucleotide-dependent pathways are present in the guinea-pig gastric antrum, and that both inhibited I(Ba) in an independent manner.

Substrate Enhances the Sensitivity of Type I Protein Kinase A to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.