Activation Of Protein Kinase C Isoforms Research Articles

Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

“Time windows” for switching kinase-phosphatase balance during inhibition of long-term potentialization of hippocampal synapses by amyloid aggregates

Phosphorylation and dephosphorylation of target proteins are crucial mechanisms for regulating cellular functions. These processes are mediated by protein kinases and phosphatases, respectively. Changes in the kinase-phosphatase balance, known as K-P balance, are well documented during the progression of amyloidosis. The overactivation of kinases or the failed activity of phosphatases is the root cause of hyperphosphorylation of the structural tau protein leading to the formation of fibrillar tangles. The data suggests bidirectional regulation of phosphorylation/dephosphorylation processes during amyloidosis-related states is consistent across different animal models and many protocols. Therefore, it is crucial to identify the “time windows” during which the K-P balance can be altered. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum located in area CA1. Baseline synaptic responses were generated by paired-pulse stimulation of the Schaffer collaterals at a frequency of 0.033 Hz using a bipolar electrode. Four 100 Hz trains were delivered 5 minutes apart to induce long-term potentiation (LTP). Serine and threonine phosphatase activity was assessed using a non-radioactive molybdate dye-based assay kit (Promega, USA) based on the manufacturer’s instructions. The procedure relies on the creation of a colored complex of phosphates with a dye containing molybdenum, followed by the measurement of optical densities of supernatants that were incubated against a control at 640 nm. The activity of protein kinase C (PKC) will be measured directly via an enzyme immunoassay (ELISA) on a plate reader, using a commercial Abcam kit (Abcam, USA). The technique relies on the binding of engineered antibodies to activated PKC, and optical densities of incubated supernatants are measured against a control at 450 nm. In this study, we identified two important time periods during the application of amyloid Aβ25-35 aggregates that affect plasticity in the CA3-CA1 synapses. Specifically, the PKC isoforms are activated during early neurochemical events (0–20 min of incubation of slices in the presence of Aβ25–35 aggregates). If the hippocampal CA3-CA1 synapses are tetanized during this period, we did not observe any inhibition of synaptic plasticity either in the early or late phase development of LTP. Biochemical analysis indicates that hippocampal slices pretreated with Aβ25–35 aggregates for 20–30 minutes exhibit increased PKC activity, whereas no such effect is observed after one hour of incubation. Simultaneously, the hour-long incubation of hippocampal slices in the presence of Aβ25–35 aggregates resulted in the disappearance of long-term potentiation and the return of responses to the pre-tetanic level of synaptic transmission during the late-LTP phase (3 hours after tetanus induction). Inhibitory analysis revealed that an abrupt rise in the activity of stress-induced phosphatase 1α (PP1α) interrupts kinase-mediated signals after LTP induction, leading to the suppression of fEPSPs in the CA3-CA1 synapses. These findings suggest that exposing hippocampal slices to Aβ25–35 aggregates for 20–30 minutes may alter the K-P balance and enhance kinase activity through PKC isoform induction. The majority of PKC isoforms exhibit a tendency to desensitize following activation. Probably, the depletion of the pool of PKCs, which occurs within the first 20–30 minutes after incubation of hippocampal slices with Aβ25–35 aggregates, along with a notable elevation in the activity of stress-induced phosphatase PP1α, is accountable for the second time window for switching the kinase-phosphatase balance, stabilized after an hour of incubation of hippocampal slices in the presence of Aβ25–35 aggregates. Incubating hippocampal slices with Aβ25–35 aggregates (for 0–30 minutes) leads to a shift in the kinase-phosphatase balance towards the activity of kinases due to the activation of PKC isoforms. After this stage, there is a significant rise in phosphatase activity, resulting from the induction of PP1α. This induces a shift in the balance between kinase and phosphatase towards dephosphorylation processes.

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Single-molecule studies reveal regulatory interactions between master kinases PDK1, AKT1, and PKC

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, whereas PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

TLR2 dimer-specific ligands selectively activate protein kinase C isoforms in Leishmania infection.

Of the thirteen Toll-like receptors (TLRs) in mice, TLR2 has a unique ability of forming heterodimers with TLR1 and TLR6. Such associations lead to selective cellular signalling and cellular responses such as cytokine expression. One of the signalling intermediates is protein kinase C (PKC); of which, eight isoforms are expressed in macrophages. Leishmania-a protozoan parasite that resides and replicates in macrophages-selectively modulates PKC-α, PKC-β, PKC-δ and PKC-ζ isoforms in macrophages. As TLR2 plays significant roles in Leishmania infection, we examined whether these PKC isoforms play selective roles in TLR2 signalling and TLR2-induced anti-leishmanial functions. We observed that the TLR2 ligands-Pam3 CSK4 (TLR1/2), PGN (TLR2/2) and FSL (TLR2/6)-differentially phosphorylated and translocated PKC-α, PKC-β, PKC-δ and PKC-ζ isoforms to cell membrane in uninfected and L.major-infected macrophages. The PKC isoform-specific inhibitors differentially altered IL-10 and IL-12 expression, Th1 and Th2 responses and anti-leishmanial effects in macrophages and in BALB/c mice. While PKC isoforms' inhibitors had insignificant effects on the Pam3CSK4-induced anti-leishmanial functions, PGN-induced pro-leishmanial effects were enhanced by PKC-(α+β) inhibitors, whereas PKC-(δ+ζ) inhibitors enhanced the anti-leishmanial effects of FSL. These results indicated that the ligand-induced TLR2 dimerization triggered differential dose-dependent and kinetic profiles of PKC isoform activation and that selective targeting of PKC isoforms using their respective inhibitors in combination significantly modulated TLR2-induced anti-leishmanial functions. To the best of our knowledge, this is the first demonstration of TLR2 dimer signalling through PKC isoforms and TLR2-induced PKC isoform-targeted anti-leishmanial therapy.

Nanoparticle-Encapsulated Bryostatin-1 Activates α-Secretase and PKC Isoforms In vitro and Facilitates Acquisition and Retention of Spatial Learning in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) animal models have revealed neuroprotective actions of Bryostatin-1 mediated by activation of novel PKC isoforms, suppression of beta-amyloid and downregulation of inflammatory and angiogenic events, making Bryostatin-1 an attractive candidate for attenuating AD-associated neural, vascular, and cognitive disturbances. To further enhance Bryostatin-1 efficacy, nanoparticle-encapsulated Bryostatin-1 formulations were prepared. We compared nano-encapsulated and unmodified Bryostatin-1 in in vitro models of neuronal PKC-d, PKC-e isoforms, α-secretase and studied nano-encapsulated Bryostatin-1 in an AD mouse model of spatial memory (BC3-Tg (APPswe, PSEN1 dE9) 85Dbo/J mice). We found that nanoencapsulated Bryostatin-1 formulations displayed activity greater or equal to that of unmodified Bryostatin-1 in PKC-δ and -ε and α-secretase activation assays. We next evaluated how treatment with a nanoencapsulated Bryostatin-1 formulation facilitated spatial learning in the Morris water maze. AD transgenic mice (6.5 to 8 months of age) were treated with nanoparticle encapsulated Bryostatin-1 formulation (1, 2.5, or 5 μg/mouse) three times the week before testing and then daily for each of the 5 days of testing. Across the acquisition phase, mice treated with nanoencapsulated Bryostatin-1 had shorter latencies, increased % time in the target zone and decreased % time in the opposite quadrant. The mice were given retention testing after a 2-week period without drug treatment. Mice treated with nanoencapsulated Bryostatin-1 had shorter latencies to find the escape platform, indicating retention of spatial memory. These data suggest that cognitive deficits associated with AD could be treated using highly potent nanoparticle-encapsulated formulations of Bryostatin-1.

Pharmacological Protein Kinase C Modulators Reveal a Pro-hypertrophic Role for Novel Protein Kinase C Isoforms in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro. Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs. Conclusion: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer’s disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease.

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. mTOR inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 KO mouse); however, the mechanism of rescue is unknown. Here we identify PKC downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice we find that rapamycin restores mitochondrial protein levels, inhibits signaling through both mTOR complexes, and reduces the abundance and activity of multiple protein kinase C (PKC) isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss, and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.Reporting SummaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article.

Protein and mRNA expression of protein kinase C (PKC) in the postmortem brain of bipolar and schizophrenic subjects

Abnormalities of protein kinase C (PKC) have been implicated in the pathophysiology of bipolar (BP) illness. This is primarily based on studies of PKC in platelets of BP patients. Whether such abnormalities of PKC activity and isoforms exist in the brain is unclear. We have therefore determined PKC activity, protein and mRNA expression of PKC isoforms in the prefrontal cortex (PFC), cingulate cortex (CING) and temporal cortex (TEMP) from BP (n = 19), schizophrenic (SZ) (n = 20) and normal control (NC) (n = 25) subjects. The brain samples were obtained from the Harvard Brain Bank, and the subjects were diagnosed according to DSM-IV criteria. Protein levels were determined using Western blot technique and mRNA levels were determined using real-time PCR (qPCR) method. We found that there was a significant decrease in the PKC activity in the cytosol and membrane fractions of PFC and TEMP obtained from BP subjects but not from SZ subjects. When we compared the expression of PKC isozymes, we found that the protein and mRNA expression of several isozymes was significantly decreased in the PFC (i.e., PKCα, PKCβI, PKCβII and PKCε) and TEMP (i.e., PKCα, PKCβI, PKCβII, PKCε and PKCγ) of BP subjects, but not in the CING. Overall, there was no difference in the mRNA or protein expression of PKC isozymes between SZ and NC subjects in any of the three brain areas we studied. Our results show that there is a region-specific decrease of certain PKC isozymes in the membrane and cytosol fractions of BP but not SZ subjects.

Role of prelimbic cortex PKC and PKM\u03b6 in fear memory reconsolidation and persistence following reactivation

The persistence of newly acquired memories is supported by the activity of PKMζ, an atypical isoform of protein kinase C (PKC). Whether the activity of conventional and atypical PKC isoforms contributes to reactivated memories to persist is still unknown. Similarly, whether memory reactivation is a prerequisite for interventions to be able to change memory persistence is scarcely investigated. Based on the above, we examined the role of conventional and atypical PKC isoforms in the prelimbic cortex in reconsolidation and persistence of a reactivated contextual fear memory in male Wistar rats. It is shown that (i) inhibiting the PKC activity with chelerythrine or the PKMζ activity with ZIP impaired the persistence of a reactivated memory for at least 21 days; (ii) ZIP given immediately after memory reactivation affected neither the reconsolidation nor the persistence process. In contrast, when given 1 h later, it impaired the memory persistence; (iii) chelerythrine given immediately after memory reactivation impaired the reconsolidation; (iv) omitting memory reactivation prevented the chelerythrine- and ZIP-induced effects: (v) the ZIP action is independent of the time elapsed between its administration and the initial memory test. The results indicate that prelimbic cortex PKC and PKMζ are involved in memory reconsolidation and persistence.

Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition.We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCβ. Pharmacological inhibition of PKCα, but not of PKCβ, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCβ, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCβ) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Protein Phosphorylation Mechanisms Underlying Chronic Pain in Sickle Cell Disease

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD.We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes.In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions. DisclosuresNo relevant conflicts of interest to declare.

Co-ordinated activation of classical and novel PKC isoforms is required for PMA-induced mTORC1 activation.

Protein kinase C (PKC) has been shown to activate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, a central hub in the regulation of cell metabolism, growth and proliferation. However, the mechanisms by which PKCs activate mTORC1 are still ambiguous. Our previous study revealed that activation of classical PKCs (cPKC) results in the perinuclear accumulation of cPKC and phospholipase D2 (PLD2) in recycling endosomes in a PLD2-dependent manner. Here, we report that mTORC1 activation by phorbol 12,13-myristate acetate (PMA) requires both classic, cPKC, and novel PKC (nPKC) isoforms, specifically PKCη, acting through distinct pathways. The translocation of mTOR to perinuclear lysosomes was detected after treatment of PKC activators, which was not colocalized with PKCα- or RAB11-positive endosomes and was not inhibited by PLD inhibitors. We found that PKCη inhibition by siRNA or bisindolylmaleimide I effectively decreased mTOR accumulation in lysosomes and its activity. Also, we identified that PKCη plays a role upstream of the v-ATPase/Ragulator/Rag pathway in response to PMA. These data provides a spatial aspect to the regulation of mTORC1 by sustained activation of PKC, requiring co-ordinated activation of two distinct elements, the perinuclear accumulation of cPKC- and PLD-containing endosomes and the nPKC-dependent translation of of mTOR in the perinuclear lysosomes. The close proximity of these two distinct compartments shown in this study suggests the possibility that transcompartment signaling may be a factor in the regulation of mTORC1 activity and also underscores the importance of PKCη as a potential therapeutic target of mTORC-related disorders.

Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells

PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations (“spiking”) at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K+ depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca2+ concentration ([Ca2+]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca2+]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca2+]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.

Inhibition of Cardiac Kir Current (IK1) by Protein Kinase C Critically Depends on PKCβ and Kir2.2.

BackgroundCardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date.Methods and ResultsIn the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC.ConclusionWe conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias.

Spatio-Temporal Regulation of PKC Isoforms Imparts Signaling Specificity.

Nishizuka and colleagues discovered protein kinase C (PKC) as a calcium-dependent, lipid cofactor-sensitive protein kinase (1). Initially known as the only physiological effector for tumor-promoting phorbol esters, this ubiquitous enzyme eventually took center stage in cellular signaling. Part of the AGC kinase branch of the kinome, PKC is a family of serine/threonine kinases comprising 11 isoforms encoded by 9 genes and grouped into 4 classes – classical (cPKCs-α, βI, βII, γ), novel (nPKCs-δ, ϵ, η, θ), atypical (aPKCs-ζ, ι/λ), and PKCμ (a form between novel and atypical isoforms) (Box 1). Their considerable structural homology, overlapping substrate specificities, and biochemical properties indicated at least partial enzymatic redundancy and rendered the task of identifying isoform-specific functions challenging. The initial phase of PKC research correlated the unique structural features of the isoforms with their functions (Box 1) (2). But, with their structural overlaps and hugely varying functions in different models, specificity of the isoforms became a confounding puzzle demanding stringent isoform-specific regulation to avoid functional redundancy. To define this stringency, subsequent PKC research focused on the upstream and downstream regulatory mechanisms (3, 4), highlighting the importance of subcellular distribution as a function of time (5). As simultaneous activation of all PKC isoforms would be energetically and spatially conflicting for decoding the message received by the cell surface receptor, cell type, and stimulus-specific selective and sequential activation deemed justified (6). The recent phase of PKC research propounds a PKC-signaling module (7, 8) wherein an inter-isoform network regulates the PKC isoforms’ activity (Box 2; Figure ​Figure1B).1B). Here, we propose activator- and cofactor-specific sequential activation of PKC isoforms in a spatio-temporal model wherein selective subcellular compartmentalization quantitatively determines the isoform-specific effector functions. Box 1 Structural features of PKC isoforms defining functional specificity. PKC serve as a paradigm for the reversible regulation of membrane localization by the concerted action of two membrane-targeting modules. C1: a cysteine-rich region of approximately 50 residues present in all PKC isozymes. In cPKCs and nPKCs, it is present as a tandem repeat C1A and C1B. aPKCs contain a single copy of the domain termed atypical because it does not bind phorbol esters. The domain contains two pulled apart β-sheet forming the ligand-binding pocket. Two zinc atoms are coordinated by two histidines and six cysteines at opposite ends of the primary sequence stabilizing the domain. In aPKCs, one face of the ligand-binding pocket is compromised, so that the module cannot bind phorbol esters or DAG. Ligand binding dramatically alters the surface properties of the module (C1B). The ligand caps the hydrophilic ligand-binding pocket, so that the top-third of the C1 domain presents a continuous hydrophobic surface thus achieving membrane targeting by simply altering membrane properties. C2: an independent membrane-targeting module that binds calcium in cPKCs, but not in nPKCs. C2 domain is a β-strand rich globular domain with loops formed by sequences at opposite ends of the primary structure. Two topological variants exist – Type I for domains that follow the C1 domain (cPKCs) and Type II for domains that precede C1 (nPKCs). In the calcium-responsive C2 domains of cPKCs, the pocket is lined by multiple aspartic acid residues that coordinate two to three calcium ions that act as a bridge between the C2 domain and the phospholipid head groups of the membrane. CR, cysteine rich; PS, pseudosubstrate; DAG, diacylglycerol; PMA, phorbol-12-myristate 13-acetate; PB1, Phox–Bem 1; C, constant regions; V, variable regions.

Thrombin promotes the expression of Ccnd1 gene in RPE cells through the activation of converging signaling pathways

The breakdown of the blood–retina barrier exposes retinal pigment epithelium (RPE) to serum components, thrombin among them. In addition to coagulation, thrombin acting through Protease-Activated Receptors (PARs 1–4) participates in a number of processes including cell proliferation, transformation, and migration. The purpose of this study was to identify interacting signaling pathways by which the activation of PAR1 by thrombin triggers cyclin D1 gene (Ccnd1) expression and the proliferation of RPE cells, characteristic of proliferative vitreoretinopathy (PVR). Our results demonstrate that thrombin induces the expression of the c-fos gene (c-fos), the activation of the (fos/jun) AP-1 site and the expression of Ccnd1, in precise correlation with the activation of CREB. Although the expression of both, c-fos and Ccnd1 requires the activation of conventional PKC isoforms and PI3K, downstream signaling from PI3K differs for both genes. Whereas the expression of c-fos requires PI3K-induced PDK1/Akt activity, that of Ccnd1 is mediated by PDK1-independent PKCζ signaling. Additionally, CREB activation may contribute to the induction of Ccnd1 expression through binding to the Ca/CRE element in the Ccnd1 gene promoter. Since cyclin D1 is a key regulator of cell cycle G1/S phase progression essential for proliferation, these findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

Mechanisms for redox-regulation of protein kinase C.

Protein kinase C (PKC) is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses. PKC isoforms are traditionally viewed as allosterically activated enzymes that are recruited to membranes by growth factor receptor-generated lipid cofactors. An inherent assumption of this conventional model of PKC isoform activation is that PKCs act exclusively at membrane-delimited substrates and that PKC catalytic activity is an inherent property of each enzyme that is not altered by the activation process. This traditional model of PKC activation does not adequately explain the many well-documented actions of PKC enzymes in mitochondrial, nuclear, and cardiac sarcomeric (non-sarcolemmal) subcellular compartments. Recent studies address this dilemma by identifying stimulus-specific differences in the mechanisms for PKC isoform activation during growth factor activation versus oxidative stress. This review discusses a number of non-canonical redox-triggered mechanisms that can alter the catalytic properties and subcellular compartmentation patterns of PKC enzymes. While some redox-activated mechanisms act at structural determinants that are common to all PKCs, the redox-dependent mechanism for PKCδ activation requires Src-dependent tyrosine phosphorylation of a unique phosphorylation motif on this enzyme and is isoform specific. Since oxidative stress contributes to pathogenesis of a wide range of clinical disorders, these stimulus-specific differences in the controls and consequences of PKC activation have important implications for the design and evaluation of PKC-targeted therapeutics.

Impaired IKs channel activation by Ca2 +-dependent PKC shows correlation with emotion/arousal-triggered events in LQT1

BackgroundThe most common inherited cardiac arrhythmia, LQT1, is due to IKs potassium channel mutations and is linked to high risk of adrenergic-triggered cardiac events. We recently showed that although exercise-triggered events are very well treated by ß-blockers for these patients, acute arousal-triggered event rate were not significantly reduced after beta-blocker treatment, suggesting that the mechanisms underlying arousal-triggered arrhythmias may be different from those during exercise. IKs is strongly regulated by β-adrenergic receptor (β-AR) signaling, but little is known about the role of α1-AR-mediated regulation. Methods and resultsHere we show, using a combination of cellular electrophysiology and computational modeling, that IKs phosphorylation and α1-AR regulation via activation of calcium-dependent PKC isoforms (cPKC) may be a key mechanism to control channel voltage-dependent activation and consequently action potential duration (APD) in response to adrenergic-stimulus. We show that simulated mutation-specific combined adrenergic effects (β+α) on APD were strongly correlated to acute stress-triggered cardiac event rate for patients while β-AR effects alone were not. ConclusionWe were able to show that calcium-dependent PKC signaling is key to normal QT shortening during acute arousal and when impaired, correlates with increased rate of sudden arousal-triggered cardiac events. Our study suggests that the acute α1-AR-cPKC regulation of IKs is important for QT shortening in “fight-or-flight” response and is linked to decreased risk of sudden emotion/arousal-triggered cardiac events in LQT1 patients.

Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/Disc Large/Zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins

Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.