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Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels.

Transient receptor potential vanilloid subtype 1 (TRPV1) is a nonselective cationic channel activated by painful stimuli such as capsaicin and noxious heat, and enriched in sensory neurons of the pain pathway. During inflammation, chemical mediators activate protein kinases (such as PKC) that phosphorylate TRPV1 and thereby enhance its function, with consequent increases in nociceptor sensitization. However, the causal relationships between TRPV1 phosphorylation and pathological pain remain unexplored. To directly investigate the roles of one specific TRPV1 phosphorylation eventin vivo, we genetically altered a major PKC phosphorylation site, mouse TRPV1 S801, to alanine. The TRPV1 expression pattern in sensory neurons of S801A knock-in (KI) mice was comparable to that in WT controls. However, sensitization of capsaicin-mediated currents after the activation of PKC was substantially impaired in sensory neurons from KI mice. Thermal hyperalgesia induced by PMA or burn injury in KI was identical to WT. Inflammatory thermal hyperalgesia was only marginally attenuated in KI mice. In contrast, PMA-evoked nocifensive responses and sensitization of capsaicin responses were significantly attenuated in the hindpaws of KI mice. Ongoing pain from inflamed masseter muscle was also reduced in KI mice, and was further inhibited by the TRPV1 antagonist AMG9810. These results suggest that PKC-mediated phosphorylation of TRPV1 S801 contributes to inflammation-mediated sensitization of TRPV1 to ligand, but not heat,in vivo. Further, this suggests that interference with TRPV1 S801 phosphorylation might represent one potential way to attenuate inflammatory pain, yet spare basal sensitivity and produce fewer side effects than more general TRPV1 inhibition.SIGNIFICANCE STATEMENTTransient receptor potential vanilloid subtype 1 (TRPV1) has been considered a potential target for pain intervention. Global inhibitors of TRPV1 function, however, produce side effects which could compromise their clinical utility. By precisely removing a unique PKC phosphorylation site (TRPV1 S801) in mice through CRISPR/Cas9 editing, we providein vivoevidence for a highly specific inhibition that leaves basal TRPV1 function intact, yet alleviates some forms of hyperalgesia. These findings support inhibition of TRPV1 S801 phosphorylation as a potential intervention for pain management.

TGF-β inhibited membrane-associated protein (TIMAP) is greatly expressed in endothelial cell lines and serves as a protein phosphatase 1 (PP1) regulatory subunit. Phosphorylation state of TIMAP, through affecting PP1 activity, has a remarkable effect on endothelial barrier function. Here we present evidence for a previously unidentified PKC phosphorylation site in TIMAP. Protein-protein interaction was detected in pulmonary endothelial cells between endogenous TIMAP and activated PKCα. PKCα phosphorylated the full length recombinant TIMAP in in vitro kinase assay and Ser331 of TIMAP was shown to be phosphorylated by PKC. Phosphorylation of TIMAP upon PKC activation in endothelial cells results in enrichment of TIMAP in the membrane, but no such change can be observed in PKC depleted cells. However, the previously identified PKA/GSK-3β induced enrichment of TIMAP at the plasma membrane was not affected in the absence of PKC. Interaction between TIMAP and the TIMAP-PP1 substrate phospho-ERM was described earlier, but now we show that binding of PKC phosphorylated TIMAP to ERM is severely reduced. This suggests an inhibitory effect of phospho-Ser331 on TIMAP-PP1 activity toward phospho-ERM. Accordingly, phospho-ERM level in the membrane fraction of the phospho-mimic S331D TIMAP mutant transfected cells was increased, but the S331A mutant overexpressing endothelial cells had a lower phospho-ERM level. Consistent with the phospho-ERM level, electric resistance measurements showed that the S331A mutation of TIMAP resulted in faster recovery from the PMA treatment. Taken together, phosphorylation of TIMAP on Ser331 by PKC represents a new mechanism of endothelial barrier regulation, through the inhibition of phospho-ERM dephosphorylation.

Protein kinase C (PKC) and substrates like SNAP-25 regulate neurotransmission. At the neuromuscular junction (NMJ), PKC promotes neurotransmitter release during synaptic activity. Thirty minutes of muscle contraction enhances presynaptic PKC isoform levels, specifically cPKCβI and nPKCe, through retrograde BDNF/TrkB signaling. This establishes a larger pool of these PKC isoforms ready to promote neuromuscular transmission. The PKC phosphorylation site in SNAP-25 has been mapped to the serine 187 (Ser-187), which is known to enhance calcium-dependent neurotransmitter release in vitro. Here, we localize SNAP-25 at the NMJ and investigate whether cPKCβI and/or nPKCe regulate SNAP-25 phosphorylation. We also investigate whether nerve and muscle cell activities regulate differently SNAP-25 phosphorylation and the involvement of BDNF/TrkB signaling. Our results demonstrate that nPKCe isoform is essential to positively regulate SNAP-25 phosphorylation on Ser-187 and that muscle contraction prevents it. TrkB and cPKCβI do not regulate SNAP-25 protein level or its phosphorylation during neuromuscular activity. The results provide evidence that nerve terminals need both pre- and postsynaptic activities to modulate SNAP-25 phosphorylation and ensure an accurate neurotransmission process.

Phosphorylation of AMPA receptor GluA1 plays important roles in synaptic potentiation. Most previous studies have been performed in the hippocampus, while the roles of GluA1 phosphorylation in the cortex remain unknown. Here we investigated the involvement of the phosphorylation of GluA1 in the LTP in the anterior cingulate cortex (ACC) using mice with a GluA1 knock-in mutation at the PKA phosphorylation site serine 845 (s845A) or CaMKII/PKC phosphorylation site serine 831 (s831A). The network LTP, which is constructed by multiple recordings of LTP at different locations within the ACC, was also investigated. We found that the expression of LTP and network LTP was significantly impaired in the s845A mice, but not in the s831A mice. By contrast, basal synaptic transmission and NMDA receptor-mediated responses were not affected. Furthermore, to uncover potential information under the current acquired data, a new method for reconstruction and better visualization of the signals was developed to observe the spatial localizations and dynamic temporal changes of fEPSP signals and multiple LTP responses within the ACC circuit. Our results provide strong evidence that PKA phosphorylation of the GluA1 is important for the network LTP expression in the ACC. SIGNIFICANCE STATEMENT Previous studies have shown that PKA and PKC phosphorylation of AMPA receptor GluA1 plays critical roles in LTP in the hippocampus, while the roles of GluA1 phosphorylation in the cortex remain unknown. In the present study, by combining a 64-channel multielectrode system and a novel analysis and visualization method, we observed the accurate spatial localization and dynamic temporal changes of network fEPSP signals and LTP responses within the ACC circuit and found that PKA phosphorylation, but not PKC phosphorylation, of the GluA1 is required for LTP in the ACC.

The role of phosphorylation in ADAM17-dependent shedding is controversial. We show that the A1 adenosine receptor stimulates exocytosis in umbrella cells by a pathway that requires phosphorylation of ADAM17–Ser-811, followed by HB-EGF shedding and EGF receptor transactivation. Preventing ADAM17 phosphorylation blocks these downstream events., Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A1 adenosine receptors (A1ARs) triggers a Gi-Gβγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A1AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17S811A mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17S811A in bladder tissues impairs A1AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain.

The nuclear receptor complex of the steroid hormone, 20-hydroxyecdysone (20E), is a heterodimer composed of EcR and USP. Our previous studies in Drosophila suggest that PKC modulates 20E signaling by phosphorylating EcR-USP. However, the exact phosphorylation sites in EcR and USP have not been identified. Using LC-MS/MS analysis, we first identified Ser35 of USP as a PKC phosphorylation site. Mutation of USP Ser35 to Ala35 in S2 cells not only eliminated USP phosphorylation, but also attenuated the 20E-induced luciferase activity, mimicking the treatment with a PKC-specific inhibitor chelerythrine chloride in Kc cells. In the larval salivary glands (SG), inhibition of PKC activity with the binary GAL4/UAS system reduced USP phosphorylation and down-regulated the 20E primary-response genes, E75B and Br-C, and RNAi knockdown of Rack1 had stronger inhibitory effects than overexpression of PKCi. Moreover, RNAi knockdown of four PKC isozyme genes expressed in the SG exhibited a variety of inhibitory effects on USP phosphorylation and expression of E75B and Br-C, with the strongest inhibitory effects occurring when aPKC was knocked down by RNAi. Taken together, we conclude that PKC-mediated USP phosphorylation at Ser35 modulates 20E signaling in Drosophila.

Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide the pathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K+transport, and are regulated by numerous receptors via Gq/11-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca2+/calmodulin (CaM) and PIP2, we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca2+/CaM and PIP2on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M1receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to “sensitize” KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose–response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP2in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca2+, to disrupt the interaction of YFP-tagged AKAP791-153with CFP-tagged KCNQ2-5. Tonic depletion of PIP2by cotransfection of a PIP2phosphatase had no effect, and sudden depletion of PIP2did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP2depletion.

KCNQ1 is co-assembled with KCNE1 subunits in the heart to form the cardiac delayed rectifier K(+) current (IKs), which is one of the main currents responsible for myocyte repolarization. The most commonly inherited form of cardiac arrhythmias, long-QT syndrome type 1 (LQT1), is due to mutations on KCNQ1. Gq-coupled receptors (GqPCRs) are known to mediate positive inotropism in human ventricular myocardium. The mechanism of IKs current modulation by GqPCRs remains incompletely understood. Here we studied the molecular mechanisms underlying Gq regulation of the IKs channel. Heterologously expressed IKs (human KCNQ1/KCNE1 subunits) was measured in Xenopus oocytes, expressed together with GqPCRs. Our data from several GqPCRs shows that IKs is regulated in a biphasic manner, showing both an activation and an inhibition phase. Receptor-mediated inhibition phase was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP2) was blocked by the lipid kinase inhibitor wortmannin. In addition, stimulation of PIP(2) production, by overexpression of phosphatidylinositol-4-phosphate-5-kinase (PIP5-kinase), decreased receptor-mediated inhibition. The receptor-mediated activation phase was inhibited by the PKC inhibitor calphostin C and by a mutation in a putative PKC phosphorylation site in the KCNE1 subunit. Our results indicate that the depletion of membrane PIP(2) underlies receptor-mediated inhibition of IKs and that phosphorylation by PKC of the KCNE1 subunit underlies the GqPCR-mediated channel activation.

GAP-43 is the major neuronal substrate of protein kinase C (PKC). Its phosphorylation status dictates the severity of pathfinding errors by GAP-43 (+/−) growth cones in vivo, as well as its modulation of actin dynamics in vitro. These experiments show that stably overexpressing cDNAs mutant at its single PKC phosphorylation site at serine41 in retinoic acid treated SH-Sy5Y neuroblastoma cells regulates intrinsic and extrinsic behaviors of growing neurons. Intrinsically, only Wt and pseudophosphorylated GAP-43Ser41Asp precipitated with F-actin and potentiated F-actin — regulated filopodia formation. GAP-43Ser41Asp inhibited neurite outgrowth whereas only unphosphorylatable GAP-43Ser41Ala precipitated neurotubulin, potentiated neurotubulin accumulation in neurites and increased outgrowth. When PI3-kinase was inhibited GAP-43Ser41Asp-mediated filopodia formation was inhibited whereas GAP-43Ser41Ala-mediated neurite extension was potentiated. Extrinsically, only Wt and GAP-43Ser41Asp potentiated both homotypic adhesion and neurite outgrowth on NCAM-expressing monolayers and promoted NCAM stability. With respect to the underlying mechanism, more F-actin and NCAM colocalized with Wt and GAP-43Ser41Asp in detergent resistant membranes (DRMs) isolated from live cells and GAP-43Ser41Asp-mediated functions were insensitive to cholesterol depletion. In contrast, GAP-43Ser41Ala-mediated functions were sensitive to cholesterol depletion. Neither GAP-43Ser41Asp nor GAP-43Ser41Ala was able to protect against growth cone collapse mediated by PIP2 inhibitors. The results show that modification of GAP-43 at its PKC phosphorylation site directs its distribution to different membrane microdomains that have distinct roles in the regulation of intrinsic and extrinsic behaviors in growing neurons.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.

To study the involvement of the protein kinase C (PKC) substrate B-50 [also known as growth-associated protein-43 (GAP-43), neuromodulin, and F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca2+ in a concentration-dependent manner (EC50 of ∼10-5M). Ca2+-induced CCK-8 release was maximal at 104M Ca2+, amounting to ∼10% of the initial 6,000 ± 550 fmol of CCK-8 content/mg of synaptosomal protein. Only 30% of the Caa+-induced CCK-8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti-B-50 antibodies were introduced into permeated synaptosomes to study their effect on Ca2+-induced CCK-8 release. The N-terminally directed antibodies (NM2), which inhibited PKC-mediated B-50 phosphorylation, inhibited Ca2+-induced CCK-8 release in a dose-dependent manner, whereas the C-terminally directed antibodies (NM6) affected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibitors PKC19–36 and 1 −(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), which inhibited B-50 phosphorylation in permeated synaptosomes, had no effect on Ca2+-induced CCK-8 release. Our data strongly indicate that B-50 is involved in the mechanism of presynaptic CCK-8 release, at a step downstream of the Ca2+ trigger. As CCK-8 is stored in large densecored vesicles, we conclude that B-50 is an essential factor in the exocytosis from this type of neuropeptide-containing vesicle. The differential effects of the monoclonal antibodies indicate that this B-50 property is localized in the N-terminal region of the B-50 molecule, which contains the PKC phosphorylation site and calmodulin-binding domain.

MinK-related peptide 2 (MiRP2) and Kv3.4 subunits assemble in skeletal muscle to create subthreshold, voltage-gated potassium channels. MiRP2 acts on Kv3.4 to shift the voltage dependence of activation, speed recovery from inactivation, suppress cumulative inactivation and increase unitary conductance. We previously found an R83H missense mutation in MiRP2 that segregated with periodic paralysis in two families and diminished the effects of MiRP2 on Kv3.4. Here we show that MiRP2 has a single, functional PKC phosphorylation site at serine 82 and that normal MiRP2-Kv3.4 function requires phosphorylation of the site. The R83H variant does not prevent PKC phosphorylation of neighboring S82; rather, the change shifts the voltage dependence of activation and endows MiRP2-Kv3.4 channels with sensitivity to changes in intracellular pH across the physiological range. Thus, current passed by single R83H channels decreases as internal pH is lowered (pK(a) approximately 7.3, consistent with histidine protonation) whereas wild-type channels are largely insensitive. These findings identify a key regulatory domain in MiRP2 and suggest a mechanistic link between acidosis and episodes of periodic paralysis.

Nervous system polycomb 1 (NSPc1) shares high homology with vertebrate PcG proteins Mel-18 and Bmi-1. The mRNA of NSPc1 is highly expressed in the developmental nervous system [Mech. Dev. 102 (2001) 219–222]. However, the functional characterization of NSPc1 protein is not clear. In the present study, using Western blotting technique, we aimed to describe the distributions of NSPc1 protein in rat tissues and cell lines. The subcellular localization of NSPc1 was examined in HeLa and SH-SY5Ycell lines, and its transcriptional repression activity was examined in COS-7 cell line. We found that the NSPc1 protein was localized mainly in the nucleus. NSPc1 remarkably repressed the transcription. Most interestingly, both the C-terminal of NSPc1 and two phosphorylation sites in the C-terminal, especially the PKC phosphorylation site at S183, were important in mediating transcription repression. Taken together, results from our study suggest that NSPc1, as a typical PcG family member, has powerful transcriptional repression ability, which may be related to the PKC signaling pathway.

The sensing of extracellular Ca 2+ concentration ([Ca 2+ ] o ) and modulation of cellular processes associated with acute or sustained changes in [Ca 2+ ] o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca 2+ ] o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca 2+ ] o activated PKC-α and PKC-e in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca 2+ ] o required influx of Ca 2+ through Ni 2+ -sensitive Ca 2+ channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -e with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca 2+ ] o . Activation of ERK1/2 by high [Ca 2+ ] o was not necessary for the [Ca 2+ ] o -regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca 2+ ] o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.

Aim: This study was performed to examine the role of Na+,K+-ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). Methods: The studies were performed on COS-7 cells transfected with rat Na+,K+-ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. Results: Ouabain-mediated inhibition of Na+,K+-ATPase resulted in a dose dependent decrease in the rate of RVD. Total Rb-86(+) uptake as well as ouabain dependent Rb-86(+) uptake, used as an index of Na+,K+-ATPase dependent K+ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS-7 cells expressing rat Na+,K+-ATPase, where Ser23 in the catalytic alpha1 subunit of rat Na+,K+-ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na+,K+-ATPase exhibited a significantly lower rate of RVD and showed no increase in Rb-86(+) uptake during RVD. Conclusion: Taken together, these results suggest that a PKC-mediated transient increase in Na+,K+-ATPase activity plays an important role in RVD.

Phorbol esters, activators of protein kinase C (PKC), have been shown to enhance synaptic transmission. One potential downstream target of PKC in the presynaptic terminal is the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) SNAP-25, which has a PKC phosphorylation site in its C-terminal coil centered at serine 187 (S187/Ser187). We examined the role of S187 in hippocampal synaptic transmission. After proteolytic cleavage of native SNAP-25 by botulinum neurotoxin E (BoNT/E), synaptic transmission was restored in a subset of transfected CA3 pyramidal cells with a toxin-resistant form of SNAP-25 containing unaltered S187 (Swt), S187 mutated to alanine (SA) or S187 mutated to glutamate (SE). We observed that phorbol-12,13-diacetate (PDAc, 10 μM) induced potentiation of neurotransmission to a similar degree for both Swt and SA (2.4-fold and 3.1-fold increase, respectively). Furthermore, basal levels of transmission mediated by SE were reduced relative to that of Swt (failure rates of 72% and 41%, respectively). Together, these data suggest that phosphorylation of SNAP-25 S187 does not mediate the observed enhancement of neurotransmission by phorbol esters at hippocampal synapses.

The effects of endothelin on the transient outward K(+) currents were compared between Kv1.4 and Kv4.3 channels in Xenopus oocytes expression system. Both transient outward K(+) currents were decreased by stimulation of endothelin receptor ET(A) coexpressed with the K(+) channels. Transient outward current of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, while that of Kv4.3 was decreased by about 60%. By mutagenesis experiments we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in I(to) by ET-1. In Kv4.3 a PKC phosphorylation site was identified which is in part responsible for the decrease in I(to). Differences in the suppression of I(to) could be ascribed to the difference in intracellular signaling including the number of phosphorylation sites. These findings might give clues for the understanding of molecular mechanism of ventricular arrhythmias in heart failure, in which endothelin is involved in the pathogenesis.

Structural changes at synapses are associated with long-term facilitation (LTF) of synaptic transmission between sensory and motor neurons inAplysia. We have cloned a cDNA encodingAplysiaadducin (ApADD), theAplysiahomolog of mammalian adducins that are regulatory components of the membrane cytoskeleton. ApADD is recovered in the particulate fraction of nervous system extracts and is localized predominantly in the submembraneous region ofAplysianeurons. ApADD is phosphorylatedin vitroby protein kinase C (PKC) at a site homologous to thein vivoPKC phosphorylation site in mammalian adducins. Phosphorylation of ApADD at this site is also detectedin vivoin the intactAplysianervous system and is increased 18 hr after serotonin-induced LTF. In contrast, there is no change in phosphorylation during short-term facilitation or 1 hr after initial LTF induction. Thus, ApADD is modulated specifically with later phases of LTF and provides an attractive candidate protein that contributes to structural changes accompanying long-lasting synaptic alteration.

The heterologous expression system will provide clues for understanding the basic mechanism of arrhythmogenicity in both inherited and acquired long QT syndrome, which are reviewed here, with emphasis on the K+ channels. Endothelin is implicated in the morphological and electrical remodeling of cardiac muscles in heart failure. The effects of endothelin on the transient outward K+ currents (Ito) were compared between Kv1.4 (rich in endocardial muscle) and Kv4.3 (rich in epicardial muscle) channels in the Xenopus oocytes expression system. Both Itos were decreased by stimulation of endothelin receptor ETA coexpressed with the K+ channels. Ito of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, whereas that of Kv4.3 was decreased by about 60%. By mutagenesis experiments, we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in Ito by ET-1. In Kv4.3 we identified a PKC phosphorylation site that is partly responsible for the decrease. Differences in the suppression of Ito could be due to the differences in intracellular signaling including the number of phosphorylation sites. These findings show some of the molecular mechanisms of ventricular arrhythmias in heart failure, resulting in dispersion and prolongation of action potential which elicit reentry and after depolarization.

The transcriptional regulation of Stat proteins is controlled through their C-terminal domains, which harbor both a tyrosine phosphorylation site, required for dimerization and subsequent nuclear translocation, and a serine phosphorylation site, required for maximum transcriptional activity. Previously, we reported that protein kinase Cdelta (PKCdelta) phosphorylates and interacts with Stat3 in an interleukin (IL)-6-dependent manner. In this study, we further characterized this interaction, and investigated the potential role of such an interaction. We show here that the catalytic domain of PKCdelta interacts with the Src homology 2 domain and part of the adjacent C-terminal transactivation domain of Stat3. This interaction, which does not seem to involve a classical phosphotyrosine SH2-mediated binding, however, significantly enhances the interaction of Stat3 and the IL-6 receptor subunit glycoprotein (gp) 130, which is the initial step for Stat3 activation by IL-6. Expression of a dominant negative PKCdelta or depletion of the endogenous PKCdelta by phorbol 12-myristate 3-acetate treatment abrogates the association of Stat3 with gp130. At the same time, PKCdelta is recruited to gp130 via association with Stat3, which may facilitate its phosphorylation on the gp130 receptor. Finally, we identified Thr-890, a putative PKC phosphorylation site on gp130, to be critical for the effect of PKCdelta. Our data indicate that PKCdelta plays important regulatory roles in IL-6 signaling.

Two novel alternatively spliced isoforms of the human two-pore-domain potassium channel TREK-2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK-2b) and 2559 bp (TREK-2c) encode proteins of 508 amino acids each. RT-PCR showed that TREK-2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK-2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK-2c in rat brain which partially overlapped with that of TREK-1. Expression of TREK-2b and TREK-2c in human embryonic kidney (HEK) 293 cells showed that their single-channel characteristics were similar. The slope conductance at negative potentials was 163 +/- 5 pS for TREK-2b and 179 +/- 17 pS for TREK-2c. The mean open and closed times of TREK-2b at -84 mV were 133 +/- 16 and 109 +/- 11 micros, respectively. Application of forskolin decreased the whole-cell current carried by TREK-2b and TREK-2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK-2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol-12-myristate-13-acetate (PMA) also reduced whole-cell current. However, removal of the putative TREK-2b-specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK-2 contributes to the diversity of two-pore-domain K+ channels.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) beta1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) alpha. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC alpha, caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PKC alpha and PLC beta1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC beta1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC beta1 mutant in which the PKC phosphorylation site Ser(887) was replaced by alanine, or a dominant-negative PKC alpha, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC beta1 by PKC alpha plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.

Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR.

Modulation of postsynaptic AMPA receptors in the brain by phosphorylation may play a role in the expression of synaptic plasticity at central excitatory synapses. It is known from biochemical studies that GluR1 AMPA receptor subunits can be phosphorylated within their C terminal by cAMP-dependent protein kinase A (PKA), which is colocalized with the phosphatase calcineurin (i.e., phosphatase 2B). We have examined the effect of PKA and calcineurin on the time course, peak open probability (PO,PEAK), and single-channel properties of glutamateevoked responses for neuronal AMPA receptors and homomeric GluR1(flip) receptors recorded in outside-out patches. Inclusion of purified catalytic subunit Cα-PKA in the pipette solution increased neuronal AMPA receptorPO,PEAK(0.92) compared with recordings made with calcineurin included in the pipette (PO,PEAK0.39). Similarly, Cα-PKA increasedPO,PEAKfor recombinant GluR1 receptors (0.78) compared with patches excised from cells cotransfected with a cDNA encoding the PKA peptide inhibitor PKI (PO,PEAK0.50) or patches with calcineurin included in the pipette (PO,PEAK0.42). Neither PKA nor calcineurin altered the amplitude of single-channel subconductance levels, weighted mean unitary current, mean channel open period, burst length, or macroscopic response waveform for recombinant GluR1 receptors. Substitution of an amino acid at the PKA phosphorylation site (S845A) on GluR1 eliminated the PKA-induced increase inPO,PEAK, whereas the mutation of a Ca2+,calmodulin-dependent kinase II and PKC phosphorylation site (S831A) was without effect. These results suggest that AMPA receptor peak response open probability can be increased by PKA through phosphorylation of GluR1 Ser845.

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr 253 in α-tubulin as a site that is phosphorylated by protein kinase C βI (PKCβI). Thr 253 is not part of a linear PKC consensus motif. Instead, Thr 253 occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCβI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this “structurally formed” consensus motif and its importance for the protein kinase–substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.

In response to the kinase activity of v-Src there is an increase in the membrane association of the novel protein kinase C (PKC) isoform PKC delta (Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367-1373). We report here that in v-Src-transformed cells PKC delta co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC delta had reduced enzymatic activity relative to the non-tyrosine-phosphorylated PKC delta from v-Src-transformed cells. The association between Src and PKC delta was dependent upon both an active Src kinase and membrane association. The association between c-Src Y527F and PKC delta was substantially enhanced by mutating a PKC phosphorylation site at Ser-12 in Src to Ala indicating that PKC delta phosphorylation of Src at Ser-12 destabilizes the interaction, possibly in a negative feedback loop. These data demonstrate that upon recruitment of PKC delta to the membrane in v-Src-transformed cells there is the formation of a Src.PKC delta complex in which PKC delta becomes phosphorylated on tyrosine and down-regulated.

K+-dependent Na+/Ca2+-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K+-dependent Na+/Ca2+-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca2+ signal dynamics in olfactory and other neurons. Synaptic Ca2+ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca2+ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca2+ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca2+ signalling by activating a signal damping and termination pathway., Activity of the K+-dependent Na+/Ca2+-exchanger, NCKX4, is stimulated by purinergic signals that depend on dual activation of two protein kinase pathways. This regulation provides a novel mechanism to shape Ca2+ signaling and thus to have important impact on neuronal processes.

Recent studies indicate a critical role for metabotropic glutamate receptor 5 (mGluR5) in the reinstatement of cocaine seeking. However, the signal transduction pathways through which mGluR5s regulate cocaine seeking have not been identified. Here, we show that intra-accumbens shell administration of an mGluR5 (9.0 μm MPEP), but not mGluR1 (50.0 μm YM 298198), antagonist before a priming injection of cocaine (10 mg/kg) attenuated the reinstatement of drug seeking in rats. Consistent with these results, intra-shell microinjection of the mGluR1/5 agonist DHPG (250 μm) promoted cocaine seeking. Intra-shell administration of a phospholipase C (PLC) inhibitor (40.0 μm U73122) or a protein kinase C (PKC) inhibitor (10.0 μm Ro 31-8220 or 30.0 μm chelerythrine chloride) attenuated cocaine seeking. Pharmacological inhibition of PKC in the shell also blocked intra-shell DHPG-induced reinstatement of cocaine seeking. In addition, cocaine priming-induced reinstatement of drug seeking was associated with increased phosphorylation of PKCγ, but not PKCα or PKCβII, in the shell. Cocaine seeking previously was linked to increased phosphorylation of GluA2 at Ser880, a PKC phosphorylation site, which promotes the endocytosis of GluA2-containing AMPA receptors via interactions with Protein Associated with C Kinase (PICK1). The present results indicated that inhibition of PICK1 (100 μm FSC-231) in the shell attenuated cocaine seeking. There were no effects of any drug treatment in the shell on sucrose seeking. Together, these findings indicate that accumbens shell mGluR5 activation promotes cocaine seeking, in part, through activation of PLC and PKCγ. Moreover, the endocytosis of shell GluA2-containing AMPARs during cocaine seeking may depend on interactions with PKCγ and PICK1.

The effects of arachidonic acid on ACh-gated channel currents were examined using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Arachidonic acid decreased ACh-evoked currents during treatment, to a greater extent in Ca(2+)-free extracellular solution. The currents were enhanced for more than 30 min after washing, reaching 150 and 170% in Ca(2+)-containing and -free extracellular solutions, respectively. The current enhancement was inhibited by the selective protein kinase C (PKC) inhibitor, GF109203X, whereas the current depression was not affected. Furthermore, arachidonic acid-evoked current depression was blocked in mutant ACh receptors with PKC phosphorylation site deletions on the alpha and delta subunits, but the long-lasting potentiation effect remained. These results indicate that arachidonic acid may decrease ACh receptor currents by a direct binding to PKC phosphorylation sites of the ACh receptors and may potentiate the currents via a novel pathway related to arachidonic acid-regulated PKC activation, but not via PKC phosphorylation of the ACh receptor itself.

The role of 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the beta-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the beta-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the beta-system is stimulated by an increased level of cAMP or following addition of N6,2'-O-dibutyryl-3',5'-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the beta-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the beta-system is in State II.

Phosphorylation of the μ opioid receptor (MOPr), mediated by several protein kinases, is a critical process in the regulation of MOPr signaling. Although G protein-coupled receptor kinases are known to play an essential role in the agonist-induced phosphorylation and desensitization of MOPr, evidence suggests that other protein kinases, especially protein kinase C (PKC), also participate in the regulation of MOPr signaling. In this study, we investigated the biochemical nature and downstream effects of PKC-mediated MOPr phosphorylation. We observed in vitro phosphorylation of the MOPr C terminus by purified PKC. Protein mass spectrometry and site-directed mutagenesis implicated Ser363 of MOPr as the primary substrate for PKC, and this was confirmed in Chinese hamster ovary cells stably expressing full-length MOPr using an antibody that specifically recognizes phosphorylated Ser363. Alanine mutation of Ser363 did not affect the affinity of MOPr-ligand binding and the efficiency of receptor G-protein coupling. However, the S363A mutation attenuated the desensitization of receptor G-protein coupling induced by phorbol 12-myristate. Our research thus has identified a specific PKC phosphorylation site in MOPr and demonstrated that PKC-mediated phosphorylation of MOPr induces receptor desensitization at the G protein coupling level.

A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

RalA and RalB are monomeric G proteins that are 83% identical in amino acid sequence but have paralogue-specific effects on cell proliferation, metastasis, and apoptosis. Using in vitro kinase assays and phosphosite-specific antibodies, here we show phosphorylation of RalB by protein kinase C (PKC) and RalA by protein kinase A. We used mass spectrometry and site-directed mutagenesis to identify S198 as the primary PKC phosphorylation site in RalB. Phorbol ester [phorbol 12-myristate 13-acetate (PMA)] treatment of human bladder carcinoma cells induced S198 phosphorylation of stably expressed FLAG-RalB as well as endogenous RalB. PMA treatment caused RalB translocation from the plasma membrane to perinuclear regions in a S198 phosphorylation–dependent manner. Using RNA interference depletion of RalB followed by rescue with wild-type RalB or RalB(S198A) as well as overexpression of wild-type RalB or RalB(S198A) with and without PMA stimulation, we show that phosphorylation of RalB at S198 is necessary for actin cytoskeletal organization, anchorage-independent growth, cell migration, and experimental lung metastasis of T24 or UMUC3 human bladder cancer cells. In addition, UMUC3 cells transfected with a constitutively active RalB(G23V) exhibited enhanced subcutaneous tumor growth, whereas those transfected with phospho-deficient RalB(G23V-S198A) were indistinguishable from control cells. Our data show that RalA and RalB are phosphorylated by different kinases, and RalB phosphorylation is necessary for in vitro cellular functions and in vivo tumor growth and metastasis. Cancer Res; 70(21); 8760–9. ©2010 AACR.

Na(+)/Ca(2+) exchanger (NCX) is one of the major mechanisms for removing Ca(2+) from the cytosol especially in cardiac myocytes and neurons, where their physiological activities are triggered by an influx of Ca(2+). NCX contains a large intracellular loop (NCXIL) that is responsible for regulating NCX activity. Recent evidence has shown that proteins, including kinases and phosphatases, associate with NCX1IL to form a NCX1 macromolecular complex. To search for the molecules that interact with NCX1IL and regulate NCX1 activity, we used the yeast two-hybrid method to screen a human heart cDNA library and found that the C-terminal region of sarcomeric mitochondrial creatine kinase (sMiCK) interacted with NCX1IL. Moreover, both sMiCK and the muscle-type creatine kinase (CKM) coimmunoprecipitated with NCX1 using lysates of cardiacmyocytes and HEK293T cells that transiently expressed NCX1 and various creatine kinases. Both sMiCK and CKM were able to produce a recovery in the decreased NCX1 activity that was lost under energy-compromised conditions. This regulation is mediated through a putative PKC phosphorylation site of sMiCK and CKM. The autophosphorylation and the catalytic activity of sMiCK and CKM are not required for their regulation of NCX1 activity. Our results suggest a novel mechanism for the regulation of NCX1 activity.

Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively spliced forms, Kv4.3-short (Kv4.3-S) and Kv4.3-long (Kv4.3-L), differ by the presence of a 19-amino acid insert downstream from the sixth transmembrane segment. The isoforms are similar kinetically; however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we expressed the Kv4.3 isoforms in Xenopus oocytes and examined changes in their inactivation properties after stimulation of PKC activity. Whereas there was no difference in open-state inactivation, there were profound differences in CSI. In Kv4.3-S, PMA reduced the magnitude of CSI by 24% after 14.4 s at −50 mV. In contrast, the magnitude of CSI in Kv4.3-L increased by 25% under the same conditions. Mutation of a putatively phosphorylated threonine (T504) to aspartic acid within a PKC consensus recognition sequence unique to Kv4.3-L eliminated the PMA response. The change in CSI was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our previously published 11-state model closely simulated our experimental data. Our data demonstrate isoform-specific regulation of CSI by PKC in Kv4.3 and show that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.

Sub-lethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn(2+) liberation mediates neuronal death pathways, we tested whether a sub-lethal increase in free Zn(2+) could also trigger neuroprotection. Neuronal free Zn(2+) transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn(2+) that contributed significantly to excitotoxicity in non-preconditioned neurons, but not in tolerant neurons, unless preconditioning-induced free Zn(2+) was chelated. Thus, preconditioning may trigger the expression of Zn(2+)-regulating processes, which, in turn, prevent subsequent Zn(2+)-mediated toxicity. Indeed, preconditioning increased Zn(2+)-regulated gene expression in neurons. Examination of the molecular signaling mechanism leading to this early Zn(2+) signal revealed a critical role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn(2+). We identified a conserved PKC phosphorylation site at serine-32 (S32) of metallothionein (MT) that was important in modulating Zn(2+)-regulated gene expression and conferring excitotoxic tolerance. Importantly, we observed increased PKC-induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn(2+) serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.

Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freund's adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser880and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca2+permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.

Profilin is a cytoskeletal protein that interacts specifically with actin, phosphoinositides and poly (l-proline). Experimental results and in silico studies revealed that profilin exists as dimer and tetramer. Profilin oligomers possess weak affinity to poly (l-proline) due to unavailability of binding sites in dimers and tetramers. Phosphorylation studies indicate that profilin dimers are not phosphorylated while teramers are preferentially phosphorylated over monomers. In silico studies revealed that PKC phosphorylation site, S137 is buried in dimer while it is accessible in tetramer.

Metabotropic glutamate receptors (mGluRs) 1–8 are G protein-coupled receptors (GPCRs) that modulate excitatory neurotransmission, neurotransmitter release, and synaptic plasticity. PKC regulates many aspects of mGluR function, including protein–protein interactions, Ca 2+ signaling, and receptor desensitization. However, the mechanisms by which PKC regulates mGluR function are poorly understood. We have now identified calmodulin (CaM) as a dynamic regulator of mGluR5 trafficking. We show that the major PKC phosphorylation site on the intracellular C terminus of mGluR5 is serine 901 (S901), and phosphorylation of this residue is up-regulated in response to both receptor and PKC activation. In addition, S901 phosphorylation inhibits mGluR5 binding to CaM, decreasing mGluR5 surface expression. Furthermore, blocking PKC phosphorylation of mGluR5 on S901 dramatically affects mGluR5 signaling by prolonging Ca 2+ oscillations. Thus, our data demonstrate that mGluR5 activation triggers phosphorylation of S901, thereby directly linking PKC phosphorylation, CaM binding, receptor trafficking, and downstream signaling.

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Pur- kinje cell synapses is thought to regulate motor learning and memory formation in the cere- bellum. Neuronal activity-evoked protein kinase C (PKC) activation is required for the in- duction of LTD. In addition, theδ2 glutamate receptor (GluRδ2), which is predominantly expressed at PF-Purkinje cell synapses, is indispensable for the induction of LTD; however, the mechanisms by which GluRδ2 regulates LTD and its relationship with PKC activation remain elusive. Interestingly, GluRδ2 is phosphorylated by PKC on serine 945 (Ser945) near its C-terminus and a postsynaptic protein S-SCAM, which could potentially regulate glutamate receptor trafficking and synaptic plasticity, binds to the extreme C-terminus of GluRδ2 in a phosphorylation-dependent manner on Ser945. Here, using a Sindbis-based vi- rus expression approach, we show that a mutant GluRδ2, in which alanine replaced Ser945 and did not undergo PKC phosphorylation, was normally localized at the postsynaptic sites of PF-Purkinje cell synapses. In addition, like wild-type GluRδ2, the phosphorylation-dis- rupted GluRδ2 successfully rescued abrogated LTD in GluR δ2-null Purkinje cells. These re- sults indicate that Ser945, a major PKC phosphorylation site of of GluRδ2, may not play a crucial role in induction of LTD in the cerebellum. (Keio J Med 57 (2) : 105-110, June 2008)

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.

In this study we showed that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) activates endogenous transient receptor potential-canonical, subtype 6 (TRPC6), channels in neuronal PC12D cells. Activation of TRPC6 channels is correlated with the formation of a multiprotein complex containing M1 mAChRs, TRPC6 channels, and protein kinase C (PKC). Formation of the M1 mAChR-TRPC6-PKC complex is transient, with highest levels reached approximately 2 min after stimulation of M1 mAChRs. PKC in the complex phosphorylates TRPC6 on a conserved serine residue in the carboxyl-terminal domain (Ser768 in the TRPC6A isoform and Ser714 in the TRPC6B isoform). The immunophilin FKBP12, the phosphatase calcineurin, and Ca2+-binding protein calmodulin are also recruited to the M1 mAChR-TRPC6-PKC complex following activation of M1 mAChRs and remain stably associated with the TRPC6 channels after M1 mAChRs and PKC have disassociated. Binding of FKBP12, calcineurin, and calmodulin to TRPC6 channels is blocked by the following: 1) inhibition of PKC; 2) mutation of the PKC phosphorylation site (Ser(7168/714)) in the channels; or 3) pretreatment with FK506 or rapamycin, immunosuppressants that directly bind FKBP12. Inhibition of FKBP12 binding blocks the dephosphorylation of TRPC6 channels and the disassociation of M1 mAChRs, without affecting disassociation of PKC. The calcineurin inhibitor cyclosporin A also blocks the dephosphorylation of TRPC6 and prevents the disassociation of M1 mAChRs. Together, these results show that activated TRPC6 channels form the center of a dynamic multiprotein complex that includes PKC and calcineurin, which respectively phosphorylate and dephosphorylate the channels. Phosphorylation of the TRPC6 channels by PKC is required for the binding of FKBP12, which in turn is required for the binding of calcineurin and calmodulin. Subsequent dephosphorylation of the channels by calcineurin is required for the disassociation of M1 mAChRs.