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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.

The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca2+o) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaRT888. However, PKC inhibition enhances signalling even in CaSs lacking Thr-888, suggesting that an additional inhibitory site exists. An apparently equivalent PKC regulatory site in metabotropic glutamate receptor-5 (Ser-839) aligns not with CaST888 but instead with CaSS875, not previously considered a PKC site. CaSS875A (non-phosphorylatable) exhibited significantly enhanced Ca2+o sensitivity of both intracellular Ca2+ mobilisation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas the phosphomimetic CaSS875D mutant exhibited a loss of function. The CaSS875A/T888A double mutant exhibited even greater Ca2+o sensitivity than CaST888 alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaSS875A/T888A double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation (NPS-2143) or Ca2+o removal. Therefore, we have now identified CaSS875 as the missing PKC phosphorylation site that, together with CaST888, shapes the CaS signalling that underpins Ca2+o homeostasis. Together with the inactive form of the CaS9s extracellular domain, these sites attenuate Ca2+o sensitivity to attain appropriate physiological Ca2+o sensing. SIGNIFICANCE STATEMENT The calcium-sensing receptor (CaS) controls whole body calcium homeostasis by regulating parathyroid hormone secretion and renal calcium reabsorption. CaS function can be inhibited upon phosphorylation by protein kinase C (PKC), principally but not exclusively at Thr-888. Here we provide evidence that Ser-875, a previously unrecognised PKC site in CaS, is an additional inhibitory phosphorylation site for the receptor.

Rationale: Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BKα and auxiliary β1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BKα is primarily plasma membrane localized, whereas the cellular location of β1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. Objective: Test the hypothesis that ET-1 inhibits BK channels by altering BKα and β1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. Methods and Results: ET-1, through activation of PKC (protein kinase C), reduced surface β1 abundance and the proximity of β1 to surface BKα in myocytes. In contrast, ET-1 did not alter surface BKα, total β1, or total BKα proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1–induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface β1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of β1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. Conclusions: ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of β1 subunits. The decrease in surface β1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.

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.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

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.

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.

Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC ϵ to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQQ209L and specific PKC isoform cDNAs. Overexpression of GNAQQ209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p2 or 100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC ϵ increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and ϵ. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and ϵ, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC ϵ. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138