PKC Phosphorylation Sites Research Articles

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome

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 invitro 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 invitro, 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.

Homologous Serine/Threonine in the CaV 2.2α1 and 2.3α1 Subunits Behave Similarly, as Stimulatory and Inhibitory PKC Sites

High voltage-gated calcium (Cav) channels are regulated by PKC isozymes. These isozymes target selected serine/threonine (Ser/Thr) PKC phosphorylation sites in the intracellular regions of Cavα1 subunits of these channels. It has been found earlier using Cav2.2α1 subunits that stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) PKC sites exist and their activation with PKC isozymes led to potentiation and depression of calcium currents (ICa) respectively. Based on the above report, it was planned to examine if the homologous sites in the Cav2.3α1 subunits behave similarly. In this regard the WT Cav2.3α1 or Ser/Thr Ala mutants of stimulatory (Thr-365, Ser-1995 and Ser-2011) or inhibitory (Ser-369) sites were expressed along with β1b and 2/δ cDNA subunits in Xenopus oocytes and the barium currents (IBa) were studied. Intracellular injection of PKC isozymes βII or e potentiated WT Cav2.3 currents. While both PKC βII and e potentiated IBa through Thr-365 (T365/S369A/S1995A/S2011A), only PKC e increased IBa through Ser-1995 (T365A/S369A/S1995/S2011A) channels. Ser-2011 failed to act as a stimulatory site contrary to its homologous site, Ser-2132 in the Cav2.2α1 subunits. However, Ser-369 acted as inhibitory site as its homolog Ser-425 in the Cav2.2α1 subunits. Both PKC βII and e inhibited IBa through Ser-369 (T365A/S369/S1995A/S2011A) channels. When both Thr-365 and Ser-369 were present (T365/S369/S1995A/S2011A), IBa was neither stimulated nor inhibited. However, stimulation was dominant when two stimulatory sites (Thr-365 & Ser-1995) were present along with Ser-369. Experiments with other mutants, including Ser/Thr Asp constructs are being studied and will be discussed.

Altered signaling pathways linked to angiotensin II underpin the upregulation of renal Na+-ATPase in chronically undernourished rats

This study has investigated the participation of altered signaling linked to angiotensin II (Ang II) that could be associated with increased Na+ reabsorption in renal proximal tubules during chronic undernutrition. A multideficient chow for rats (basic regional diet, BRD) was used, which mimics several human diets widely taken in developing countries. The Vmax of the ouabain-resistant Na+-ATPase resident in the basolateral membranes increased >3-fold (P<0.001) accompanied by an increase in Na+ affinity from 4.0 to 0.2mM (P<0.001). BRD rats had a >3-fold acceleration of the formation of phosphorylated intermediates in the early stage of the catalytic cycle (in the E1 conformation) (P<0.001). Immunostaining showed a huge increase in Ang II-positive cells in the cortical tubulointerstitium neighboring the basolateral membranes (>6-fold, P<0.001). PKC isoforms (α, ε, λ, ζ), Ang II type 1 receptors and PP2A were upregulated in BRD rats (in %): 55 (P<0.001); 35 (P<0.01); 125, 55, 11 and 30 (P<0.001). PKA was downregulated by 55% (P<0.001). With NetPhosK 1.0 and NetPhos 2.0, we detected 4 high-score (>0.70) regulatory phosphorylation sites for PKC and 1 for PKA in the primary sequence of the Na+-ATPase α-subunit, which are located in domains that are key for Na+ binding and catalysis. Therefore, chronic undernutrition stimulates tubulointerstitial activity of Ang II and impairs PKC- and PKA-mediated regulatory phosphorylation, which culminates in an exaggerated Na+ reabsorption across the proximal tubular epithelium.

Nuclear Import of Transcription Factor BR-C Is Mediated by Its Interaction with RACK1

The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

Abstract 590: RARRES1 is a modulator of angiogenesis and autophagy

Abstract Retinoic Acid Receptor Responder (RARRES1) is a putative tumor suppressor gene that exerts its tumor suppressor function via unknown mechanisms. Determining the molecular mechanisms involved in the function of RARRES1 in important biological processes, angiogenesis and autophagy, is the focus of our study and may delineate molecular targets for therapeutic intervention. Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a vital requirement for the growth and metastasis of tumors. It constitutes a pivotal step in cancer progression. For the tumors to grow and have the propensity to metastasize they have to undergo the “angiogenic switch”. This switch depends on the dynamic balance between the pro-angiogenic factors (such as HIF-1α and VEGF) and the anti-angiogenic factors (such as thrombospondin, VEGI). Stimuli for angiogenesis include hypoxia, inflammation or genetic variations in tumor suppressors. We sought to determine if RARRES1 expression could modulate angiogenesis. Our results indicate that overexpression of RARRES1 in HUVECs severely impairs in vitro angiogenesis. The data also indicate reduced levels of angiogenic markers and signaling molecules, phospho-VEGF-R and phospho-p38 MAPK. The results demonstrate that RARRES1 expression significantly lowers H2O2-induced ROS production, a known inducer of angiogenesis. Bioinformatics searches of RARRES1 revealed a potential MAPK binding site, the Erk-D domain and multiple PKC phosphorylation sites. PKC signaling (ERK phosphorylation) has been implicated in cellular autophagy. It is an evolutionarily conserved self-degradative process that plays a housekeeping role by eliminating damaged organelles and misfolded or aggregated proteins via the lysosomal degradation pathway. Deregulation of autophagy has been reported in various cancers. Interestingly, our data indicates that RARRES1 expression induces phospho-ERK in multiple cancer cell lines. We hypothesized that the increased phospho-ERK levels in RARRES1 expressing cells would result in autophagy induction. Our results demonstrate that RARRES1 expression induces the expression of autophagy markers, ATG3, beclin and LC-3β. In summary, the data presented here indicate that (a) loss of RARRES1 results in increased ROS production and angiogenesis by p38 MAPK activation and (b) autophagy defects in RARRES1-deficient cancer cells facilitates tumor progression. Our study implicates that RARRES1 may be a novel anti-angiogenic molecule that induces autophagic response. Future studies will determine the mechanism between these important cell-biological processes and if RARRES1 may serve as a target for therapeutic intervention both in cancer and in angiogenesis-related disorders. Citation Format: Arpita Roy, Okjin Jean Kim, Malathi Ramalinga, Solomon Lynch, Stephen Byers, Deepak Kumar. RARRES1 is a modulator of angiogenesis and autophagy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 590. doi:10.1158/1538-7445.AM2014-590

A1 adenosine receptor-stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation.

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 ADAM17(S811A) mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17(S811A) 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.

PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity

Transient receptor potential canonical 6 (TRPC6) is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R), TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG) and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

Phospholemman-Dependent Regulation of Na/K-Atpase Modulates Constriction and Relaxation in Aortic Smooth Muscle

The Na/K ATPase (NKA) plays a role in modulating vascular tone through both NO-dependent and independent pathways. Phospholemman (PLM) is a muscle-specific regulator of Na/K ATPase and, in cardiac muscle, links PKA, PKC, and NO-signaling pathways to NKA stimulation. PLM is expressed in vascular smooth muscle (VSM) and we hypothesized that its phosphorylation may modulate the vascular responses to agonists. We tested this using wild-type (WT), PLM knock-out (KO), and mutant PLM knock-in mice (3SA) in which the PKC and PKA phosphorylation sites (S63, S68, S69) are mutated to alanines. Agonist-induced constriction and relaxation were measured in aortic rings in an isometric wire myograph. Vascular NKA activity was assessed by K-induced relaxation (a surrogate measure of Na/K ATPase activation) and PLM phosphorylation by immunoblotting. In WT aortae, PLM phosphorylation (S63 and S68) was significantly increased in response to phenylephrine (PE) and K-induced relaxation was significantly higher in WT than KO mice (85±7% vs 58±4% of PE-induced pre-constriction; p<0.01), suggesting PLM regulates NKA activity in VSM. The dose-response curve for PE-induced constriction was profoundly shifted up and to the left in 3SA aortae compared to WT suggesting PLM phosphorylation normally limits constriction. Ouabain (300μM) completely abolished this difference. Pretreatment with L-NAME (300μM) also potentiated constrictor responses to PE to a greater extent in WT than 3SA vessels. Relaxation induced by the NO donor spermine NONOate was blunted in vessels from 3SA mice: 67±6% vs 84±2% (U46619-induced constriction; p<0.01 cf WT). In summary, isolated aortic rings from mice expressing unphosphorylatable PLM showed markedly elevated constriction in response to PE and attenuated relaxation in response to an NO donor. Thus, PLM phosphorylation regulates the activity of vascular NKA and this contributes to modulation of aortic constriction and relaxation.

Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 μM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.

Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP

TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP–PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cβ. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4–1.2nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cβ effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cβ activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.

Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca2+]i through G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca2+]i and are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+ bowl and the regulator of K+ conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+ bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.

PKC-Mediated USP Phosphorylation at Ser35 Modulates 20-Hydroxyecdysone Signaling in Drosophila

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.

Cloning, characterization and expression of estrogen receptor beta in the male half-smooth tongue sole, Cynoglossus semilaevis

A full-length sequence encoding the estrogen receptor beta was isolated from half-smooth tongue sole, Cynoglossus semilaevis (hstsERβ) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. The hstsERβ cDNA clone was found to contain 1,791 nucleotides including an open reading frame that encodes 578 amino acids. The deduced hstsERβ protein consisted of six nuclear receptor-characteristic domains. Based on a phylogenetic analysis, the hstsERβ C and E domains are highly conserved compared to other fishes. The potential phosphorylation sites for PKC, CK-2 and PTK are also found in this protein. Highest amino acid identities were found for hstsERβ with common carp (Cyprinus carpio) ERβ (76%) and Japanese flounder (Paralichthys olivaceus) ERβ (76%). Tissue expression analysis confirmed that the hstsERβ was widely distributed and predominantly expressed in testis, brain and liver. Seasonal changes in the testis, brain and liver expression profiles of hstsERβ were examined by RT-PCR; the present results suggest that level of hstsERβ in brain increased to the highest then decreases with gonadal growth; whereas in the testis and liver, the hstsERβ mRNA level dropped to lowest then slightly increased.

The C-Type Natriuretic Peptide Induces Thermal Hyperalgesia through a Noncanonical Gβγ-dependent Modulation of TRPV1 Channel

Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain essentially unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-Gβγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is coexpressed in transient receptor potential vanilloid-1 (TRPV1)-expressing mouse dorsal root ganglia (DRG) neurons. NPR-C can be coimmunoprecipitated with Gα(i), and C-type natriuretic peptide (CNP) treatment induced translocation of protein kinase Cε (PKCε) to the plasma membrane of these neurons, which was inhibited by pertussis toxin pretreatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons and increased their firing frequency, an effect that was absent in DRG neurons from TRPV1(-/-) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pretreatment of DRG neurons with the specific inhibitors of Gβγ, phospholipase C-β (PLCβ), or PKC, but not of protein kinase A, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Furthermore, CNP injection into mouse hindpaw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγ or TRPV1 and was also absent in TRPV1(-/-) mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity.

Proteomic Analysis of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor Complexes

The AMPA receptor (AMPA-R) is a major excitatory neurotransmitter receptor in the brain. Identifying and characterizing the neuronal proteins interacting with AMPA-Rs have provided important information about the molecular mechanisms underlying synaptic transmission and plasticity. In this study, to identify more AMPA-R interactors in vivo, we performed proteomic analyses of AMPA-R complexes from the brain. AMPA-R complexes were isolated from the brain through various combinations of biochemical techniques for solubilization, enrichment, and immunoprecipitation. Mass spectrometry analyses of these isolated complexes identified several novel components of the AMPA-R complexes as well as some previously identified components. The identification of these novel components helps to further define the complex mechanisms involved in the regulation of AMPA receptor function and synaptic plasticity.

Mutations in lipopolysaccharide-binding protein (LBP) gene change the susceptibility to clinical mastitis in Chinese Holstein

Mastitis is an unsolved human challenge all dairy farms facing with, which leads to immeasurable economic loss to the farmers. LBP gene plays a vital role in the innate immune recognition of Gram-negative bacterium that is a major cause of bovine clinical mastitis, but little is known about LBP mutations and their effects on cows' susceptibility to clinical mastitis. In this study, PCR-SSCP method was adopted to analyze SNPs of LBP gene in Chinese Holstein for the first time. 17 SNPs were found in the promoter core region, exon1, exon2, exon3, exon4 and exon8. The mutation g.-81C → T in promoter leads to an AP-2 binding site lost. Two mutations, g.11T → C (4 Leu → Ser) and g.68G → C (23Gly → Ala) in signal peptide brought about molecular secondary structural change, meanwhile, g.11T → C made a Big-1 domain lost, and there was an N-myristoylation site at the g.68G/C locus. The three mutations above were in complete linkage disequilibrium in allele A. In mature LBP protein, five mutations were found: g.3034G → A(36Asp → Asn), g.3040A → G(38Asn → Asp), g.3056T → C(43Ile → Thr) in allele D; g.4619G → A(67Ala → Thr) in allele F; 19975G → A (282Val → Met) in allele J. And SNPs in allele D and F were in complete linkage disequilibrium, also in which 38Asn → Asp and 67Ala → Thr influenced the protein secondary structure. Prediction of the 3-D structure shows mutations 36Asp → Asn, 38Asn → Asp and 43 Ile → Thr were on the concave surface of LBP protein at barrel-N, 67Ala → Thr was in the apolar pocket at barrel-N. Motif analysis shows 36Asp → Asn causes loss of a CK2 phosphorylation site, 67 Ala → Thr forms a new PKC phosphorylation site. And 43Ile → Thr, 67Ala → Thr made hydrophobic amino acids to be hydrophilic amino acids. Interestingly, the morbidity of AB (mixed type g.-81C/T, g.11T/C, g.68G/C), CD (mixed type g.3034G/A, g.3040A/G, g.3056T/C) and EF (mixed type g.4619G/A) genotype cows are significant higher than others in this study (P < 0.01), and it can be deduced that the SNPs in these 3 genotypes might affect the secretion of LBP protein and regulate the binding ability of LBP protein to LPS. Taken together, it is revealed that these SNPs may hold the secret of susceptibility to clinical mastitis in Chinese Holstein.

Estrogen Receptor β and Its Domains Interact with Casein Kinase 2, Phosphokinase C, and N-Myristoylation Sites of Mitochondrial and Nuclear Proteins in Mouse Brain

The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.

Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes (42.5)

Abstract CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that increase Oct-2 and IgG1 mRNA and protein expression, independent of class switch recombination. One of the most proximal intermediates identified is PLCγ2. Since the cytoplasmic domain of CD86 lacks tyrosine residues reported to bind PLCγ2, we used a proteomics-based identification approach to show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and prohibitin-2 (Phb2), bind to CD86. Although basal expression of Phb1/2 and association with CD86 was low in resting B cells, expression and association increased after CD40 engagement. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by shRNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine PKC-phosphorylation sites, but did not affect Phb1/2 binding to CD86. Furthermore, Phb1/2 reduction and CD86 truncation revealed that the CD86-induced phosphorylation of PLCγ2 required Phb1/2 alone; whereas, IκBα and NF-κB (p65) phosphorylation required both Phb1/2 and the CD86 cytoplasmic domain. Together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell to regulate IgG1 production and is the first evidence to define the molecular basis of membrane-proximal CD86 signal transduction in B cells.

Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation

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.