Serine Phosphorylation Sites Research Articles

The protective role of YAP1 on ER stress-induced cell death in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (VSMCs) has been implicated in the progression of atherosclerosis, especially in vascular remodelling and plaque rupture. Although it is known that Yes-associated protein 1 (YAP1) is a critical molecule that regulates cell proliferation, differentiation and apoptosis, the role of YAP1 in VSMCs apoptosis remains unknown. In this study, we investigated whether YAP1 modulates VSMC apoptosis induced by endoplasmic reticulum (ER) stress. In cultured VSMC, tunicamycin caused cell death accompanied by an increase in caspase-3 processing and C/EBP homologous protein (CHOP) expression. YAP1 protein expression was downregulated by tunicamycin and the phosphorylation of YAP1 at the Ser127 site was significantly increased by tunicamycin. Tunicamycin further decreased cell viability followed by an increase in caspase-3 processing in the absence of YAP1 when compared with treatment only with tunicamycin or siYAP1. On the other hand, overexpression of a constitutively active YAP1 (YAP1-5SA), which lacks five serine phosphorylation sites, significantly prevented the caspase-3 processing and restored the decrease in cell viability induced by tunicamycin. Overexpression of YAP1-5SA significantly inhibited tunicamycin-induced caspase-8 processing without affecting phosphorylation of p-38 and Akt. Furthermore, the overexpression of YAP1-5SA significantly restored the decrease in ANKRD1 expression induced by tunicamycin. The inhibition of tunicamycin-induced caspase-3 cleavage by YAP1-5SA was markedly attenuated in ANKRD1-knockdown cells. These results demonstrate that ER stress can alter intracellular YAP1 protein expression in VSMCs and that YAP1 is protective against VSMC apoptosis induced by ER stress through inhibiting caspase8/3 activation mediated in part by upregulation of ANKRD1.

Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

Signaling via the transient receptor potential (TRP) ion channel C6 plays a pivotal role in hereditary and sporadic glomerular kidney disease. Several studies have identified gain-of-function mutations of TRPC6 and report induced expression and enhanced channel activity of TRPC6 in association with glomerular diseases. Interfering with TRPC6 activity may open novel therapeutic pathways. TRPC6 channel activity is controlled by protein expression and stability as well as intracellular trafficking. Identification of regulatory phosphorylation sites in TRPC6 and corresponding protein kinases is essential to understand the regulation of TRPC6 activity and may result in future therapeutic strategies. In this study, an unbiased phosphoproteomic screen of human TRPC6 identified several novel serine phosphorylation sites. The phosphorylation site at serine 14 of TRPC6 is embedded in a basophilic kinase motif that is highly conserved across species. We confirmed serine 14 as a target of MAPKs and proline-directed kinases like cyclin-dependent kinase 5 (Cdk5) in cell-based as well as in vitro kinase assays and quantitative phosphoproteomic analysis of TRPC6. Phosphorylation of TRPC6 at serine 14 enhances channel conductance by boosting membrane expression of TRPC6, whereas protein stability and multimerization of TRPC6 are not altered, making serine 14 phosphorylation a potential drug target to interfere with TRPC6 channel activity.-Hagmann, H., Mangold, N., Rinschen, M. M., Koenig, T., Kunzelmann, K., Schermer, B., Benzing, T., Brinkkoetter, P. T. Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

The antiviral role of heat shock protein 27 against red spotted grouper nervous necrosis virus infection in sea perch

Heat shock protein 27 (HSP27), functioning as a stress induced protective protein, has been reported to participate in various biological processes, including apoptosis, thermal protection, and virus infection. In this study, a HSP27-like gene from the seawater fish sea perch, designated as LjHSP27, was characterized. The 1361 bp full-length cDNA of LjHSP27 encoded a 221 amino acid protein containing a conserved α-crystallin domain, two variable amino- and carboxy-terminal extensions, a WD/EPF motif, two serine phosphorylation sites, and two putative actin binding regions. Phylogenetic analysis showed that LjHSP27 shared the closest genetic relationship with HSP27 of the Asian seabass Lates calcarifer. LjHSP27 mRNA was ubiquitously expressed in all tissues examined, but significantly up-regulated in spleen and kidney and down-regulated in brain post red spotted grouper nervous necrosis virus (RGNNV) infection. In vitro, LjHSP27 transcript was remarkably reduced post RGNNV infection, but rapidly increased after polyinosinic-polycytidylic acid treatment. Up-regulation and down-regulation of LjHSP27 inhibited and promoted RGNNV replication in cultured LJB cells, respectively. Luciferase assay indicated that LjHSP27 could enhance the promoter activities of zebrafish interferon (IFN)1 and IFN3, suggesting its potential role in innate immune responses. Moreover, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, as indicated by the up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes, while KNK437 caused down-regulation of LjHSP27 dramatically led to opposite results, suggesting that LjHSP27 might exert its anti-RGNNV activities by regulating the apoptosis signaling pathway. Our results would provide a new insight into the underlying molecular mechanism of HSP and RGNNV interaction.

Selective Phosphorylation of AMPA Receptor Contributes to the Network of Long-Term Potentiation in the Anterior Cingulate Cortex.

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.

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases.

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.

Nucleotide variants of the Nramp1 gene in pigs and their effects on disease resistance

The Nramp1 gene is an important factor associated with susceptibility to infectious diseases caused by intracellular pathogens. The objective of this study was to determine the structures and sequences of Nramp1 in Tibetan, Gansu Black, Large White, Yorkshire, and Duroc pigs. Sequence analysis of the Nramp1 gene in the five pig breeds revealed the presence of 11 nucleotide variants in intronic regions, 2 nucleotide variants in the control region, 10 nucleotide variants and one deletion in the 3’ non-coding region, and 15 nucleotide variants in the exons. However, only 4 nucleotide variants resulted in amino acid changes. Further analysis predicted that the Tibetan pig Nramp1 protein harbors 10 transmembrane domains. The analysis also predicted 10 serine phosphorylation sites, 3 threonine phosphorylation sites, and 4 tyrosine phosphorylation sites in the Nramp1 protein of Tibetan pigs. Predictions of the Tibetan pig Nramp1 tertiary structure revealed 7 putative a -helices and 5 putative b-sheets. Predictions of the Yorkshire pig Nramp1 protein revealed significant differences compared with the Tibetan pig Nramp1 protein. The results indicated that differences in transmembrane domains and tertiary structures of the Nramp1 protein of the Tibetan and Yorkshire pig breeds could explain differences in the disease resistance of these two breeds

Abstract 1370: EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling

Abstract Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Phosphomimetic Spred1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of EGFR may disrupt Spred1-NF1 binding. To identify the Spred1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1370. doi:10.1158/1538-7445.AM2017-1370

Genome-wide identification, characterization, and expression analysis of the SWEET gene family in cucumber

SWEETs (sugars will eventually be exported transporters) are a novel class of recently identified sugar transporters that play important roles in diverse physiological processes. However, only a few species of the plant SWEET gene family have been functionally identified. Up till now, there has been no systematic analysis of the SWEET gene family in Cucurbitaceae crops. Here, a genome-wide characterization of this family was conducted in cucumber (Cucumis sativus L.). A total of 17 CsSWEET genes were identified, which are not evenly distributed over the seven cucumber chromosomes. Cucumber SWEET protein sequences possess seven conserved domains and two putative serine phosphorylation sites. The phylogenetic tree of the SWEET genes in cucumber, Arabidopsis thaliana, and Oryza sativa was constructed, and all the SWEET genes were divided into four clades. In addition, a number of putative cis-elements were identified in the promoter regions of these CsSWEET genes: nine types involved in phytohormone responses and eight types involved in stress responses. Moreover, the transcript levels of CsSWEET genes were analyzed in various tissues using quantitative real-time polymerase chain reaction. A majority (70.58%) of the CsSWEET genes were confined to reproductive tissue development. Finally, 18 putative watermelon ClaSWEET genes and 18 melon CmSWEET genes were identified that showed a high degree of similarity with CsSWEET genes. The results from this study provided a basic understanding of the CsSWEET genes and may also facilitate future research to elucidate the function of SWEET genes in cucumber and other Cucurbitaceae crops.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis. Randomized controlled trial. Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model. Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Corrigendum: MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop.

Scientific Reports 6: Article number: 33583; published online: 26 September 2016; updated: 01 December 2016

Concerted Action of PGC-1-related Coactivator (PRC) and c-MYC in the Stress Response to Mitochondrial Dysfunction

PGC-1-related coactivator (PRC) has a dual function in growth-regulated mitochondrial biogenesis and as a sensor of metabolic stress. PRC induction by mitochondrial inhibitors, intracellular ROS, or topoisomerase I inhibition orchestrates an inflammatory program associated with the adaptation to cellular stress. Activation of this program is accompanied by the coordinate expression of c-MYC, which is linked kinetically to that of PRC in response to multiple stress inducers. Here, we show that the c-MYC inhibitor 10058-F4 blocks the induction of c-MYC, PRC, and representative PRC-dependent stress genes by the respiratory chain uncoupler, carbonyl cyanide m-chlorophenyl hydrazine (CCCP). This result, confirmed by the suppression of PRC induction by c-MYC siRNA silencing, demonstrates a requirement for c-MYC in orchestrating the stress program. PRC steady-state expression was markedly increased upon mutation of two GSK-3 serine phosphorylation sites within the carboxyl-terminal domain. The negative control of PRC expression by GSK-3 was consistent with the phosphor-inactivation of GSK-3β by CCCP and by the induction of PRC by the GSK-3 inhibitor AZD2858. Unlike PRC, which was induced post-translationally through increased protein half-life, c-MYC was induced predominantly at the mRNA level. Moreover, suppression of Akt activation by the Akt inhibitor MK-2206 blocked the CCCP induction of PRC, c-MYC, and representative PRC stress genes, demonstrating a requirement for Akt signaling. MK-2206 also inhibited the phosphor-inactivation of GSK-3β by CCCP, a result consistent with the ability of Akt to phosphorylate, and thereby suppress GSK-3 activity. Thus, PRC and c-MYC can act in concert through Akt-GSK-3 signaling to reprogram gene expression in response to mitochondrial stress.

RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements

SummaryReceptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

TGF-β regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms.

Members of the TGF-β superfamily are important regulators of chondrocyte function. Sox9, a key transcriptional regulator of chondrogenesis, is required for TGF-β-mediated regulation of specific cartilage genes. TGF-β can signal through a canonical, Smad-mediated pathway or non-conical pathways, including p38. Here we show that both pathways are activated in chondrocytes after treatment with TGF-β and that TGF-β stabilizes Sox9 protein and increases phosphorylation of Sox9. Mutagenesis of potential serine phosphorylation sites on Sox9 was used to demonstrate that serine 211 is required to maintain normal basal levels of Sox9 as well as mediate increased Sox9 levels in response to TGF-β. The serine 211 site is in a motif that is targeted by p38 kinase. We used siRNA and pharmacological agents to show that p38 and Smad3 independently regulate the phosphorylation and stability of Sox9. Previously, we demonstrated that Papss2 is a downstream transcriptional target of Sox9 and TGF-β. Here we show that p38 is required for TGF-β-mediated regulation of Papss2 mRNA. Together the results suggest a new mechanism for TGF-β-mediated gene regulation in chondrocytes via p38 and phosphorylation and stabilization of Sox9. Understanding how TGF-β regulates Sox9 may lead to identification of therapeutic targets for OA.

Prediction of Protein Phosphorylation Sites by Integrating Secondary Structure Information and Other One-Dimensional Structural Properties.

Studies on phosphorylation are important but challenging for both wet-bench experiments and computational studies, and accurate non-kinase-specific prediction tools are highly desirable for whole-genome annotation in a wide variety of species. Here, we describe a phosphorylation site prediction webserver, PhosphoSVM, that employs Support Vector Machine to combine protein secondary structure information and seven other one-dimensional structural properties, including Shannon entropy, relative entropy, predicted protein disorder information, predicted solvent accessible area, amino acid overlapping properties, averaged cumulative hydrophobicity, and subsequence k-nearest neighbor profiles. This method achieved AUC values of 0.8405/0.8183/0.7383 for serine (S), threonine (T), and tyrosine (Y) phosphorylation sites, respectively, in animals with a tenfold cross-validation. The model trained by the animal phosphorylation sites was also applied to a plant phosphorylation site dataset as an independent test. The AUC values for the independent test data set were 0.7761/0.6652/0.5958 for S/T/Y phosphorylation sites, respectively. This algorithm with the optimally trained model was implemented as a webserver. The webserver, trained model, and all datasets used in the current study are available at http://sysbio.unl.edu/PhosphoSVM .

MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

Molecular and Genetic Characterization of HIV-1 Tat Exon-1 Gene from Cameroon Shows Conserved Tat HLA-Binding Epitopes: Functional Implications.

HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA.

Abstract 1874: Oncogenic EGFR signaling inhibits the Spred1-NF1 interaction to sustain constitutive Ras signaling

Abstract Spred proteins negatively regulate Ras/MAPK signaling following mitogen stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Loss-of-function Spred1 and NF1 mutations occur across multiple cancer types including melanoma, non-small cell lung carcinoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that oncogenic EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of oncogenic EGFR may disrupt Spred1-NF1 binding. Our findings provide one potential mechanism by which oncogenic EGFR signaling disrupts negative feedback to allow for constitutive Ras signaling. Furthermore, this work may elucidate a novel kinase therapeutic target for restoring NF1 mediated inhibition of Ras. Citation Format: Evan Markegard, Ellen L. Mercado, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. Oncogenic EGFR signaling inhibits the Spred1-NF1 interaction to sustain constitutive Ras signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1874.

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins.

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

Regulation of scleraxis transcriptional activity by serine phosphorylation

Cardiac fibroblasts are the major extracellular matrix producing cells in the heart. Our laboratory was the first to demonstrate that the transcription factor scleraxis induces collagen 1α2 expression in both cardiac fibroblasts and myofibroblasts. Here we identify a novel post-translational mechanism by which scleraxis activity is regulated and determine its effect on transcription of genes targeted by scleraxis. Putative serine phosphorylation sites on scleraxis were revealed by in silico analysis using motif prediction software. Mutation of key serine residues to alanine, which cannot be phosphorylated, significantly attenuated the expression of fibrillar type I collagen and myofibroblast marker genes that are normally induced by scleraxis. Down-regulation of collagen 1α2 expression was due to reduced binding of the non-phosphorylated scleraxis mutant to specific E-box DNA-binding sites within the promoter as determined by chromatin immunoprecipitation in human cardiac myofibroblast cells and by electrophoretic mobility shift assay. This is the first evidence suggesting that scleraxis is phosphorylated under basal conditions. The phosphorylation sequence matched that targeted by Casein Kinase 2, and inhibition of this kinase activity disrupted the ability of scleraxis to modulate the expression of its target genes while also attenuating TGFβ-induced expression of type I collagen and myofibroblast phenotype conversion marker genes. These results demonstrate a novel mechanism for regulation of scleraxis activity, which may prove to be tractable for pharmacologic manipulation.