Activating Phosphorylation Site Research Articles

Two activating phosphorylation sites of Pbs2 MAP2K in the yeast HOG pathway are differentially dephosphorylated by four PP2C phosphatases Ptc1–Ptc4

To cope with an increased external osmolarity, thebudding yeastSaccharomyces cerevisiaeactivates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.

AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.

AKT is a central signaling protein kinase that plays a role in the regulation of cellular survival metabolism and cell growth, as well as in pathologies such as diabetes and cancer. Human AKT consists of three isoforms (AKT1-3) that may fulfill different functions. Here, we report that distinct subcellular localization of the isoforms directly influences their activity and function. AKT1 is localized primarily in the cytoplasm, AKT2 in the nucleus, and AKT3 in the nucleus or nuclear envelope. None of the isoforms actively translocates into the nucleus upon stimulation. Interestingly, AKT3 at the nuclear envelope is constitutively phosphorylated, enabling a constant phosphorylation of TSC2 at this location. Knockdown of AKT3 induces moderate attenuation of cell proliferation of breast cancer cells. We suggest that in addition to the stimulation-induced activation of the lysosomal/cytoplasmic AKT1-TSC2 pathway, a subpopulation of TSC2 is constitutively inactivated by AKT3 at the nuclear envelope of transformed cells.

Effects of Glycogen Synthase Kinase-3β (GSK3β) Inhibition in Glioblastoma Using Novel Compounds

Abstract AIMS GSK3β is a constitutively conserved serine/threonine kinase involved in a number of biological processes including cell cycle progression, apoptosis, cellular metabolism, proliferation and differentiation. High expression levels in the brain make this kinase a potential target for treatment of neurological disorders. The aim was to investigate the anti-proliferative and cytotoxic effects of four novel highly selective, type-II GSK3β inhibitors identified by in silico analysis of naturally occurring compounds. METHOD We demonstrate the effects on the cell viability of immortalised cell lines (grade 4 glioblastoma U87MG, foetal astrocytes SVGp12) and short-term glioblastoma patient derived cultures (PD301 and PD304). The compounds were incubated with cells for 24, 48 and 72h and then assayed for cell viability using MTS. The concentration of inhibitors was determined from previous IC50 experiments on purified GSK3β enzyme. The changes in activity status of GSK3β pre- and post-treatment were also determined using immunohistochemistry or Western Blotting. RESULTS Two of the compounds caused significant decrease in cell viability at 24h and 3uM as shown by decrease in viability of 20.5% relative to control. This decrease was larger in the glioma cell lines relative to the glial non-cancerous control (13%). Western blot analysis revealed that activating phosphorylation sites were detectable. More work is needed to determine if this is the mechanism by which reduction in cell viability occurs. CONCLUSION Two of the selected novel compounds have demonstrated ability to decrease glioblastoma cell viability. Future studies will include analysis of the compounds in relation to the BBB-glioma in vitro model.

Rapamycin Suppresses Penile NADPH Oxidase Activity to Preserve Erectile Function in Mice Fed a Western Diet.

The mechanistic target of rapamycin (mTOR) is a nutrient-sensitive cellular signaling kinase that has been implicated in the excess production of reactive oxygen species (ROS). NADPH oxidase-derived ROS have been implicated in erectile dysfunction pathogenesis. The objective of this study was to determine if mTOR is an activator of NADPH oxidase in the penis and to determine the functional relevance of this pathway in a translationally relevant model of diet-induced erectile dysfunction. Male mice were fed a control diet or a high-fat, high-sucrose Western style diet (WD) for 12 weeks and treated with vehicle or rapamycin for the final 4 weeks of the dietary intervention. Following the intervention, erectile function was assessed by cavernous nerve-stimulated intracavernous pressure measurement, in vivo ROS production was measured in the penis using a microdialysis approach, and relative protein contents from the corpus cavernosum were determined by Western blot. Erectile function was impaired in vehicle treated WD-mice and was preserved in rapamycin treated WD-mice. Penile NADPH oxidase-mediated ROS were elevated in WD-mice and suppressed by rapamycin treatment. Western blot analysis suggests mTOR activation with WD by increased active site phosphorylation of mTOR and p70S6K, and increased expression of NADPH oxidase subunits, all of which were suppressed by rapamycin. These data suggest that mTOR is an upstream mediator of NADPH oxidase in the corpus cavernosum in response to a chronic Western diet, which has an adverse effect on erectile function.

PPP6C negatively regulates oncogenic ERK signaling through dephosphorylation of MEK

SUMMARYFlux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.

Age-dependent increase in activity of eukaryotic elongation factor 2 kinase in mesenteric arteries from spontaneously hypertensive rats.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) negatively regulates protein translation through the phosphorylation of its specific substrate, eEF2. We previously found that expression of eEF2K was increased in arteries from 13–15-week-old spontaneously hypertensive rats (SHR) as well as in left ventricles of cardiac hypertrophy models. Furthermore, we demonstrated that eEF2K mediates the development of essential hypertension and pulmonary arterial hypertension in animal models. Protein expression changes with age during development of hypertension in SHR. In the present study, we examined whether activity and expression of eEF2K change in isolated mesenteric arteries dependent on the age. After superior mesenteric arteries were isolated from 4–10-week-old Wistar Kyoto rats (WKY) and SHR, Western blotting was performed. The phosphorylation of eEF2K at Ser500, an activating phosphorylation site, was increased in the arteries from 10-week-old SHR, whereas the phosphorylation of eEF2K at Ser366, an inactivating phosphorylation site, was increased in the arteries from 4–5-week-old SHR compared with WKY. The expression of eEF2K was increased in the arteries from 10-week-old SHR compared with WKY. The phosphorylation of eEF2 at Thr56 was decreased in the arteries from 4–5-week-old SHR, whereas it was increased in the arteries from 10-week-old SHR compared with WKY. We for the first time revealed that eEF2K activity is lower in prehypertensive stage but higher in hypertensive stage in SHR, suggesting that an inhibition of eEF2K activity may be a potential therapeutic strategy for the treatment of essential hypertension.

Virtual screening of black rice anthocyanins as antiobesity through inhibiting TLR4 and JNK pathway

Obesity is one of metabolic syndrome and have several complications including type 2 diabetes mellitus and inflammations. To reduce the complications and prevent the obesity, some natural constituents has been tested. This study proved the screening of cyanidin, cyanidin-3-O-glucoside, peonidin and peonidin-3-O-glucoside for inhibiting TLR4 and JNK protein using in-silico study. Four black rice anthocyanins, cyanidin, cyanidin-3-O-glucoside, peonidin and peonidin-3-O-glucoside as ligands taken out from PubChem database. Toll-like receptor 4 (TLR4) and c-Jun NH2-terminal kinase as target protein retrieved from protein data bank. Both of anthocyanins and proteins were docked by Hex 8.0 software and examined using Discovery studio v.19. All black rice anthocyanins interacted with TLR4 and JNK proteins. Cyanidin-3-O-glucoside, cyanidin, peonidin-3-O-glucoside and peonidin inhibit TLR4 in substrate residues site, while those anthocyanins directly inhibited the JNK protein in ATP active sites, gatekeeper residues, and phosphorylation sites. Those data indicated that four kind of anthocyanins that detected on black rice have functional role as anti-obesity. Further in-vivo or in-vitro research are required to support this experiment.

Abstract 2137: A high proportion of protein phosphorylation sites identified in CPTAC breast cancer and lung adenocarcinoma may result from direct phosphorylation by CDK1

Abstract Cyclin-dependent kinase 1 (CDK1) is a serine/threonine kinase that drives the G2/M phase of the cell cycle, and increased CDK1 kinase activity is a frequent feature of several tumor types. In the current study, we have used the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets to identify potential target phosphorylation sites associated with CDK1 activity in breast and lung adenocarcinoma and to experimentally study some of their biologic effects. Among the global protein phosphorylation sites detected in CPTAC, 80% and 81% of the identified phosphorylation sites in breast cancer and lung adenocarcinoma were found to correlate well with the level of an activating phosphorylation site in CDK1, t161. Positive correlations (r > 0.6) were present in most paired samples of tumor tissue and normal tissue. In addition, 42% and 40% of all phosphorylation sites in both of these tumor types were found to contain the minimum CDK1 consensus motif Ser/Thr-Pro (S/T-P). This result suggests that many of the sites with this motif are directly phosphorylated by CDK1, while most of the sites that lack this sequence are likely to be phosphorylated by other kinases. In both tumor types, increased phosphorylation of proteins in several signaling pathways, including those for glycolysis and the cell cycle, were found to be associated with phosphorylation of CDK1 t161. To study a possible direct effect of CDK1-dependent phosphorylation on a protein involved in cell cycle progression, we selected Epithelial Cell Transforming 2 (ECT2), which is a Rho family-specific GEF (guanine nucleotide exchange factor) with a role in G2/M. In agreement with the proteomic data, mass spectrometry analysis showed elevated ECT2 phosphorylation on T359, S367, T373, S376 and S861 in the G2/M phase that was sensitive to treatment with a CDK1 inhibitor. Biological experiments confirmed that phosphorylation of these sites enhanced the pro-oncogenic and GEF activity of ECT2. Additional experimental mechanistic analysis is needed to demonstrate whether specific phosphorylations are attributable to direct or indirect effects of CDK1. Citation Format: Dunrui Wang, Xiaolan Qian, Sarah Eng, Lisa Jenkins, Marian Durkin, Douglas Lowy. A high proportion of protein phosphorylation sites identified in CPTAC breast cancer and lung adenocarcinoma may result from direct phosphorylation by CDK1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2137.

Functional analysis of an R311C variant of Ca2+ -calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder.

Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+ -CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+ -CaM. The effects of the R311C mutation on CaMKK2 activity and Ca2+ -CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+ -dependent downstream signaling and cell viability, respectively. The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+ -CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+ -CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.

Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K.

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11‐Pbs2‐Hog1 MAPK cascade and the Ssk2/Ssk22‐Pbs2‐Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr‐518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser‐514 and Thr‐518 under optimal osmostress conditions. Mono‐phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2‐Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone‐to‐Hog1 crosstalk in the absence of osmostress. We also report that the rapid‐and‐transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

UNC5C‑knockdown enhances the growth and metastasis of breast cancer cells by potentiating the integrinα6/β4 signaling pathway.

Unc-5 Netrin Receptor C (UNC5C) is a netrin-1 dependence receptor that mediates the induction of apoptosis in the absence of netrin-1. The present study found that UNC5C is heterogeneously expressed in breast cancer cell lines. By knocking down UNC5C in SK-BR-3 and ZR-75-30 cells and overexpressing UNC5c in MDA-MB-231 cells, it was demonstrated that UNC5C exerts an inhibitory effect on the growth and metastasis of breast cancer cells. The mechanism involved a UNC5C-knockdown-induced enhancement of matrix metalloproteinase (MMP)3, MMP7, MMP9 and MMP10 expression via activation of the PI3K/AKT, ERK and p38 MAPK signaling pathways. Notably, UNC5C directly interacted with integrin α6, which is involved in the growth and metastasis of breast cancer cells. Additionally, UNC5C-knockdown enhanced the phosphorylation of FAK and SRC, which are key kinases in the netrin-1/Unc5C and netrin-1/integrin α6/β4 signaling pathways. This suggests that netrin-1 functions as an integrator for both the netrin-1/Unc5C and netrin-1/integrin α6/β4 signaling pathways. UNC5C-knockdown potentiated netrin-1/integrin α6/β4 signaling. Given that UNC5C-knockdown inhibited integrin-liked protein kinase phosphorylation at Thr-173, at least in SK-BR-3 cells, this may be an inhibitory phosphorylation site rather than activating phosphorylation site for relaying integrin signaling.

Kif2a Scales Meiotic Spindle Size in Hymenochirus boettgeri.

Size is a fundamental feature of biological systems that affects physiology at all levels. For example, the dynamic, microtubule-based spindle that mediates chromosome segregation scales to a wide range of cell sizes across different organisms and cell types. Xenopus frog species possess a variety of egg and meiotic spindle sizes, and differences in activities or levels of microtubule-associated proteins in the egg cytoplasm between Xenopus laevis and Xenopus tropicalis have been shown to account for spindle scaling [1]. Increased activity of the microtubule severing protein katanin scales the X.tropicalis spindle smaller compared to X.laevis [2], as do elevated levels of TPX2, a protein that enriches the cross-linking kinesin-5 motor Eg5 at spindle poles [3]. To examine the conservation of spindle scaling mechanisms more broadly across frog species, we have utilized the tiny, distantly related Pipid frog Hymenochirus boettgeri. We find that egg extracts from H.boettgeri form meiotic spindles similar in size to X.tropicalis but that TPX2 and katanin-mediated scaling is not conserved. Instead, the microtubule depolymerizing motor protein kif2a functions to modulate spindle size. H.boettgeri kif2a possesses an activating phosphorylation site that is absent from X.laevis. Comparison of katanin and kif2a phosphorylation sites across a variety of species revealed strong evolutionary conservation, with X.laevis and X.tropicalis possessing distinct and unique alterations. Our study highlights the diversity and complexity of spindle assembly and scaling mechanisms, indicating that there is more than one way to assemble a spindle of a particular size.

Phosphorylation-Dependent Inhibition of Akt1.

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

Hyperhomocysteinemia, Suppressed Immunity, and Altered Oxidative Metabolism Caused by Pathogenic Microbes in Atherosclerosis and Dementia.

Many pathogenic microorganisms have been demonstrated in atherosclerotic plaques and in cerebral plaques in dementia. Hyperhomocysteinemia, which is a risk factor for atherosclerosis and dementia, is caused by dysregulation of methionine metabolism secondary to deficiency of the allosteric regulator, adenosyl methionine. Deficiency of adenosyl methionine results from increased polyamine biosynthesis by infected host cells, causing increased activity of ornithine decarboxylase, decreased nitric oxide and peroxynitrate formation and impaired immune reactions. The down-regulation of oxidative phosphorylation that is observed in aging and dementia is attributed to deficiency of thioretinaco ozonide oxygen complexed with nicotinamide adenine dinucleotide and phosphate, which catalyzes oxidative phosphorylation. Adenosyl methionine biosynthesis is dependent upon thioretinaco ozonide and adenosine triphosphate (ATP), and the deficiency of adenosyl methionine and impaired immune function in aging are attributed to depletion of thioretinaco ozonide from mitochondrial membranes. Allyl sulfides and furanonaphthoquinones protect against oxidative stress and apoptosis by increasing the endogenous production of hydrogen sulfide and by inhibiting electron transfer to the active site of oxidative phosphorylation. Diallyl trisulfide and napabucasin inhibit the signaling by the signal transducer and activator of transcription 3 (Stat3), potentially enhancing immune function by effects on T helper lymphocytes and promotion of apoptosis. Homocysteine promotes endothelial dysfunction and apoptosis by the unfolded protein response and endoplasmic reticulum stress through activation of the N-methyl D-aspartate (NMDA) receptor, causing oxidative stress, calcium influx, apoptosis and endothelial dysfunction. The prevention of atherosclerosis and dementia may be accomplished by a proposed nutritional metabolic homocysteine-lowering protocol which enhances immunity and corrects the altered oxidative metabolism in atherosclerosis and dementia.

Abstract 204: Protective Roles of Eukaryotic Elongation Factor 2 Kinase on Rat Cardiomyoblast Death Under Glucose-deprived Condition

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K; also known as calmodulin-dependent protein kinase III) has both active and inactive phosphorylation sites. An intracellular energy sensor, AMP-activated protein kinase (AMPK) was reported to activate eEF2K via increasing dephosphorylation at Ser366 (inactive site). Activated eEF2K phosphorylates and inactivates a specific substrate, eEF2, which results in the inhibition of protein translation consuming high energy. Glucose depletion (GD) is one of the primary causes for cardiomyocyte death in the developed cardiac hypertrophy. We have recently found that the expression and dephosphorylation of eEF2K (Ser366) and eEF2 phosphorylation were significantly increased in left ventricle of several cardiac hypertrophy models. However, it is almost unknown whether eEF2K/eEF2 signals affect GD-induced cardiomyocyte death. The aim of this study was to explore it. GD was induced by incubating H9c2 cells in a glucose-free medium. H9c2 cell viability, apoptotic-like nuclear condensation or protein expression was examined using a cell counting assay, DAPI staining or Western blotting, respectively. GD induced H9c2 cell death (p<0.01, n=6) and caspase-3 fragmentation (p<0.05, n=10-12). In addition, GD significantly increased phosphorylation of AMPK (p<0.05, n=6-8) and eEF2 (p<0.01, n=4-8) as well as eEF2K dephosphorylation at Ser366 (p<0.01, n=4-8). eEF2K gene knockdown (eEF2K KD) by siRNA transfection significantly increased GD-induced H9c2 cell death (p<0.05, n=7) and caspase-3 fragmentation (p<0.01, n=9). Moreover, eEF2K KD significantly facilitated GD-induced increase of nuclear condensation (44.0±3.3%, eEF2K siRNA vs. 30.9±2.4%, control siRNA p<0.01, n=5). AMPK KD did not affect GD-induced H9c2 cell death and eEF2K dephosphorylation. In conclusion, we for the first time revealed in H9c2 cells that activated eEF2K might play protective roles in GD-induced apoptosis via the inhibition of caspase-3 fragmentation, whereas AMPK activation is not directly related to the regulation of eEF2K/eEF2 signals in GD condition. The present results suggest eEF2K as a novel pharmacotherapeutic target for cardiac dysfunction.

Backbone Assignment of Phosphorylated Cytoplasmic Domain B of Mannitol Transporter IIMtl in Thermoanaerobacter Tengcongensis

The cytoplasmic domains A and B of the mannitol transporter enzyme <TEX>$II^{Mtl}$</TEX> are covalently linked in Escherichia coli, but separately expressed in Thermoanaerobacter Tengcongensis. The phosphorylation of domain B (<TEX>$TtIIB^{Mtl}$</TEX>) substantially increases the binding affinity to the domain A (<TEX>$TtIIA^{Mtl}$</TEX>) in T. Tengcongensis. To understand the structural basis of the enhanced domain-domain interaction by protein phosphorylation, we obtained NMR backbone assignments of the phospho-<TEX>$TtIIB^{Mtl}$</TEX> using a standard suite of triple resonance experiments. Our results will be useful to monitor chemical shift changes at the active site of phosphorylation and the binding interfaces.

Homocysteine, Infections, Polyamines, Oxidative Metabolism, and the Pathogenesis of Dementia and Atherosclerosis.

Hyperhomocysteinemia is a risk factor for development of dementia and Alzheimer's disease (AD), and low blood levels of folate and cobalamin are associated with hyperhomocysteinemia and AD. In elderly subjects with cognitive decline, supplementation with folate, cobalamin, and pyridoxal demonstrated reduction of cerebral atrophy in gray matter regions vulnerable to the AD process. Multiple pathogenic microbes are implicated as pathogenic factors in AD and atherosclerosis, and the deposition of amyloid-β (Aβ), phosphorylation of tau protein, neuronal injury, and apoptosis in AD are secondary to microbial infection. Glucose utilization and blood flow are reduced in AD, and these changes are accompanied by downregulation of glucose transport, Na, K-ATPase, oxidative phosphorylation, and energy consumption. Thioretinaco ozonide, the complex formed from thioretinamide, cobalamin, ozone, and oxygen is proposed to constitute the active site of oxidative phosphorylation, catalyzing synthesis of adenosine triphosphate (ATP) from nicotinamide adenine dinucleotide (NAD+) and phosphate. Pathogenic microbes cause synthesis of polyamines in host cells by increasing the transfer of aminopropyl groups from adenosyl methionine to putrescine, resulting in depletion of intracellular adenosyl methionine concentrations in host cells. Depletion of adenosyl methionine causes dysregulation of methionine metabolism, hyperhomocysteinemia, reduced biosynthesis of thioretinamide and thioretinaco ozonide, decreased oxidative phosphorylation, decreased production of nitric oxide and peroxynitrite, and impaired host response to infectious microbes, contributing to the pathogenesis of dementia and atherosclerosis.

Abstract 196: The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members

Abstract Introduction: Phosphatase of regenerating liver 3 (PRL-3) is a dual specificity phosphatase and its up-regulation in cancer cells is related to poor prognosis. We have previously described PRL-3 as a downstream target of IL-6 in multiple myeloma (MM) by demonstrating that it was upregulated in response to this cytokine. The Src kinase family (SFK) is composed of 8 members and regulates proto-oncogenic cellular pathways. As it has been reported that the SFK members Lyn, Fyn and Hck are important in signal transduction of IL-6 signals in MM cells and that Src is reported to be the major kinase regulated by PRL-3 in colon cancer, we evaluated effects of PRL-3 on activation of Fyn, Lyn, Hck and Src in MM. Methods: By retroviral transduction in the IL-6-dependent MM cell line INA-6,we generated functional PRL-3(INA-PRL-3) overexpressing cells, and control cells expressing catalytically inactive mutant PRL-3 (C104S) or empty vector (Mock). We measured global tyrosine (Y) phosphorylation (P) by immunoblotting using (P-Y1000) antibody. We measured the expression of 4 SFK members by qRT PCR and immunoblotting. Activity of SFK members was evaluated using MILLIPLEX MAP8-plex Human SFK kit. We confirmed MILLIPLEX result for Src by immunoblotting with antibody against P-Y416 Src (activating phosphorylation site). We also measured Src activity after inhibiting PRL-3 by PRL-3 inhibitor I or shRNA. Finally, we showed the influence of PRL-3 on cell growth and cell sensitivity to two Src inhibitors (PP2 and Su6656) by using CellTiter-Glo Luminescent Cell Viability Assay. Results: INA-PRL-3 had more global P-Y than C104S and Mock cells in the absence of IL-6 and showed a pattern of P-Y more similar to that of the parental INA-6 cells grown in the presence of IL-6. C104S and INA-PRL-3 cells showed lowest and highest activity, respectively, of Lyn and Src. Stimulation of cells with IL-6 increased active Lyn and Src but still C104S had lowest activity. We did not observe significant difference in activity of Fyn and Hck. Measuring total amount of 4 SFK members showed up-regulation of total Hck and Fyn in C104S cells in mRNA and protein level. This indicated that inactive PRL-3 in C104S cells leads to less SFK activity compared to both INA-PRL-3 and Mock cells. Increased total amount of Hck and Fyn in C104S could be the result of diminished negative feedback regulation of expression of SFK members by their active forms. INA-PRL-3 showed more P-Y416 Src also by immunoblotting and its inhibition by PRL-3 inhibitor or shRNA decreased Src activity. INA-PRL-3 had higher growth rate than both C104S and Mock cells. This difference was more prominent in the absence of IL-6 and high SFK activity in INA-PRL-3 could be one of the possible reasons for this. INA-PRL-3 also showed high sensitivity to both PP2 and Su6656. Conclusion: Inhibition of PRL-3 and SFK members could be considered as a possible treatment for MM. Citation Format: Pegah Abdollahi, Esten Nymoen Vandsemb, Magnus Aassved Hjort, Kristine Misund, Toril Holien, Torstein Baade Rø, Tobias Schmidt Slørdahl, Magne Børset. The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members. [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 196.

Alternative splice variant of the thiazide-sensitive NaCl cotransporter: a novel player in renal salt handling.

The thiazide-sensitive NaCl cotransporter (NCC) is an important pharmacological target in the treatment of hypertension. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms. Only the third isoform has been extensively investigated. The aim of the present study was, therefore, to establish the abundance and localization of the almost identical isoforms 1 and 2 (NCC1/2) in the human kidney and to determine their functional properties and regulation in physiological conditions. Immunohistochemical analysis of NCC1/2 in the human kidney revealed that NCC1/2 localizes to the apical plasma membrane of the distal convoluted tubule. Importantly, NCC1/2 mRNA constitutes ∼ 44% of all NCC isoforms in the human kidney. Functional analysis performed in the Xenopus laevis oocyte revealed that thiazide-sensitive (22)Na(+) transport of NCC1 was significantly increased compared with NCC3. Mimicking a constitutively active phosphorylation site at residue 811 (S811D) in NCC1 further augmented Na(+) transport, while a nonphosphorylatable variant (S811A) of NCC1 prevented this enhanced response. Analysis of human urinary exosomes demonstrated that water loading in human subjects significantly reduces the abundance of NCC1/2 in urinary exosomes. The present study highlights that previously underrepresented NCC1/2 is a fully functional thiazide-sensitive NaCl-transporting protein. Being significantly expressed in the kidney, it may constitute a unique route of renal NaCl reabsorption and could, therefore, play an important role in blood pressure regulation.

Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran

Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA+ strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002–0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran.