scholarly journals Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

2009 ◽  
Vol 418 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Bei You ◽  
Guijun Yan ◽  
Zhiling Zhang ◽  
Lin Yan ◽  
Jing Li ◽  
...  
Keyword(s):  
Troponin I ◽  
Molecular Conformation ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Binding Assays ◽  
Interacting Protein ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
293 Cells

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143. Substitution of Thr31 with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr51, Thr129 or Thr143 with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr31. Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.

scholarly journals Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

2007 ◽  
Vol 405 (3) ◽  
pp. 591-596 ◽  
Author(s):  
Jun-ichi Saito ◽  
Shinnosuke Toriumi ◽  
Kenjiro Awano ◽  
Hidenori Ichijo ◽  
Keiichi Sasaki ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Feedback Regulation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Regulation Mechanism ◽  
H2o2 Treatment ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
293 Cells

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cϵ mutant enhanced AP-1 activity. Exogenous PP2Cϵ associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr845in vitro. In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cϵ maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cϵ and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

scholarly journals Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Yoko Nakano ◽  
Botond Banfi ◽  
Algirdas J. Jesaitis ◽  
Mary C. Dinauer ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Targeting ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Regulatory Subunits ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz–Jeghers cancer syndrome

2002 ◽  
Vol 362 (2) ◽  
pp. 481-490 ◽  
Author(s):  
Gopal P. SAPKOTA ◽  
Jérôme BOUDEAU ◽  
Maria DEAK ◽  
Agnieszka KIELOCH ◽  
Nick MORRICE ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Wild Type ◽  
Cancer Syndrome ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Increased Risk ◽  
In Cells

Peutz—Jeghers syndrome is an inherited cancer syndrome, which results in a greatly increased risk of developing tumours in those affected. The causative gene encodes a nuclear-localized protein kinase, termed LKB1, which is predicted to function as a tumour suppressor. The mechanism by which LKB1 is regulated in cells is not known, and nor have any of its physiological substrates been identified. Recent studies have demonstrated that LKB1 is phosphorylated in cells. As a first step towards identifying the roles that phosphorylation of LKB1 play, we have mapped the residues that are phosphorylated in human embryonic kidney (HEK)-293 cells, as well as the major in vitro autophosphorylation sites. We demonstrate that LKB1 expressed in HEK-293 cells, in addition to being phosphorylated at Ser431, a previously characterized phosphorylation site, is also phosphorylated at Ser31, Ser325 and Thr366. Incubation of wild-type LKB1, but not a catalytically inactive mutant, with manganese-ATP in vitro resulted in the phosphorylation of LKB1 at Thr336 as well as at Thr366. We were unable to detect autophosphorylation at Thr189, a site previously claimed to be an LKB1 autophosphorylation site. A catalytically inactive mutant of LKB1 was phosphorylated at Ser31 and Ser325 in HEK-293 cells to the same extent as the wild-type enzyme, indicating that LKB1 does not phosphorylate itself at these residues. We show that phosphorylation of LKB1 does not directly affect its nuclear localization or its catalytic activity in vitro, but that its phosphorylation at Thr336, and perhaps to a lesser extent at Thr366, inhibits LKB1 from suppressing cell growth.

Abstract 1573: Phosphorylation of Cardiac Troponin I by Mammalian Sterile20-like Kinase Mst1

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bei You ◽  
Guijun Yan ◽  
Zhiling Zhang ◽  
Jian-Ping Jin ◽  
Jianxin Sun
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Molecular Conformation ◽  
Phosphorylation Site ◽  
Cardiac Troponin I ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Free Form ◽  
Spectrometric Analysis ◽  
Troponin Complex

Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of human heart cDNA library with a dominant negative Mst1 (K59R) as bait, cardiac troponin I (cTnI) was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiac myocytes, in which cTnI interacted with full length Mst1, but not with its N-terminal kinase fragment. In vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1 in its free form or in reconstituted troponin complex. In contrast, cardiac TnT was phosphorylated by Mst1 only when incorporated in the troponin complex. Mass spectrometric analysis indicated that Mst1 phosphorylates cTnI at Thr 31 , Thr 51 , Thr 129 , and Thr 143 . Substitution of Thr 31 with Ala substantially reduced Mst1-mediated cTnI phosphorylation by approximately 90%, while replacement of either Thr 51 orThr 129 , or Thr 143 with Ala reduced Mst1-catalyzed cTnI phosphorylation by approximately 60%, suggesting that Thr 31 is a preferential phosphorylation site for Mst1. Protein epitope analysis and binding assays showed that Mst1 mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. Our results suggest that Mst1 is a novel mediator of cTnI and/or cTnT phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions. This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

2013 ◽  
Vol 305 (7) ◽  
pp. F943-F956 ◽  
Author(s):  
Rodrigo Alzamora ◽  
Mohammad M. Al-Bataineh ◽  
Wen Liu ◽  
Fan Gong ◽  
Hui Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Phosphorylation Site ◽  
Intercalated Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
Amp Activated Protein Kinase

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.

scholarly journals Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1131 ◽  
Author(s):  
Federico Di Marco ◽  
Francesco Trevisani ◽  
Pamela Vignolini ◽  
Silvia Urciuoli ◽  
Andrea Salonia ◽  
...  
Keyword(s):  
Indoleacetic Acid ◽  
Antioxidant Properties ◽  
Antioxidant Compounds ◽  
Kidney Cells ◽  
Hek 293 ◽  
293 Cells ◽  
High Concentrations ◽  
Β Carotene ◽  
Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

scholarly journals Restoration of Silencing in Saccharomyces cerevisiae by Tethering of a Novel Sir2-Interacting Protein, Esc8

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore
Keyword(s):  
Protein A ◽  
Dominant Negative ◽  
Class I ◽  
Dominant Negative Effect ◽  
Rna Pol Ii ◽  
Interacting Protein ◽  
Nucleosome Remodeling ◽  
Negative Effect ◽  
Close Homolog

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

2009 ◽  
Vol 297 (2) ◽  
pp. G361-G370 ◽  
Author(s):  
Eikichi Ihara ◽  
Lori Moffat ◽  
Meredith A. Borman ◽  
Jennifer E. Amon ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Conformational Changes ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

scholarly journals Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

2007 ◽  
Vol 406 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Nobuhito Ono ◽  
Ingrid Van der Heijden ◽  
George L. Scheffer ◽  
Koen Van de Wetering ◽  
Elizabeth Van Deemter ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kidney Cells ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Drug Conjugates ◽  
293 Cells ◽  
Boar Sperm ◽  
Alternatively Spliced

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

scholarly journals Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

1999 ◽  
Vol 19 (10) ◽  
pp. 6500-6508 ◽  
Author(s):  
Nanette J. Pazdernik ◽  
David B. Donner ◽  
Mark G. Goebl ◽  
Maureen A. Harrington
Keyword(s):  
Sequence Similarity ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Death Domain ◽  
Interacting Protein ◽  
C Terminus ◽  
Catalytically Active ◽  
Induced Apoptosis

ABSTRACT The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-κB, events critical for proper immune system function. A screen for upstream activators of NF-κB identified a novel serine-threonine kinase capable of activating NF-κB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-κB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-κB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-κB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

Sign in / Sign up

Export Citation Format

Share Document