Alteration in Phosphorylation of P20 Is Associated With Insulin Resistance

Wang, Yu; Xu, Aimin; Ye, Ji-Ming; Kraegen, Edward W.; Tse, Cynthia A.; Cooper, Garth J. S.

doi:10.2337/diabetes.50.8.1821

Cited by 28 publications

(25 citation statements)

References 48 publications

(52 reference statements)

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“…11,16,20 Thus, the observed protective effects of Hsp20 on myocyte apoptosis could be mediated by either Hsp20 or its phosphorylated form. To determine the phosphorylation status of Hsp20 in Ad.Hsp20-infected myocytes before and after ISO treatment for 24 hours, we used 2-dimensional gel electrophoresis and LC-MS/MS analysis.…”

Section: Overexpressed Hsp20 Protein Is Partly Phosphorylated At Ser1mentioning

confidence: 99%

“…Hsp20 has initially been isolated from skeletal muscle in mixed complexes with ␣B-crystallin and Hsp25, which implies that all 3 proteins might be involved in similar processes, at least in skeletal muscle, and probably also in heart. 10 It has also been reported that Hsp20 is expressed in cardiac muscle, stomach, intestine, bladder, and blood, 10,11 and it is the only small heat-shock protein that contains a cGMP/cAMP-dependent protein kinase (PKG/PKA) consensus phosphorylation site (RRAS). 10 Stable overexpression of Hsp20 in Chinese hamster ovary (CHO) cells resulted in enhanced survival, after a heat shock, similar to ␣B-crystallin.…”

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confidence: 99%

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Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits β-Agonist-Induced Cardiac Apoptosis

Fan

Chu

Mitton

et al. 2004

Circulation Research

112

144

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Abstract-Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained ␤-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to ␤-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against ␤-agonist-induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.

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Section: Overexpressed Hsp20 Protein Is Partly Phosphorylated At Ser1mentioning

confidence: 99%

mentioning

confidence: 99%

Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits β-Agonist-Induced Cardiac Apoptosis

Fan

Chu

Mitton

et al. 2004

Circulation Research

112

144

View full text Add to dashboard Cite

show abstract

“…Sequence analysis revealed high amino acid similarity, between the mouse cardiac p20 and the human and rat slow-twitch skeletal muscle p20, 15,16 which is a heat shock-related protein and can be phosphorylated by the cAMP-PKA pathway. 17 Interestingly, phosphorylation of p20 was also induced by insulin in skeletal muscle, 18 which might be associated with insulin resistance. It is noteworthy that adenovirus-mediated overexpression of the cardiac p20 in adult rat cardiomyocytes increased cell contractility and intracellular Ca 2ϩ transient amplitudes, indicating that p20 is involved in the regulation of myocardial contractility.…”

Section: Heat Shock Proteinsmentioning

confidence: 99%

Phosphoproteome Analysis of Cardiomyocytes Subjected to β-Adrenergic Stimulation

Chu

Egnaczyk

Zhao

et al. 2004

Circulation Research

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Abstract-Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac ␤-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32 P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 mol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190Ϯ23%) and succinyl CoA synthetase (160Ϯ16%), whereas the phosphorylation of pyruvate dehydrogenase (48Ϯ10%), NADH-ubiquinone oxidoreductase (46Ϯ6%), heat shock protein 27 (18Ϯ3%), ␣B-crystallin (20Ϯ3%), and an unidentified 26-kDa protein (29Ϯ7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (Ϸ20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser 16 in p20 on ␤-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to ␤-adrenergic signaling. ␤-Adrenergic receptors and their signaling effectors are key mediators of neurohormonal influence over the cardiovascular system, modulating the strength, velocity, and frequency of cardiac contraction and relaxation. 1 At the subcellular level, the cardiac responses to ␤-adrenergic agonists are associated with phosphorylation of several downstream target phosphoproteins. Multiple studies have indicated that the most critical substrates for the positive inotropic and lusitropic ␤-agonist effects are phospholamban in the sarcoplasmic reticulum (SR) and troponin I (TnI) in the myofilaments, both phosphorylated through the cAMPdependent protein kinase A (PKA) pathway. 2-4 Phosphorylation of phospholamban relieves its inhibitory effects on SERCA2, with subsequent acceleration of SR Ca 2ϩ transport, and phosphorylation of TnI reduces the myofilament sensiti...

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“…Two-dimensional Gel Electrophoresis, Immunoblotting, and Carbohydrate Detection-The proteins secreted by either adipocytes or 3T3-L1 preadipocytes were separated by two-dimensional gel electrophoresis as described previously (24). The separated proteins were stained with either silver or Coomassie Brilliant Blue R-250.…”

Section: Differentiation Of 3t3-l1 Cells and Concentration Of Proteinmentioning

confidence: 99%

Hydroxylation and Glycosylation of the Four Conserved Lysine Residues in the Collagenous Domain of Adiponectin

Wang¹,

Xu²,

Knight

et al. 2002

Journal of Biological Chemistry

Self Cite

319

272

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It has recently been shown that the fat-derived hormone adiponectin has the ability to decrease hyperglycemia and to reverse insulin resistance. However, bacterially produced full-length adiponectin is functionally inactive. Here, we show that endogenous adiponectin secreted by adipocytes is post-translationally modified into eight different isoforms, as shown by two-dimensional gel electrophoresis. Carbohydrate detection revealed that six of the adiponectin isoforms are glycosylated. The glycosylation sites were mapped to several lysines (residues 68, 71, 80, and 104) located in the collagenous domain of adiponectin, each having the surrounding motif of GXKGE(D). These four lysines were found to be hydroxylated and subsequently glycosylated. The glycosides attached to each of these four hydroxylated lysines are possibly glucosylgalactosyl groups. Functional analysis revealed that full-length adiponectin produced by mammalian cells is much more potent than bacterially generated adiponectin in enhancing the ability of subphysiological concentrations of insulin to inhibit gluconeogenesis in primary rat hepatocytes, whereas this insulin-sensitizing ability was significantly attenuated when the four glycosylated lysines were substituted with arginines. These results indicate that full-length adiponectin produced by mammalian cells is functionally active as an insulin sensitizer and that hydroxylation and glycosylation of the four lysines in the collagenous domain might contribute to this activity.In addition to serving as an energy storage depot for triglycerides, adipose tissue is now recognized as an active endocrine organ that can secret a variety of biologically active molecules (adipocytokines) in response to extracellular signals (1-4). Some of these adipocytokines, such as leptin, tumor necrosis factor-␣, and resistin, have been shown to play critical roles in the regulation of systemic energy homeostasis, and altered expression and/or secretion of these adipocytokines may contribute to the causation of insulin resistance, type II diabetes, and its complications such as cardiovascular diseases.Adiponectin (also called ACRP30, adipoQ, and GBP28) is a protein exclusively secreted by adipocytes and was originally cloned by four research groups using different approaches (5-8). Several recent studies suggest that adiponectin might be a critical, long sought after hormone that links obesity, insulin resistance, and type II diabetes (9 -11). The adiponectin gene is located in chromosome 3q27, a susceptibility locus for type II diabetes and related metabolic syndromes (12-14). Circulating adiponectin is abundant in humans as well as rodents, with plasma circulating levels in the microgram/ml range, accounting for ϳ0.01% of the total plasma protein (15, 16). mRNA expression and the secretion level of adiponectin are dramatically decreased in a variety of animal models of insulin resistance as well as in obese humans and type II diabetic patients from different ethnic groups (15)(16)(17)(18)(19)(20). Notably, treatmen...

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Alteration in Phosphorylation of P20 Is Associated With Insulin Resistance

Cited by 28 publications

References 48 publications

Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits β-Agonist-Induced Cardiac Apoptosis

Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits β-Agonist-Induced Cardiac Apoptosis

Phosphoproteome Analysis of Cardiomyocytes Subjected to β-Adrenergic Stimulation

Hydroxylation and Glycosylation of the Four Conserved Lysine Residues in the Collagenous Domain of Adiponectin

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