Multiple Classes of Sulfhydryls Modulate the Skeletal Muscle Ca2+ Release Channel

Aghdasi, Bahman; Zhang, Jia-Zheng; Wu, Yifan; Reid, Michael B.; Hamilton, Susan L.

doi:10.1074/jbc.272.6.3739

Cited by 87 publications

(106 citation statements)

References 35 publications

Supporting

Mentioning

100

Contrasting

Unclassified

Order By: Relevance

“…To understand the role of thiols in redox-signaling events related to cell survival/apoptosis, we have studied the effect of thiol alkylation on activation of Akt by PDGF-BB in VSMC. NEM has been used extensively as a specific thiol-alkylating agent (36,37). To determine thiol alkylation by NEM, growth-arrested VSMC were treated with and without 20 M NEM for 30 min, and free thiols were measured using 5,5Ј-dithiobis(2-dinitrobenzoic acid) reagent (35).…”

Section: Resultsmentioning

confidence: 99%

N-Ethylmaleimide Inhibits Platelet-derived Growth Factor BB-stimulated Akt Phosphorylation via Activation of Protein Phosphatase 2A

Yellaturu¹,

Bhanoori²,

Neeli³

et al. 2002

Journal of Biological Chemistry

125

View full text Add to dashboard Cite

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BBinduced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger Nacetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.The cellular redox state plays an important role in the regulation of gene expression in prokaryotes and eukaryotes (1-4). The following observations support this notion: 1) Oxidants regulate the activities of several transcription factors, including activator protein-1, nuclear factor kappa B, and p53 (5-7); 2) Oxidants are capable of activating several early response events, including stimulation of protein tyrosine phosphorylation, activation of mitogen-activated protein kinases and induction of expression of proto-oncogenes (8 -11); 3) Oxidants are produced acutely in response to various agents, including growth factors and cytokines in several cell types (12, 13), and a requirement for their production in the mitogenic effects of receptor tyrosine kinase and G protein-coupled receptor agonists has been demonstrated (14, 15); and 4) In addition to producing oxidants, cells also possess enzymatic and non-enzymatic mechanisms for their removal (16 -18), and this feature attests to the role of oxidants as second messenger molecules (19). Despite the growing body of information on the role of oxidants in the regulation of gene expression, the mechanisms by which these molecules transmit the extracellular signals from the plasma membrane to the nucleus are less clear. Thiols play a critical role in the reduction/oxidation reactions as w...

show abstract

Section: Resultsmentioning

confidence: 99%

N-Ethylmaleimide Inhibits Platelet-derived Growth Factor BB-stimulated Akt Phosphorylation via Activation of Protein Phosphatase 2A

Yellaturu¹,

Bhanoori²,

Neeli³

et al. 2002

Journal of Biological Chemistry

125

View full text Add to dashboard Cite

show abstract

“…The intrinsic Zn# + might be essential for function of RyRs. It has been shown that RyR1s can be modulated by multiple classes of thiol groups [18]. Thus, as another mechanism, Zn# + might have its effect through binding to thiol groups on RyR1s.…”

Section: Interaction Of the Effects Of Zn 2 + And Other Ryr Modulatorsmentioning

confidence: 99%

Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions

Xia

Cheng

Wang

et al. 2000

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…The identification of the cysteines involved in these modulations is difficult because RYR1 has 100 cysteines per subunit. However, the functionally important cysteines appear to be hyper-reactive (21)(22)(23). We have previously shown that the N terminus of RYR1 contains sulfhydryls that react rapidly with NEM (19).…”

Section: Cam Protects Both Cysteines Required For Oxidation-inducedmentioning

confidence: 99%

A Role for Cysteine 3635 of RYR1 in Redox Modulation and Calmodulin Binding

Moore

Zhang

Hamilton

1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit crosslink are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [ 3 H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.Reactive oxygen species and nitric oxide (NO) 1 are produced by skeletal muscle even at rest, but their levels are dramatically increased by muscle activity (1-5). Both reactive oxygen species and NO alter muscle function (6), possibly by altering excitation-contraction coupling (7). One of the proteins involved in excitation-contraction coupling, the skeletal muscle Ca 2ϩ -release channel, is modulated by both oxidants (8 -10) and NO (6,11). This regulation appears to be controlled, at least in part, by the binding of calmodulin (CaM) to RYR1 (12, 13). Ca 2ϩ -free CaM is a partial agonist of RYR1, whereas Ca 2ϩ -CaM is an inhibitor (14). In addition to these direct functional effects, CaM bound to RYR1 protects the channel from oxidation-induced intersubunit cross-linking (12), and conversely, oxidation can block CaM binding to RYR1. In contrast to the effects of oxidation, alkylation of RYR1 with NEM rapidly destroys the ability of RYR1 to bind 35 S-Ca 2ϩ -free CaM (apoCaM) but does not alter its ability to bind 35 S-Ca 2ϩ -CaM. Alkylation also prevents oxidation-induced intersubunit crosslinking. Our studies suggest that there is only one CaM site per subunit of RYR1 at either high or low Ca 2ϩ (13). Some indication of the location of the CaM binding site has been obtained from examination of sites on RYR1 protected from tryptic cleavage by CaM (13). Treatment of RYR1 with trypsin rapidly destroys its ability to bind CaM, but CaM bound to RYR1 can protect its binding site from tryptic digestion. The sites protected by Ca 2ϩ -CaM or apoCaM are at amino acids 3630 and 3637, suggesting that either the binding sites for CaM in both the Ca 2ϩ -free and Ca 2ϩ -bound forms are physically close to this region of RYR1 or that the binding of both forms of CaM produces a conformational change that buries this region of RYR1. The latter possibility seems unlikely because the functional effects of apoCaM are opposite those of Ca 2ϩ -CaM. The sequence between these two sites is AVVACFR, suggesting that cysteine 3635 may be one of the cysteines that, in the absence of CaM, can form the intersubunit disulfide bond. This intersubunit contact site is likely to represent an important site for regulating RYR1 activity. In the current study, we demonstrate that cysteine 3635 is one...

show abstract

Multiple Classes of Sulfhydryls Modulate the Skeletal Muscle Ca2+ Release Channel

Cited by 87 publications

References 35 publications

N-Ethylmaleimide Inhibits Platelet-derived Growth Factor BB-stimulated Akt Phosphorylation via Activation of Protein Phosphatase 2A

N-Ethylmaleimide Inhibits Platelet-derived Growth Factor BB-stimulated Akt Phosphorylation via Activation of Protein Phosphatase 2A

Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions

A Role for Cysteine 3635 of RYR1 in Redox Modulation and Calmodulin Binding

Contact Info

Product

Resources

About