Asymmetric dimethylarginine stimulates Akt1 phosphorylation via heat shock protein 70-facilitated carboxyl-terminal modulator protein degradation in pulmonary arterial endothelial cells

Xutong Sun, Manuela Kellner, Ankit Desai, Ting Wang, Qing Lu, Archana Kangath, Ning Qu, Christina Klinger, Sohrab Fratz, Jason Yuan, Jeffrey R. Jacobson, Joe GN Garcia, Ruslan Rafikov, Jeffrey R. Fineman, Stephen M. Black

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Abstract

Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominantnegative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxylterminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMPprotein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMPlevelsmay be an avenue to treat pulmonary hypertension.

Original languageEnglish (US)
Pages (from-to)275-287
Number of pages13
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume55
Issue number2
DOIs
Publication statusPublished - Aug 1 2016

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Keywords

  • Asymmetric dimethylarginine; Akt1; mitochondrion; proteasome

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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