Activation of Akt during Simulated Ischemia/Reperfusion in Cardiac Myocytes

doi:10.1006/bbrc.2000.2522

Biochemical and Biophysical Research Communications

Volume 270, Issue 3, 21 April 2000, Pages 947-952

https://doi.org/10.1006/bbrc.2000.2522 Get rights and content

Abstract

In the present study we have investigated whether Akt was activated during simulated ischemia (SI) and simulated ischemia/reperfusion (SI/R) in neonatal rat cardiomyocytes. Akt was phosphorylated on both S473 and T308 residues after 10 min of simulated SI/R and remained elevated for 60 min before returning to basal levels after 2 h. No phosphorylation was observed during SI alone. SI/R-stimulated Akt activation was inhibited by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, the tyrosine kinase inhibitor genistein and the Src tyrosine kinase inhibitor PP2, indicating a requirement for tyrosine kinase activity in Akt activation. Furthermore, SB203580, a p38 MAPK inhibitor, partially inhibited Akt activation. SI/R also induced the phosphorylation of PHAS-I, a downstream Akt target, in a wortmannin-dependent manner. These results demonstrate for the first time that SI/R stimulates Akt activation via PI3-K-and Src tyrosine kinase-dependent pathways, whereas p38 MAPK appears to be involved in maintaining Akt activation.

References (32)

Y. Seko et al.
Hypoxia and hypoxia/reoxygenation activate p65^PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes
Biochem. Biophys. Res. Commun.
(1997)
Y. Seko et al.
Hypoxia and hypoxia/reoxygenation activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes
Biochem. Biophys. Res. Commun.
(1996)
Y. Seko et al.
Hypoxia induces activation and subcellular translocation of focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes
Biochem. Biophys. Res. Commun.
(1999)
H. Konishi et al.
Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27
FEBS Lett.
(1997)
M. Takata et al.
Requirement for Akt (protein kinase B) in insulin-induced activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-I)
J. Biol. Chem.
(1999)
S.R. Datta et al.
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery
Cell
(1997)
G. Rena et al.
Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B
J. Biol. Chem.
(1999)
T.F. Franke et al.
The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase
Cell
(1995)
D.R. Alessi et al.
Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase
FEBS Lett.
(1996)
R.P. Doornbos et al.
Protein kinase C zeta is a negative regulator of protein kinase B activity
J. Biol. Chem.
(1999)

J.C. Bode et al.

The mitogen activated protein (MAP) kinase p38 and its upstream activator MAPK kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity

J. Biol. Chem.

(1999)

R.E. Rhoads

Signal transduction pathways that regulate eukaryotic protein synthesis

J. Biol. Chem.

(1999)

M.A. Bogoyevitch et al.

Stimulation of the stress-activated mitogen activated protein kinase subfamilies in perfused heart

Circ. Res.

(1996)

Y. Seko et al.

Hypoxia and hypoxia/reoxygenation activate Raf-1, mitogen-activated protein kinase kinase, mitogen-activated protein kinase, and S6 kinase in cultured rat cardiac myocytes

Circ. Res.

(1996)

Y. Mizukami et al.

Mitogen-activated protein kinase translocates to the nucleus during ischemia and is activated during reperfusion

Biochem. J.

(1997)

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Regular ArticleActivation of Akt during Simulated Ischemia/Reperfusion in Cardiac Myocytes

Abstract

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Stimulation of the stress-activated mitogen activated protein kinase subfamilies in perfused heart

Circ. Res.

Hypoxia and hypoxia/reoxygenation activate Raf-1, mitogen-activated protein kinase kinase, mitogen-activated protein kinase, and S6 kinase in cultured rat cardiac myocytes

Circ. Res.

Mitogen-activated protein kinase translocates to the nucleus during ischemia and is activated during reperfusion

Biochem. J.

Regular Article
Activation of Akt during Simulated Ischemia/Reperfusion in Cardiac Myocytes