Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na+/K+-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

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Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A

Journal of Applied Physiology ◽

10.1152/jappl.1997.82.1.219 ◽

1997 ◽

Vol 82 (1) ◽

pp. 219-225 ◽

Cited By ~ 124

Author(s):

W. W. Winder ◽

H. A. Wilson ◽

D. G. Hardie ◽

B. B. Rasmussen ◽

C. A. Hutber ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Maximal Velocity ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Rat Muscle ◽

Amp Activated Protein Kinase ◽

Kinase A

Winder, W. W., H. A. Wilson, D. G. Hardie, B. B. Rasmussen, C. A. Hutber, G. B. Call, R. D. Clayton, L. M. Conley, S. Yoon, and B. Zhou. Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J. Appl. Physiol. 82(1): 219–225, 1997—This study was designed to compare functional effects of phosphorylation of muscle acetyl-CoA carboxylase (ACC) by adenosine 3′,5′-cyclic monophosphate-dependent protein kinase (PKA) and by AMP-activated protein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Functional effects of phosphorylation were determined by measuring ACC activity at different concentrations of each of the substrates and of citrate, an activator of the enzyme. The maximal velocity ( V max) and the Michaelis constants ( K m) for ATP, acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA. Phosphorylation by AMPK increased the K m for ATP and acetyl-CoA. Sequential phosphorylation by PKA and AMPK, first without label and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant ( K a) for citrate activation was increased to the same extent by AMPK phosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with no apparent functional effects on the enzyme. AMPK appears to be the more important regulator of muscle ACC.

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