Orthovanadate stimulates cyclic guanosine monophosphate-inhibited cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads through activation of particulate myelin basic protein kinase by protein tyrosine kinase.
Involvement of protein kinases in the stimulation of cGMP-inhibited cAMP phosphodiesterase (PDE) activity by orthovanadate (vanadate) was studied. When the fat pads were incubated with 2 mM vanadate or 10 nM insulin, the stimulation of myelin basic protein kinase (MBPK) activity in the particulate by vanadate reached a maximum at 60 min. In contrast, insulin showed a transient increase at 20 min. A 60-min incubation of the fat pads with vanadate stimulated all activities of protein tyrosine kinase (PTK), MBPK, and PDE in the particulate, in a similar dose-dependent manner. Amiloride, a PTK inhibitor, inhibited the stimulations of three enzymes by vanadate in a similar concentration range. Enzyme fractions, which were separated from the solubilized particulate, were subjected to the immunoblot analysis. A fraction of MBPK was identified to contain a major protein of mol wt (44K) and a minor one (42K), both of which are immunoreactive with a mitogen-activated protein kinase (MAPK) antibody. The partially purified PDE activity was stimulated by the addition of the partially purified MBPK. The further stimulation was observed with the PTK-activated MBPK. These results suggest that vanadate stimulates in part the PDE activity through the activation of the particulate MBPK, probably MAPKs, by PTK sensitive to vanadate.
Ueki
H
H
Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
Mitsugi
S
S
Kawashima
Y
Y
Motoyashiki
T
T
Morita
T
T
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D006152:Cyclic GMP / Q000494:pharmacology*; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D005994:Glycerophosphates / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D008297:Male; D008954:Models, Biological; D017680:Potassium Compounds / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014638:Vanadates / Q000494:pharmacology*
D005994:Glycerophosphates; D017680:Potassium Compounds; D014638:Vanadates; C033320:potassium fluoride; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D005459:Fluorides; C031463:beta-glycerophosphoric acid