Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase.
Phosphorylase b kinase (PhK) is a key enzyme involved in the conversion of glycogen to glucose in skeletal muscle and ultimately an increase in intracellular ATP. Since apoptosis is an ATP-dependent event, we investigated the regulation of skeletal muscle PhK during apoptosis. Incubation of PhK with purified caspase-3 in vitro resulted in the highly selective cleavage of the regulatory alpha subunit and resulted in a 2-fold increase in PhK activity. Edman protein sequencing of a stable 72 kD amino-terminal fragment and a 66 kD carboxy-terminal fragment revealed a specific caspase-3 cleavage site within the alpha subunit at residue 646 (DWMD G). Treatment of differentiated C2C12 mouse muscle myoblasts with the inducers of apoptosis staurosporine, TPEN, doxorubicin, or UV irradiation resulted in the disappearance of the alpha subunit of PhK as determined by immunoblotting, as well as a concurrent increase in caspase-3 activity. Moreover, induction of apoptosis by TPEN resulted in increased phosphorylase activity and sustained ATP levels throughout a 7 h time course. However, induction of apoptosis with staurosporine, also a potent PhK inhibitor, led to a rapid loss in phosphorylase activity and intracellular ATP, suggesting that PhK inhibition by staurosporine impairs the ability of apoptotic muscle cells to generate ATP. Thus, these studies indicate that PhK may be a substrate for caspase regulation during apoptosis and suggest that activation of this enzyme may be important for the generation of ATP during programmed cell death.
Hilder
Thomas L
TL
Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514-7365, USA.
Carlson
Gerald M
GM
Haystead
Timothy A J
TA
Krebs
Edwin G
EG
Graves
Lee M
LM
D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004317:Doxorubicin / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D051379:Mice; D018482:Muscle, Skeletal / Q000201:enzymology*; D032446:Myoblasts / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014466:Ultraviolet Rays
D004791:Enzyme Inhibitors; D005029:Ethylenediamines; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine