Inactivation and subcellular redistribution of Ca2+/calmodulin-dependent protein kinase II following spinal cord ischemia.
Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II alpha and beta subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the alpha and beta subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (< or = 25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.
Department of Neurosciences, University of California, San Diego, La Jolla.
D A
DA
Shackelford
R Y
RY
Yeh
J A
JA
Zivin
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003600:Cytosol / Q000201:enzymology; D015151:Immunoblotting; D007511:Ischemia / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011489:Protein Denaturation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D015424:Reperfusion; D013116:Spinal Cord / Q000098:blood supply*
D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases