Autophosphorylation of calmodulin-stimulated protein kinase II in intact synaptosomes.
The major phosphoproteins observed after lysis of synaptosomes and incubation in the presence of [gamma-32P]ATP and calmodulin are the autophosphorylated 50-kDa and 60-kDa subunits of calmodulin-stimulated protein kinase II (CMK II). However, when intact synaptosomes are preincubated with 32Pi, these subunits are hardly labeled even after depolarization. The aim of this study was to determine the extent to which methodological factors contribute to this discrepancy. The distribution of CMK II between the outside and the inside of synaptosomes was determined by incubating intact and lysed synaptosomes with [gamma-32P]ATP. Some 38% of the 50-kDa subunit was found on the inside of synaptosomes, and at this location it would be accessible to ATP generated within synaptosomes during the preincubation with 32Pi and could be autophosphorylated. The rest (62%) was on the outside of the synaptosomes, presumably associated with postsynaptic densities, where it could not be autophosphorylated. The effect of preincubation at 37 degrees C on CMK II autophosphorylation was determined by incubating intact synaptosomes for 45 min. This reduced calmodulin-stimulated autophosphorylation of the 50-kDa subunit in lysed synaptosomes by 38% and in intact synaptosomes by 29%. Thus, 9% of the 50-kDa autophosphorylation activity within synaptosomes was lost by thermal inactivation during preincubation. The extent of this loss of activity depended on the synaptosomal protein concentration during preincubation. CMK II activity against its major endogenous substrate synapsin I and an exogenous peptide substrate was also decreased by preincubation. The effect of the ionic environment on CMK II autophosphorylation was determined by incubating lysed synaptosomes with [gamma-32P]ATP in the absence or presence of ions at concentrations that mimic the extra or intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Dunkley
P R
PR
Faculty of Medicine, University of Newcastle, New South Wales, Australia.
Côtè
A
A
Harrison
S M
SM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D016704:Synapsins; D013574:Synaptosomes / Q000201:enzymology*; D013816:Thermodynamics
D002147:Calmodulin; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D010455:Peptides; D010710:Phosphates; D010750:Phosphoproteins; D016704:Synapsins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases