The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase.
Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.
J A
JA
Rostas
V A
VA
Brent
J W
JW
Heath
R L
RL
Neame
D A
DA
Powis
R P
RP
Weinberger
P R
PR
Dunkley
D000255:Adenosine Triphosphate; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002469:Cell Separation; D002540:Cerebral Cortex / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology*; D013574:Synaptosomes / Q000201:enzymology* / Q000648:ultrastructure
D002147:Calmodulin; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D011494:Protein Kinases