In situ protein phosphorylation in hippocampal tissue slices.
We have studied the subcellular distribution of phosphoproteins in intact hippocampal slices and examined factors that regulate their phosphorylation and dephosphorylation in situ. The presence of Ca2+ in slice equilibration and prelabeling buffers and high-K+-induced depolarization markedly increased 32Pi incorporation into endogenous proteins. Ca2+-stimulatory effects were significantly reduced by Ca2+-channel blockers and the calmodulin antagonist W-13. Certain proteins were dephosphorylated in situ, and their dephosphorylation was dependent on both Ca2+ and depolarization. A number of proteins phosphorylated in situ was similar to those previously characterized in synaptic fractions phosphorylated in vitro. Many phosphoproteins were identified on the basis of molecular weight, isoelectric point, immunoreactivity, and phosphopeptide mapping; these included the 87 kDa substrate of protein kinase C, synapsin I, the 50 and 60 kDa subunits of Ca2+/calmodulin-dependent protein kinase II (CKII), tubulin, B-50, the alpha-subunit of pyruvate dehydrogenase and myelin basic proteins. CKII phosphorylation in situ appeared similar but not identical to its in vitro counterpart. Phosphopeptide mapping analysis of in situ labeled substrate proteins indicated that cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases were all active in slice preparations under basal conditions. Increased 32Pi labeling of hippocampal proteins following tissue depolarization appeared to be associated with increased activity of endogenous protein kinases since depolarization did not result in 32Pi-labeling of any new phosphoproteins.
Yip
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Department of Neurobiology and Anatomy, University of Texas Health Science Center, Houston 77225.
Kelly
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PT
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015180:Electrophoresis, Gel, Two-Dimensional; D004594:Electrophysiology; D006624:Hippocampus / Q000378:metabolism* / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011188:Potassium; D002118:Calcium