A synaptosomal protein kinase is regulated by phosphatidylinositol 4-phosphate and Mg2+.
Triton X-100 extracts of purified rat brain synaptosomes exhibited marked phosphorylation of an endogenous Mr 87,000 polypeptide following chromatography on DEAE-cellulose. The protein kinase catalyzing this reaction was insensitive to cyclic AMP, Ca2+, calmodulin, and phorbol esters. However, phosphatidylinositol 4-phosphate (PIP) proved to be a potent inhibitor of the Mr 87,000 polypeptide phosphorylation at submicromolar concentrations, whereas phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were less potent inhibitors. Unsaturated fatty acids could also mimic the effects of PIP at levels above 4 micrograms/ml. The inhibitory effect of PIP largely reflected a profound increase in the apparent Km for Mg2+ such that increasing Mg2+ levels could partially offset the action of PIP. The PIP-sensitive protein kinase was enriched in hypotonic lysates of synaptosomes from which it was partially purified by DEAE-cellulose, hydroxylapatite, and gel permeation chromatography. This purification separated the enzyme from its Mr 87,000 substrate; however, the presence of this polypeptide in heat-inactivated alkali extracts of rat brain provided an exogenous source of substrate which could be used to assay enzyme activity. The relevance of these data to a possible role for PIP and Mg2+ in cellular signaling is discussed.
Sahyoun
N E
NE
Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
McDonald
O B
OB
Misra
U K
UK
D000818:Animals; D001921:Brain / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D015636:Magnesium Chloride; D008970:Molecular Weight; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000494:pharmacology*; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology*
D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C037178:phosphatidylinositol 4-phosphate; D015636:Magnesium Chloride; D011494:Protein Kinases; D008274:Magnesium