A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulin-dependent protein kinase II phosphorylation.
D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.
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Institute of Interdisciplinary Research, Free University of Brussels, Campus Erasme, Bldg. C, 808 Route de Lennik, B-1070 Brussels, Belgium. dcommuni@ulb.ac.be
Gevaert
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Demol
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Vandekerckhove
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Erneux
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D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000819:agonists; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D013058:Mass Spectrometry; D019319:Okadaic Acid / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology; D018909:src Homology Domains; D000099182:Phosphoinositide 5-Phosphatases
D002118:Calcium; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018076:DNA, Complementary; D000072181:Inositol Polyphosphate 5-Phosphatases; D019319:Okadaic Acid; D010455:Peptides; D010744:Phosphoric Monoester Hydrolases; D011994:Recombinant Proteins; D014544:Uridine Triphosphate; D000099182:Phosphoinositide 5-Phosphatases