A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulindependent protein kinase II phosphorylation
D. Communi et al., A novel receptor-mediated regulation mechanism of type I inositol polyphosphate 5-phosphatase by calcium/calmodulindependent protein kinase II phosphorylation, J BIOL CHEM, 276(42), 2001, pp. 38738-38747
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 ty
pe I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensi
tive inhibition by 70-85% of Ins(1,4,5)P-3 and Ins(1,3,4,5)P4 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 t
ype I 5-phosphatase. Immunodetection as well as mass spectrometric peptide
mass fingerprinting and post-source decay (PSD) sequence data analysis afte
r immunoprecipitation permitted unambiguous identification of the major nat
ive 5-phosphatase isoform hydrolyzing Ins(1,4,5)P-3 and Ins(1,3,4,5)P4 as t
ype I inositol polyphosphate 5-phosphatase. In ortho P-32-preincubated cell
s, the phosphorylated 43 kDa-enzyme could be identified after receptor acti
vation by immunoprecipitation followed by electrophoretic separation. Phosp
horylation of type I 5-phosphatase was blocked after cell preincubation in
the presence of Ca2+/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62)
. In vitro phosphorylation of recombinant type I enzyme by Ca2+/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 mech
anism of type I 5-phosphatase by phosphorylation in intact cells.