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Reversible phosphorylation as a controlling factor for sustaining calcium oscillations in HeLa cells: Involvement of calmodulin-dependent kinase II and a calyculin A-inhibitable phosphatase.

Abstract：

:The role of reversible phosphorylation in histamine-induced Ca2+ oscillations in HeLa cells has been investigated by using various activators and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, which proved to be a useful and convenient tool. Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kinase II (CaMK II), only CaMK II was essential. When added during oscillations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca2+ spike before halting the process. Introduction of the Ca2+/calmodulin-independent catalytic domain of CaMK II into the cells forestalled their response to histamine. These results show that intracellular Ca2+ cannot oscillate when CaMK II is locked in either the inactive or the stimulated state. External Ca2+ electroporated into cells preloaded with the catalytic domains was quickly removed (but not when the cells were pretreated with the endoplasmic reticulum Ca(2+)-ATPase inhibitor, tapsigargin), indicating that the ATP-driven Ca2+ pump was somehow activated by CaMK II. Protein phosphatase inhibitors calyculin A and okadaic acid abolished ongoing oscillations and, when added at low concentrations, prolonged the interspike interval. Immunoprecipitation experiments with 32P(i)-labeled cells provided the first evidence that inositol 1,4,5-trisphosphate receptor (IP3R) was phosphorylated by CaMK II in vivo. The extent of phosphorylation was increased in the presence of histamine, significantly enhanced by calyculin A, and greatly reduced by W-7. Our observations are consistent with the concept that repetitive phosphorylation-dephosphorylation cycles regulating IP3R and Ca2+ pumps are a controlling factor for sustained Ca2+ oscillations in HeLa, and possibly other, cells.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Zhu DM,Tekle E,Chock PB,Huang CY

doi

10.1021/bi952471h

subject

Has Abstract

pub_date

1996-06-04 00:00:00

pages

7214-23

issue

eissn

0006-2960

issn

1520-4995

pii

bi952471h

journal_volume

pub_type

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Reversible phosphorylation as a controlling factor for sustaining calcium oscillations in HeLa cells: Involvement of calmodulin-dependent kinase II and a calyculin A-inhibitable phosphatase.