Calcium/Calmodulin-dependent Phosphorylation and Activation of Human Cdc25-C at the G2/M Phase Transition in HeLa Cells*
- Rajnikant Patel,
- Mark Holt‡,
- Rada Philipova§,
- Stephen Moss¶,
- Howard Schulman‖,
- Hiroyoshi Hidaka** and
- Michael Whitaker§‡
- From the Department of Biochemistry, University of Leicester, University Road, Leicester, United Kingdom LE1 7RH, the§Department of Physiological Sciences. Medical School, Framlington Place, Newcastle-upon-Tyne, United Kingdom NE2 4HH, the¶Medical Research Council Laboratory of Molecular Cell Biology and the Department of Pharmacology, University College London, Gower Street, London, United Kingdom WC1E 6BT, the ‖Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5401, and the **Department of Pharmacology, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-Ku, Nagoya 466, Japan
Abstract
The human tyrosine phosphatase (p54cdc25-c) is activated by phosphorylation at mitosis entry. The phosphorylated p54cdc25-c in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54cdc25-c, we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54cdc25-c. We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54cdc25-c. Our in vitro experiments show that CaM kinase II can phosphorylate p54cdc25-c and increase its phosphatase activity by 2.5–3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2phase. In the KN-93-arrested cells, p54cdc25-c is not phosphorylated, p34cdc2 remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54cdc25-c.
Footnotes
-
↵* This work was supported by a Wellcome Trust Program grant (to M. J. W.) and by a Wellcome Trust fellowship (to R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ Present address: Dept. of Physiology, University College London, Gower St., London, United Kingdom WC1E 6BT.
-
↵‡ To whom correspondence should be addressed.
-
↵2 R. Patel, M. Holt, R. Philipova, S. Moss, H. Schulman, H. Hidaka, and M. Whitaker, unpublished data.
- Abbreviations:
- CaM
-
calmodulin
- CaM kinase II
-
calcium/calmodulin-dependent protein kinase II
- PAGE
-
polyacrylamide gel electrophoresis
- OA
-
okadaic acid
- PBS
-
phosphate-buffered saline
- Pipes
-
1,4-piperazinediethanesulfonic acid
-
- Received July 27, 1998.
- Revision received November 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
