Calcium/Calmodulin-dependent Phosphorylation and Activation of Human Cdc25-C at the G2/M Phase Transition in HeLa Cells

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. 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. calmodulin calcium/calmodulin-dependent protein kinase II polyacrylamide gel electrophoresis okadaic acid phosphate-buffered saline 1,4-piperazinediethanesulfonic acid Calcium, an intracellular second messenger, is known to be required for cells to traverse the cell cycle checkpoints at G1/S, at entry into mitosis (G2/M phase), and at mitosis exit (reviewed in Refs. 1Whitaker M.J. Patel R. Development. 1990; 108: 525-542Crossref PubMed Google Scholar, 2Lu K.P. Means A.R. Endocr. 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J. 1993; PubMed Scopus Google Scholar). HeLa cells at the G1/S phase and phase cells the G1/S phase J. J. PubMed Scopus Google by the cells the with phosphate-buffered saline The cells at for at and the cell in of and of the or the of protein HeLa cell The by with of protein for at with The with a to the human protein or a at The by the with of protein for at and the in and okadaic The in of and the not on an gel in to of the M. G. J. 1993; PubMed Scopus Google Scholar). of as M. Patel R. Whitaker M. J. 1991; PubMed Scopus Google Scholar). the at a of for the and of A The for or of the an the in J. A The tyrosine phosphorylation of the p34cdc2 by with an The to the protein A to for the as M. Patel R. Whitaker M. J. 1991; PubMed Scopus Google Scholar). The level of p34cdc2 in the by with the p34cdc2 protein the of a of the protein K. 1990; PubMed Scopus Google Scholar). the in of and the by the of of and for at of and the for The with of and with for at The for at and at for The in of with of and on for of to the at for The in of The for at to and for at The to and into of at the for at The and of The protein and as in M. G. J. 1993; PubMed Scopus Google of by CaM kinase II in a of of and of of and of CaM kinase The at for of the by the of of of by the p34cdc2 protein kinase in a of of and of The at for by the of of of by protein kinase and protein kinase as J. Biol. Full Text PDF PubMed Google Scholar). The tyrosine phosphatase activity of or phosphorylation by CaM kinase II a B as M. Patel R. Whitaker M. J. 1991; PubMed Scopus Google Scholar). The with the of The for at with and for H1 kinase activity as L. R. J. L. J. 8: PubMed Scopus Google Scholar). H1 kinase activity by the histone H1 the in and or by the in in of and for to the tyrosine phosphorylation of The H1 kinase activity of the p34cdc2 HeLa cells as by J. J. PubMed Scopus Google Scholar). The and acid of phosphorylation by CaM kinase II as J. Biol. Full Text PDF PubMed Google Scholar). KN-93 as a in in and at KN-93 and to HeLa cells the at an the cells in the cells in or in of The cells in and in the for with and of the an cell acid in at a of acid to HeLa cells an The cells the and in and okadaic The cell in of HeLa cells on with a of The cells and G1/S phase as The peptide into the cells and the G1/S phase The cells the as by M. PubMed Scopus Google Scholar). The AC3-I peptide in and at a of of the cells, a into the peptide The and at a of cells in experiments with of peptide of cell as in Rev. PubMed Scopus Google Scholar). into the this of the cells as by microinjection the cells the on a a to a for the cells the and with in and cells a The and other of and The human protein and shown in A a be on a The with a to the human protein the tyrosine phosphatase activity of the we with the B to We have shown that at the p34cdc2 is phosphorylated on tyrosine and that B is in the M. Patel R. Whitaker M. J. 1991; PubMed Scopus Google Scholar, J. Patel R. Whitaker M.J. PubMed Scopus Google Scholar). shown in B a of p34cdc2 on the results of an K. M. 1991; PubMed Scopus Google Scholar). a for CaM kinase II in we the protein with of CaM kinase II The by a and by Our results in that a protein phosphorylated by CaM kinase II in a calcium/calmodulin-dependent to a of of In the of phosphorylation we the known of the and of CaM kinase II Curr. Cell Biol. 1993; PubMed Scopus Google Scholar). 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M. M. Cell. 1990; Full Text PDF PubMed Scopus Google its activity In the of the level of tyrosine phosphorylation of p34cdc2 with that of and not with the of KN-93 on the of we a to human to and cell cells and and phase The shown in that the a protein and that the protein in the KN-93-arrested cells at a level with that in the phase cells G1/S B shown for to that KN-93 not cell cycle by the of In cells, okadaic acid is known to activation of the p34cdc2 cells to into a by and K. J. K. M. J. 1990; PubMed Scopus Google Scholar). 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PubMed Scopus Google or a peptide in a for an In and cells with AC3-I or to a of of the cells, in as and the cells and the number of cells The results experiments shown in The AC3-I peptide the of HeLa cells in to as by with not and to We that the cells in into phase by the of that the of the cells In the cells with the peptide to as by a increase in cell number We the of AC3-I for CaM kinase II protein kinase CaM kinase and CaM kinase CaM kinase the as to be protein kinase protein as the or We have that AC3-I no on the activity of CaM kinase of to as or CaM kinase of to peptide as J. Biol. 1993; Full Text PDF PubMed Google CaM kinase II activity with an of as AC3-I a for CaM kinase II protein kinase or CaM kinase and at CaM kinase The results we have in this suggest that CaM kinase II or a CaM kinase activity is required at the phase in HeLa is with a requirement for CaM kinase II at mitosis entry in J. M. J. 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Cell. 1993; PubMed Scopus Google and activates in an of this that phosphorylation be to by of the of phosphorylation be to We that studies have cell is a for of PubMed Scopus Google is of the phosphorylated on the protein the as phosphorylated on the Our that II may be the that triggers the phosphorylation and activation of at mitosis entry in We for the B protein for the and the and for with for the human p34cdc2 for the for the protein and for the AC3-I inhibitor