Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts.
AU - Lindsay, Howard D.
AU - Whitaker, Michael J.
AU - Ford, Christopher C.
PY - 1995/11
Y1 - 1995/11
N2 - Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translational modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.
AB - Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translational modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.
KW - BAPTA
KW - calcium
KW - cell division cycle
KW - cyclin
KW - CaM kinase
M3 - Journal article
VL - 108
SP - 3557
EP - 3568
JO - Journal of Cell Science
JF - Journal of Cell Science
SN - 0021-9533
IS - 11
ER -