Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10814964 | Cellular Signalling | 2014 | 9 Pages |
Abstract
Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase-anaphase transition.
Keywords
PP4PBSPP2APP1SDSFCSpp6CaMKIIEGTAPVDFPAGEDAPIshRNADMSOSmall interfering RNAshort hairpin RNAsiRNAAspartic acidEDTAethylene glycol tetraacetic acidEthylenediaminetetraacetic acidpolyacrylamide gel electrophoresisempty vectorANOVAone-way analysis of variancethreoninestandard error of the meanDimethyl sulfoxidesodium dodecyl sulphatefoetal calf serumPhosphate buffered salineProtein phosphorylationMetaphaseSEMwild-typeValineprotein phosphatase 1protein phosphatase 2AProtein phosphatase 4Protein phosphatase 6Polyvinylidene fluorideCell cycle
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Authors
Alexander Hoffman, Helen Carpenter, Richard Kahl, Lauren F. Watt, Phillip W. Dickson, John A.P. Rostas, Nicole M. Verrills, Kathryn A. Skelding,