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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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated., (2010 Elsevier Inc. All rights reserved.)

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is controlled by PACAP, acutely by phosphorylation at Ser40 and chronically by protein synthesis. Using bovine adrenal chromaffin cells we found that PACAP, acting via the continuous activation of PACAP 1 receptors, sustained the phosphorylation of TH at Ser40 and led to TH activation for up to 24 h in the absence of TH protein synthesis. The sustained phosphorylation of TH at Ser40 was not mediated by hierarchical phosphorylation of TH at either Ser19 or Ser31. PACAP caused sustained activation of PKA, but did not sustain activation of other protein kinases including ERK, p38 kinase, PKC, MAPKAPK2 and MSK1. The PKA inhibitor H89 substantially inhibited the acute and the sustained phosphorylation of TH mediated by PACAP. PACAP also inhibited the activity of PP2A and PP2C at 24 h. PACAP therefore sustained TH phosphorylation at Ser40 for 24 h by sustaining the activation of PKA and causing inactivation of Ser40 phosphatases. The PKA activator 8-CPT-6Phe-cAMP also caused sustained phosphorylation of TH at Ser40 that was inhibited by the PKA inhibitor H89. Using cyclic AMP agonist pairs we found that sustained phosphorylation of TH was due to both the RI and the RII isotypes of PKA. The sustained activation of TH that occurred as a result of TH phosphorylation at Ser40 could maintain the synthesis of catecholamines without the need for further stimulus of the adrenal cells or increased TH protein synthesis.