Regulation of Ca2+/calmodulin kinase II by a small C-terminal domain phosphatase.

Ca2+/ Calmodulin (CaM)-dependent protein kinase II (CaMKII) is a ubiquitous protein that has numerous functions in fundamental cellular processes including metabolism, cell cycle control [1], cell shape variation, gene transcription [2] and the regulation of ion channel and cytoskeletal function [3–5]. CaM kinase II is activated by Ca2+-bound CaM. There are 4 isoforms, alpha, beta, delta and gamma. In vitro studies have shown that CaM kinase II can autophosphorylate at several sites [6, 7]. Thr286 (numbering according to alpha isoform, Thr287 for beta, delta and gamma) is the best explored site and has been linked to the generation of a Ca2+/CaM-independent persistence of kinase activity. There are other known autophosphorylation sites (e.g. Thr253, Ser279, Thr305/306 and Ser314; numbering according to alpha isoform) but the in vivo functions of these sites are less well understood. Previously, we reported the identification of six variants of CaM kinase II gamma in differentiated vascular smooth muscle (dVSM) [8]. CaMKII gamma G-2 has a novel association domain that is distinct from the common association domain found in all other CaMKII gamma variants. We have previously shown that CaMKII gamma G-2 translocates to cortical adhesion plaques upon activation and regulates contractility of smooth muscle through an ERK-mediated pathway [9]. Phosphorylated CaM kinase II is believed to be regenerated to its unphosphorylated state by the action of phosphatases in the cell. Accordingly, dephosphorylation of Thr286 by phosphatases in neurons has been reported [10]. A PP1 is reported to dephosphorylate CaM kinase II associated with postsynaptic densities, but the soluble form of CaM kinase II is reported to be dephosphorylated by a PP2A [11, 12]. PP1, PP2A [11, 12] and PP2C decrease autonomous activity of CaM kinase II but PP2B does not inhibit autonomous activity [10]. A PP2C has also been reported to dephosphorylate CaM kinase II in vitro [13], but its in vivo function has not been determined. The nature of the relevant CaMKII phosphatase in other cell types is not known. C-terminal domain (CTD) phosphatases are known to be involved in the dephosphorylation of the C-terminal domain of RNA polymerase II. CTD phosphatases consist of a phosphatase catalytic domain and a Breast Cancer 1 C-terminal (BRCT) domain. However, small CTD phosphatases (SCPs) lack the BRCT domain (figure1A). To date, three isoforms (SCP1-3) have been identified in the human. The only known functions of SCPs are that human SCP1 can dephosphorylate the C-terminal domain of the largest RNA polymerase II subunit in vitro [14] and that some human SCP isoforms are suggested to function as global silencers of neuronal genes [15]. Figure 1 A: Diagrammatic presentation of the domains (as indicated) of CTD phosphatase and small CTD phosphatase. B: Alignment of Ferret (Mustela putorius furo, Mpf) SCP3 with Human (Hs) SCP3. The identical residues are shaded gray, similar residues are shaded ... The present study was initiated to identify new CaMKII binding proteins. We have used the novel association domain of CaMKII gamma G-2 as a bait in a yeast 2-hybrid assay and here present the identification and characterization of a small CTD phosphatase (SCP) homologue in smooth muscle that binds to this novel association domain. SCP3 is a PP2C type phosphatase that is primarily expressed in vascular smooth muscle tissues. This phosphatase dephosphorylates CaMKII gamma G-2 but, as we show here, the dephosphorylation is site specific in that Thr287 is protected from dephosphorylation. We show that SCP3 regulates phosphorylation of CaMKII gamma G-2 at sites that are not involved in the autonomous activation of the kinase but that have been shown to regulate other functions such as targeting of the kinase.