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Transporting the Calcium Signal to the Nucleus

CaMKIIγ delivers Ca2+/CaM from voltage-gated calcium channels to the nucleus. Local calcium signals mediated by activity-dependent opening of voltage-gated calcium channels stimulate specific gene expression responses in a process called excitation-transcription coupling (see Malik et al.). An outstanding question is how this local calcium signal is transmitted from the plasma membrane to the nucleus. Ma et al. mechanistically linked the calcium-stimulated kinase events involving CaMKII (calcium-calmodulin kinase II) isoforms at the channels to the CaMKII-mediated events in the nucleus. CaMK enzymes are regulated by phosphorylation and bind to the calcium-binding protein calmodulin (CaM). The affinity of CaMKII isoforms for Ca2+-bound CaM (Ca2+/CaM) is increased by phosphorylation of Thr286 or Thr287, and nuclear localization of some isoforms is inhibited by phosphorylation of a serine adjacent to the nuclear localization signal. Image analysis of either immunofluorescence experiments or green fluorescent protein (GFP)-tagged protein revealed that CaMKIIγ migrated from the cytosol to the nucleus in superior cervical ganglion neurons or cortical neurons exposed to depolarizing conditions. Knockdown of CaMKIIγ reduced the depolarization-induced (i) nuclear accumulation of phosphorylated CaMKII (detected with an antibody specific for Thr286- or Thr287-phosphorylated CaMKII, but not specific for a particular isoform); (ii) accumulation of nuclear CaM; and (iii) phosphorylation of the nuclear-localized transcription factor CREB and expression of a CREB target gene. A kinase-deficient mutant of CaMKIIγ rescued CREB phosphorylation in neurons in which CaMKIIγ was knocked down, but mutants (A303R or T287A) that could not bind CaM did not. However, the T287A mutant form translocated to the nucleus in response to depolarization, indicating that transclocation was independent of CaM binding. Inhibition of the phosphatase calcineurin (CaN, also known as PP2B), which is associated with the voltage-gated calcium channels, prevented depolarization-induced nuclear accumulation of CaMKIIγ, and inhibition of the phosphatase PP2A, which functions in the nucleus and is required for CaM release from CaMKIIγ, prevented the depolarization-induced increase in phosphorylated CREB. An S334E mutant (mimicking the phosphorylated form) remained cytosolic in response to depolarization, but accumulated at sites positive for CaN and voltage-gated calcium channel subunits, suggesting that dephosphorylation of this residue is necessary for release of CaMKIIγ from the channel-associated signaling complex. A T287E mutant, mimicking the phosphorylated form, remained dispersed in the cytosol in depolarizing conditions even when combined with the S334E mutation, suggesting that phosphorylation of Thr287 occurred after CaMKIIγ reached the channel complex and Ser334 dephosphorylation occurred at the channel complex. Although CaMKIIγ accumulated in the nucleus in response to depolarization in neurons in which CaMKIIβ was knocked down, CREB phosphorylation was blocked. In vitro experiments showed that CaMKIIβ phosphorylated CaMKIIγ at Thr287. The data are consistent with a model in which CaMKIIβ phosphorylates CaMKIIγ on Thr287, which increases the affinity for Ca2+/CaM and enables loading of CaMKIIγ at the channel complex. Dephosphorylation at the Ser334 by the calcium-activated CaN unmasks the nuclear localization signal so that Ca2+/CaM-loaded CaMKIIγ translocates to nucleus, where dephosphorylation of Thr287 by PP2B releases Ca2+/CaM, which then activates the CaMK events that activate CREB. H. Ma, R. D. Groth, S. M. Cohen, J. F. Emery, B. Li, E. Hoedt, G. Zhang, T. A. Neubert, R. W. Tsien, γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression. Cell 159, 281–294 (2014). [PubMed] Z. A. Malik, I. S. Stein, M. F. Navedo, J. W. Hell, Mission CaMKIIγ: Shuttle calmodulin from membrane to nucleus. Cell 159, 235–237 (2014). [PubMed]