Sisters Acts: Converging Signaling Between CaMKII and CaMKIV, Two Members of the Same Family.

Calcium (Ca2+) is a universal second messenger that regulates a number of diverse cellular processes including cell proliferation, development, motility, secretion, learning and memory1, 2. A variety of stimuli, such as hormones, growth factors, cytokines, and neurotransmitters induce changes in the intracellular levels of Ca2+. The most ubiquitous and abundant protein that serves as a receptor to sense changes in Ca2+ concentrations is Calmodulin (CaM), thus mediating the role as second messenger of this ion. The Ca2+/CaM complex initiates a plethora of signaling cascades that culminate in alteration of cell functions. Among the many Ca2+/CaM binding proteins, the multifunctional protein kinases CaMKII and CaMKIV play pivotal roles in the cell.


I. INTRODUCTION
The general structure of CaMKs includes an Nterminal kinase domain, an autoregulatory domain, an overlapping CaM-binding domain and, in phosphorylase kinase and CaMKII, a C-terminal association domain that is essential for multimerization and targeting. The best characterized CaM Kinase is CaMKII 3 .CaMKII is a multimeric enzyme composed of 12 subunits and it is encoded by 4 separate genes () with at least 24 peptides generated by alternate splicing 4,5 and at least one isoform expressed in every cell type 6 . CaMKII has a distinct mechanism of regulation that differs from the others CaM kinases. One catalytic subunit phosphorylates the autoinhibitory domain of the adjacent subunit on T286 (in the  isoform). This event requires that both the catalytic subunit and the substrate subunit are bound to Ca 2+ /CaM 7,8 . T286 phosphorylation then results in 20-80% Ca 2+ /CaM-independent activity 4,[9][10][11][12][13] . Autophosphorylation of T286 increases affinity for CaM by decreasing the rate of CaM dissociation. CaM is trapped by autophosphorylation, so that even when Ca 2+ levels are reduced, the kinase is fully active until CaM dissociates (several hundreds of seconds 13 ). This could serve as a mechanism to increase the sensitivity of CaMKII to the changes in intracellular Ca 2+ concentration 7,13 .

Anderson et al., Journal of Molecular and Cellular Cardiology, 2011
CaMKIV is a serine/threonine protein kinase that has been localized also in the nucleus 14 . Its expression is tissue-specific, with expression restricted primarily to distinct regions of the brain, T-lymphocytes, and postmeiotic germ cells, 15, 16 although it has been found in other cell types 17 , being especially enriched in cerebellar granule cells. CaMKIV (one gene, two splice variants) 18 is a monomeric enzyme, and apart from activation by Ca 2+ /CaM, shows very different modes of regulation by phosphorylation compared to CaMKII. CaMKIV has an "activation loop" phosphorylation site that is absent in CaMKII. Binding of Ca2+/CaM to CaMKIV exposes this activation loop site to allow phosphorylation by the upstream CaMKK, when it is simultaneously activated by Ca 2+ /CaM 19 . Phosphorylation of the activation loop in CaMKIV primarily increases its Ca 2+ /CaM-dependent activities. Università degli Studi di Salerno II. CaMK-MEDIATED ACTIVATION OF TRANSCRIPTION.

CaMKII and CREB
As CaM kinases II and IV have quite similar substrate specificity determinants, it is not completely surprising that they sometimes phosphorylate the same proteins. One such in vitro substrate for these kinases is the cAMPresponse element binding protein, CREB. CaMKII can phosphorylate CREB at Ser133 residue leading to the speculation that CaMKII mediates the Ca 2+ requirement for expression of the immediate early genes 5 . However, while the truncated form of CaMKII can stimulate CREBmediated transcription in some cells, it is inhibitory in others. Sun et al. 20 discovered that in addition to Ser-133, CaMKII also phosphorylated a second residue on CREB, Ser-142. Indeed, phosphorylation of Ser-142 was not only inhibitory, but this modification was also dominant and could reverse the activation of CREB resulting from its phosphorylation on Ser-133 by PKA. This phosphorylation seems to be destabilizing for the association between CREB and CBP 21 . Interestingly, the nature of the effect of CaMKII on transcription is both cell and promoter dependent.

CaMKIV AND CREB
CaMKIV shows very strong nuclear localization 22,23 , and many studies support the idea that it is responsible for Ca 2+ -dependent stimulation of transcription through phosphorylation of CREB and serum response factor (SRF) 5,22,24 . Activation by CaMKIV occurs via direct phosphorylation of the activating serines of these transcription factors, Ser133 (CREB), Ser63 (ATF-1), and Ser103(SRF), respectively 25 . CaMKIV phosphorylates CREB Ser133, the same site that is phosphorylated by PKA. Transfected CaMKIV alone is a relatively poor stimulator of transcriptional activation by CREB: indeed, cotransfection of CaMKK with CaMKIV gives a 14-fold enhancement of transcription 26 . Studies in cultured hippocampal neurons indicate that CaMKIV regulates CREB-dependent gene transcription in response to electrical stimulation or KCl depolarization 27 . This role of CaMKIV in CREB-mediated transcription has been confirmed in transgenic mice that express an inactive form of CaMKIV only in T cells in the thymus 27 . Overexpression of inactive CaMKIV would be expected to function in a dominant negative manner. These thymic T cells have a reduced ability, upon stimulation, to phosphorylate CREB, induce transcription of FosB and produce interleukin 2 (IL-2) 28 . There is also good evidence for involvement of CaMKIV in transcriptional regulation of the BDNF gene through phosphorylation of a CREB family member 29 . These observations provide a mechanism that would permit the Ca 2+ signaling pathway to be either antagonistic or additive with the cAMP pathway for activation of CREB, depending on the relative activity of specific CaM kinases.

III. CaMKs MEDIATED REGULATION OF APOPTOSIS
Bok et al 30 45 . These data suggest that CaMKII and ERK are essential mediators of cell proliferation 46,47 . The role of CaMKII in cell proliferation is not a restricted mechanism, but it is a general phenomenon that may be relevant for the biological effects of many growth factors and hormones.
V. CaMKs MEDIATED REGULATION OF DIFFERENTIATED FUNCTIONS.

SURVIVAL
The multifunctional CaMKs family proteins are involved in the control of differentiation and survival of neurons and hematopoietic stem cells 48 55 suggesting the involvement of a Ca 2+ -dependent pathway in the regulation of DC migration. The role of a Ca 2+dependent pathway in the mechanism regulating DC maturation is suggested by the opposite effects induced by Ca 2+ ionophores or chelation of extracellular Ca 2+ on this process 56 . The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. Although isolated Camk4 −/− DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive.

CARDIAC HYPERTROPHY
CaMKII has been implicated in several key aspects of acute cellular Ca 2+ regulation related to cardiac excitationcontraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum 57 proteins, including the ryanodine receptors (RyR2) and phospholamban (PLB) 57 . Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte Ca 2+ homeostasis 58 . CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy 59 and heart failure 60 , and in cardiac tissue from patients with dilated cardiomyopathy 61 .Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy. Hypertrophy develops in transgenic mice that overexpress CaMKIV 62 , but this isoform is not detectable in the heart and CaMKIV knockout mice still develop hypertrophy following transverse aortic constriction (TAC) 63

VI. CaMKs AND INFLAMMATION
Sepsis is a special type of host inflammatory response to bacterial infection that originates from massive and widespread release of pro-inflammatory mediators.
Bacterial endotoxins, such as LPS, are the major offending factors in sepsis that activate TLR-mediated signaling to generate inflammatory response that is amplified in a self-sustaining manner. There are meny evidences of a correlation between multifunctional CaM kinases and TLR-4 signaling. CaMKII directly phosphorylates components of TLR signaling, and promotes cytokine production in macrophages 71 . Complement activation is also a recognized factor in the pathogenesis of sepsis. Inhibition of the complement cascade decreases inflammation and improves mortality in animal models 51 . Differentiation and survival of antigen presenting dendritic cells (DC) uponTLR-4 activation requires CaMKIV 72 . DC from CaMKIV−/−mice failed to survive upon LPS-mediated TLR-4 induction. However, ectopic expression of CaMKIV was able to rescue this defect. In another study, the selective inhibition of CaMKII interfered with terminal differentiation of monocyte-derived DCs by preventing up-regulation of co-stimulatory and MHC II molecules as well as secretion of cytokines induced by TLR-4 agonists 73 . Thus, CaM kinases seem to play a general role in inflammatory processes VII. CONCLUSIONS CaMKs define a family of ser-thr kinases that direct a wide range of cellular processes and cell fate decisions. Since their discovery, much of the focus has been on their regulation of memory and learning. In recent years, studies on CaMKII and CaMKIV signaling in a number of cell models have established the importance of the Ca 2+-CaM-CaMKK-CaMKs pathways in effecting proliferation, survival, differentiation and associated molecular events. Intriguing new findings also indicate that, although the two kinases might share some substrates, there is specificity in the pathways they contribute, thus reflecting both shared and unique properties. The emergence of ERK as a critical CaMKII regulatory target for cell proliferation has united membrane proximal regulatory events orchestrated by the Ras activated cascade with key transcriptional CaMKs targets. Ca 2+ is ubiquitously present in the cells, hence its compartimentalization and the regulation of its downstream kinases need to be finely tuned, in order to efficiently regulate biological functions. The involvement of CaMKII and CaMKIV in pathways that regulate functions as different as proliferation, survival and differentiation imply numerous cross-talks and their harmonization. Both kinases require Ca 2+ increases to be activated, although other events are required to support their differential activation. Subcellular compartimentalization provides another tool to distinctively activate CaMKII and CaMKIV depending upon the cell's needs. It is possible, though, to hypothesize a further mechanism of counter-regulation between the two kinases: insights into the regulation and impact of a crosstalk between CaMKII and CaMKIV signaling might bring in new highlights for biological functions, and their disruption in human diseases.