Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance

The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure, characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn, these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2.     

Calmodulin-dependent protein kinase II

Three multifunctional protein kinases, cyclic AMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase II, are involved in signal transduction in response to their respective second messengers, cyclic AMP, diacylglycerol, and Ca2+. This review will summarize the key findings on calmodulin-dependent protein kinase II.     

Calmodulin-dependent protein kinase II

One of the most important mechanisms for regulating neuronal functions is through second messenger cascades that control protein kinases and the subsequent phosphorylation of substrate proteins. Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is the most abundant protein kinase in mammalian brain tissues, and the alpha-subunit of this kinase is the major protein and enzymatic molecule of synaptic junctions in many brain regions. CaM-kinase II regulates itself through a complex autophosphorylation mechanism whereby it becomes calcium-independent following its initial activation. This property has implicated CaM-kinase II as a potential molecular switch at central nervous system (CNS) synapses. Recent studies have suggested that CaM-kinase II is involved in many diverse phenomena such as epilepsy, sensory deprivation, ischemia, synapse formation, synaptic transmission, long-term potentiation, learning, and memory. During brain development, the expression of CaM-kinase II at both protein and mRNA levels coincides with the active periods of synapse formation and, therefore, factors regulating the genes encoding kinase subunits may play a role in the cell-to-cell recognition events that underlie neuronal differentiation and the establishment of mature synaptic functions. Recent findings have demonstrated that the mRNA encoding the alpha-subunit of CaM-kinase II is localized in neuronal dendrites. Current speculation suggests that the localized translation of dendritic mRNAs encoding specific synaptic proteins may be responsible for producing synapse-specific changes associated with the processing, storage, and retrieval of information in neural networks.     

Calmodulin-dependent glycogen synthase kinase

A cAMP-independent glycogen synthase kinase has been purified from rabbit liver. This kinase is completely dependent on the presence of calmodulin and Ca2+ for activity. Half-maximal activation required about 0.1 microM calmodulin. Complete inhibition was obtained in the presence of ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid or trifluoperazine. This calmodulin-dependent synthase kinase does not phosphorylate phosphorylase, myosin light chain, casein, or histone. It rapidly incorporates 0.4 to 0.5 mol of 32P/mol of synthase subunit into the NH2-terminal domain, resulting in partial inactivation of glycogen synthase. These results indicate the existence of a calmodulin-dependent kinase which may be specific for glycogen synthase.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.     

Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) is a critical regulator of energy balance at both the cellular and whole-body levels. Two upstream kinases have been reported to activate AMPK in cell-free assays, i.e., the tumor suppressor LKB1 and calmodulin-dependent protein kinase kinase. However, evidence that this is physiologically relevant currently only exists for LKB1. We now report that there is a significant basal activity and phosphorylation of AMPK in LKB1-deficient cells that can be stimulated by Ca2+ ionophores, and studies using the CaMKK inhibitor STO-609 and isoform-specific siRNAs show that CaMKKbeta is required for this effect. CaMKKbeta also activates AMPK much more rapidly than CaMKKalpha in cell-free assays. K(+)-induced depolarization in rat cerebrocortical slices, which increases intracellular Ca2+ without disturbing cellular adenine nucleotide levels, activates AMPK, and this is blocked by STO-609. Our results suggest a potential Ca(2+)-dependent neuroprotective pathway involving phosphorylation and activation of AMPK by CaMKKbeta.