CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.

The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Oxidative Stress Orchestrates MAPK and Nitric-Oxide Synthase Signal.

Reactive oxygen species (ROS) are not only harmful to cell survival but also essential to cell signaling through cysteine-based redox switches. In fact, ROS triggers the potential activation of mitogen-activated protein kinases (MAPKs). The 90 kDa ribosomal S6 kinase 1 (RSK1), one of the downstream mediators of the MAPK pathway, is implicated in various cellular processes through phosphorylating different substrates. As such, RSK1 associates with and phosphorylates neuronal nitric oxide (NO) synthase (nNOS) at Ser847, leading to a decrease in NO generation. In addition, the RSK1 activity is sensitive to inhibition by reversible cysteine-based redox modification of its Cys223 during oxidative stress. Aside from oxidative stress, nitrosative stress also contributes to cysteine-based redox modification. Thus, the protein kinases such as Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) and II (CaMKII) that phosphorylate nNOS could be potentially regulated by cysteine-based redox modification. In this review, we focus on the role of post-translational modifications in regulating nNOS and nNOS-phosphorylating protein kinases and communication among themselves.

Reactive sulfur species impair Ca2+/calmodulin-dependent protein kinase II via polysulfidation.

We previously reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is inhibited by S-nitrosylation of Cys6 in cells. Herein, we show that polysulfidation of CaMKII at Cys6 limits its enzyme activity following reactive sulfur species (RSS) stimulus. In vitro incubation of CaMKII with the RSS donor, Na2S4, induced the inhibition of the enzyme via its polysulfidation. Treatment with dithiothreitol reversed the polysulfidation and the subsequent inhibition. The inhibition of CaMKII by Na2S4 is competitive with ATP but not with the peptide substrate Syntide-2. In transfected cells expressing CaMKII, the enzyme activity decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKII (C6A) were resistant to this treatment. In addition, the endogenous CaMKII was inhibited by treatment with Na2S4 in RAW264.7 murine macrophage cells. These results suggest a novel regulation of CaMKII by RSS via its Cys6 polysulfidation in cells.

The active-site cysteine residue of Ca2+/calmodulin-dependent protein kinase I is protected from irreversible modification via generation of polysulfidation.

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na2S3 resulted in a dose-dependent inhibition of the phosphorylation at Thr177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na2S3-induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys179 by RSS, thereby protecting it from irreversible modification.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase ß.

The Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKß that exhibits higher basal activity (autonomous activity), activation of the CaMKKß/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKß appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKß activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKß at multiple residues by CaMKKß-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKß activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKß indicated that Thr144 phosphorylation by activated AMPK converts CaMKKß into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKß mutant. CaMKKß mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKß in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKß regulates the CaMKKß/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKß.

Reactive sulfur species inactivate Ca2+/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue.

Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na2S n (n = 2-4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na2S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr196 and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with either Na2S4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr196 of endogenous CaMKIV was also inhibited by treatment either with Na2S4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S-polysulfidation of its Cys198 during the response to ER stress.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic ß-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB.

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory ß-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of ß-adrenergic receptors (ß-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic ß-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic ß-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by ß-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as ß-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic ß-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.

Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKß phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 µm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKß chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKß/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

Nuclear calcium signaling induces expression of the synaptic organizers Lrrtm1 and Lrrtm2.

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2-4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca(2+)/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2.

Cytoplasmic retention of protein phosphatase 2A inhibitor 2 (I2PP2A) induces Alzheimer-like abnormal hyperphosphorylation of Tau.

Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2 (PP2A)). In AD brain, I2 (PP2A) is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2 (PP2A) containing a conserved basic motif, (179)RKR(181), which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2 (PP2A) in which the NLS was inactivated by (179)RKR(181) → AAA along with (168)KR(169) → AA mutations. In this model, the mutant NLS (mNLS)-I2 (PP2A) (I2 (PP2A)AA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2 (PP2A) activated CAMKII and GSK-3ß, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2 (PP2A) with PP2A and GSK-3ß but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2 (PP2A)-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology.

Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIß and IV and Possible Involvement of CREB Phosphorylation.

Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca2+/calmodulin-dependent protein kinase (CaMK) IIα, IIß and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIß and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIß and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

Persulfide Signaling in Stress-Initiated Calmodulin Kinase Response.

Significance: Calcium ion (Ca2+)/calmodulin (CaM)-dependent protein kinases (CaMKs) are activated by phosphorylation of a crucial threonine residue either by itself (CaMKII) or by upstream kinases, CaMK kinases (CaMKKs) (CaMKI and CaMKIV). CaMKs, present in most mammalian tissues, can phosphorylate many downstream targets, thereby regulating numerous cellular functions. Recent Advances: Aside from canonical post-translational modifications, cysteine-based redox switches in CaMKs affect their enzyme activities. In addition to reactive oxygen species (ROS) and reactive nitrogen species (RNS), reactive sulfur species (RSS) are also recognized as key signaling molecules, regulating protein function through polysulfidation, formation of polysulfides [-S-(S)n-H] on their reactive cysteine residues. To comprehend the biological significance of RSS signaling-related CaMK regulation, here we introduce a novel concept defining CaMKs as RSS targets in stress responses. The stress responses include an irreversible electrophile attack for CaMKI, inflammation for CaMKII, and endoplasmic reticulum stress for CaMKIV. Critical Issues: Development of various human diseases is associated with increased ROS, RNS, and RSS generation. Therefore, depending on specific pathophysiology, RSS could have very particular effects on CaMK functions. Future Directions: How multiple sources and mutual reactions of ROS, RNS, and RSS are coordinated is obscure. Elucidating the mechanisms through applications of enzymology, chemical biology, and mass spectrometry enables to uncover the complexities of redox regulation of CaMK cascades.

Redox regulation of Ca2+/calmodulin-dependent protein kinase IV via oxidation of its active-site cysteine residue.

We have recently reported that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) is inactivated by reactive sulfur species via polysulfidation of the active-site Cys residue. Here, we show that hydrogen peroxide (H2O2) limit CaMKIV activity at the same active-site Cys residue through oxidation and downstream signaling in cells. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), which induces its full activity. In vitro incubation of CaMKIV with H2O2 resulted in reversible inhibition of CaMKK-induced phospho-Thr196 and the consequent inactivation of CaMKIV. In contrast, mutated CaMKIV (C198V) was refractory to the H2O2-induced enzyme inhibition. In transfected cells expressing CaMKIV, Ca2+ ionophore-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with H2O2, whereas cells expressing mutant CaMKIV (C198V) were resistant to H2O2 treatment. Modification of free thiol with N-ethylmaleimide revealed that Cys198 in CaMKIV is a target for S-oxidation. Additionally, the Ca2+ influx-induced phospho-Thr196 of endogenous CaMKIV was also inhibited upon treatment with H2O2 in Jurkat T-lymphocytes and cerebellar granule cells. Phosphorylation of cyclic AMP response element-binding protein (CREB) at Ser133, which is downstream of CaMKIV, was also decreased upon treatment with H2O2. Thus, our results indicate that oxidation stress regulates cellular function by decreasing the activity of CaMKIV through Cys198 oxidation.