14-3-3 protein inhibits CaMKK1 by blocking the kinase active site with its last two C-terminal helices.

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) inhibitors: a novel approach in small molecule discovery.

The calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) plays a key role in regulation of intracellular calcium levels and signaling pathways. It is involved in activation of downstream signaling pathways that regulate various cellular processes. Dysregulation of CAMKK2 activity has been linked to various diseases including cancer, suggesting that CAMKK2 inhibitors might be beneficial in oncological, metabolic and inflammatory indications. The most pressing issues in small molecule discovery are synthesis feasibility, novel chemical structure and desired biological characteristics. To circumvent this constraint, we employed 'DrugspaceX' for rapid lead identification, followed by repositioning seven FDA-approved drugs for CAMKK2 inhibition. Further, first-level transformation (Set1 analogues) was performed in 'DrugspaceX', followed by virtual screening. The t-SNE visualization revealed that the transformations surrounding Rucaparib, Treprostinil and Canagliflozin are more promising for developing CAMKK2 inhibitors. Second, using the top-ranked Set1 analogues, Set2 analogues were generated, and virtual screening revealed the top-ranked five analogues. Among the top five Set2 analogues, DE273038_5 had the lowest docking score of -11.034 kcal/mol and SA score of 2.59, retaining the essential interactions with Hotspot residues LYS194 and VAL270 across 250 ns simulation period. When compared to the other four compounds, the ligand effectiveness score was 0.409, and the number of rotatable penalties was only three. Further, DE273038_5 after two rounds of transformations was discovered to be novel and had not been previously described in other databases. These data suggest that the new candidate DE273038_5 is likely to have inhibitory activity at the CAMKK2 active site, implying potential therapeutic use.Communicated by Ramaswamy H. Sarma.

Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases.

Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKß lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKß inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKß interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.

Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase.

Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and ß) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

Stabilization of Protein-Protein Interactions between CaMKK2 and 14-3-3 by Fusicoccins.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates several key physiological and pathophysiological processes, and its dysregulation has been implicated in obesity, diabetes, and cancer. CaMKK2 is inhibited through phosphorylation in a process involving binding to the scaffolding 14-3-3 protein, which maintains CaMKK2 in the phosphorylation-mediated inhibited state. The previously reported structure of the N-terminal CaMKK2 14-3-3-binding motif bound to 14-3-3 suggested that the interaction between 14-3-3 and CaMKK2 could be stabilized by small-molecule compounds. Thus, we investigated the stabilization of interactions between CaMKK2 and 14-3-3γ by Fusicoccin A and other fusicoccanes-diterpene glycosides that bind at the interface between the 14-3-3 ligand binding groove and the 14-3-3 binding motif of the client protein. Our data reveal that two of five tested fusicoccanes considerably increase the binding of phosphopeptide representing the 14-3-3 binding motif of CaMKK2 to 14-3-3γ. Crystal structures of two ternary complexes suggest that the steric contacts between the C-terminal part of the CaMKK2 14-3-3 binding motif and the adjacent fusicoccane molecule are responsible for differences in stabilization potency between the study compounds. Moreover, our data also show that fusicoccanes enhance the binding affinity of phosphorylated full-length CaMKK2 to 14-3-3γ, which in turn slows down CaMKK2 dephosphorylation, thus keeping this protein in its phosphorylation-mediated inhibited state. Therefore, targeting the fusicoccin binding cavity of 14-3-3 by small-molecule compounds may offer an alternative strategy to suppress CaMKK2 activity by stabilizing its phosphorylation-mediated inhibited state.

Effects of Single-Nucleotide Polymorphisms in Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2): A Comprehensive Study.

Calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a protein kinase that belongs to the serine/threonine kinase family. It phosphorylates kinases like CAMK1, CAMK2, and AMP, and this signaling cascade is involved in various biological processes including cell proliferation, apoptosis, and proliferation. Also, the CAMKK2 signaling activity is required for the healthy activity of the brain which otherwise can cause diseases like bipolar disorders and anxiety. The current study is based on in silico bioinformatics analysis that combines sequence- and structure-based predictions to mark a SNP as damaging or neutral. The combined results from sequence-based, evolutionary conservation-based, and consensus-based tools have predicted a total of 18 nsSNPs as deleterious, and these nsSNPs were further subjected to structure-based analysis. The six mutant models of V195A, V249M, R311C, F366Y, P389T, and W445C showed a higher deviation from the wildtype protein model and hence were further taken for docking studies. The molecular docking analysis has predicted that these mutations will also be disruptive to the protein-protein interactions between CAMKK2 and PRKAG1 which will create an evident reduction in the kinase activity. The current study has enlightened us that a few of the significant mutations are prime candidates in CAMKK2 which could be the fundamental cause of various bipolar and psychiatric disorders. This is the first detailed study that predicts the deleterious nsSNPs in CAMKK2 and contributes positively in providing a better understanding of disease mechanisms.

In Depth Analysis of Kinase Cross Screening Data to Identify CAMKK2 Inhibitory Scaffolds.

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates CAMK1, CAMK4, AMPK, and AKT, leading to numerous physiological responses. The deregulation of CAMKK2 is linked to several diseases, suggesting the utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work, we demonstrate that STO-609, frequently described as a selective inhibitor for CAMKK2, potently inhibits a significant number of other kinases. Through an analysis of literature and public databases, we have identified other potent CAMKK2 inhibitors and verified their activities in differential scanning fluorimetry and enzyme inhibition assays. These inhibitors are potential starting points for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

Binding and structural analyses of potent inhibitors of the human Ca2+/calmodulin dependent protein kinase kinase 2 (CAMKK2) identified from a collection of commercially-available kinase inhibitors.

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.

Structural Analysis of Inhibitor Binding to CAMKK1 Identifies Features Necessary for Design of Specific Inhibitors.

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

CaMKK2 kinase domain interacts with the autoinhibitory region through the N-terminal lobe including the RP insert.

BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca⁠2+/calmodulin-dependent kinase (CaMK) family, functions as an upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase. Thus, CaMKK2 is involved in the regulation of several key physiological and pathophysiological processes. Previous studies have suggested that Ca2+/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear. METHODS: In this study, hydrogen-deuterium exchange coupled to mass spectrometry, time-resolved fluorescence spectroscopy, small-angle x-ray scattering and chemical cross-linking were used to characterize Ca2+/CaM binding-induced structural changes in CaMKK2. RESULTS: Our data suggest that: (i) the CaMKK2 kinase domain interacts with the autoinhibitory region (AID) through the N-terminal lobe of the kinase domain including the RP insert, a segment important for targeting downstream substrate kinases; (ii) Ca2+/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca2+/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca2+/CaM transiently interacts with the CaMKK2 kinase domain. CONCLUSIONS: Interactions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca2+/CaM binding the autoinhibitory region inhibits CaMKK2 by both blocking access to the RP insert and by affecting the structure of the ATP-binding pocket. GENERAL SIGNIFICANCE: Our results corroborate the hypothesis that Ca2+/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2ß1γ1 Heterotrimer.

Here, we describe the crystallization protocol for AMPK, including protein production and purification. AMPK can be readily crystallized in the presence of PEG to give diffracting crystals to a resolution of between 2.5 and 3.5 Å using synchrotron radiation. This method allows for visualization of drugs or small molecules that bind to the ADaM site, CBS sites, ATP binding site, and the newly identified C2 binding sites in the γ-subunit via co-crystallization with phosphorylated AMPK (pT172) α2ß1γ1 isoform or α2/1ß1γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these parameters.

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.

Flufenamic acid protects against intestinal fluid secretion and barrier leakage in a mouse model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation.

Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC.

Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines.

The heavy metal cadmium is a widespread environmental contaminant that has gained public attention due to the global increase in cadmium-containing electronic waste. Human exposure to cadmium is linked to the pathogenesis of osteoporosis. We previously reported cadmium induces apoptosis and decreases alkaline phosphatase mRNA expression via extracellular signal-regulated protein kinase (ERK) activation in Saos-2 bone-forming osteoblasts. This study examines the mechanisms of cadmium-induced osteotoxicity by investigating roles of Ca+2/calmodulin-dependent protein kinase (CAMK) pathways. Saos-2 or MG-63 cells were treated for 24 or 48h with 5µM CdCl2 alone or in combination with calmodulin-dependent phosphodiesterase (PDE) inhibitor CGS-9343ß; calmodulin-dependent kinase kinase (CAMKK) inhibitor STO-609; or calmodulin-dependent kinase II (CAMKII) inhibitor KN-93. CGS-9343ß protected against cadmium-induced toxicity and attenuated ERK activation; STO-609 enhanced toxicity and exacerbated ERK activation, whereas KN-93 had no detectable effect on cadmium-induced toxicity. Furthermore, CGS-9343ß co-treatment attenuated cadmium-induced apoptosis; but CGS-9343ß did not recover cadmium-induced decrease in ALP activity. The major findings suggest the calmodulin-dependent PDE pathway facilitates cadmium-induced ERK activation leading to apoptosis, whereas the CAMKK pathway plays a protective role against cadmium-induced osteotoxicity via ERK signaling. This research distinguishes itself by identifying pleiotropic roles for CAMK pathways in mediating cadmium's toxicity in osteoblasts.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli.

Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.

To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKß mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKß mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKß is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

Constitutive activation of CaMKKα signaling is sufficient but not necessary for mTORC1 activation and growth in mouse skeletal muscle.

Skeletal muscle loading/overload stimulates the Ca²âº-activated, serine/threonine kinase Ca²âº/calmodulin-dependent protein kinase kinase-α (CaMKKα); yet to date, no studies have examined whether CaMKKα regulates muscle growth. The purpose of this study was to determine if constitutive activation of CaMKKα signaling could stimulate muscle growth and if so whether CaMKKα is essential for this process. CaMKKα signaling was selectively activated in mouse muscle via expression of a constitutively active form of CaMKKα using in vivo electroporation. After 2 wk, constitutively active CaMKKα expression increased muscle weight (~10%) and protein content (~10%), demonstrating that activation of CaMKKα signaling can stimulate muscle growth. To determine if active CaMKKα expression stimulated muscle growth via increased mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, [³H]phenylalanine incorporation into proteins was assessed with or without the mTORC1 inhibitor rapamycin. Constitutively active CaMKKα increased protein synthesis ~60%, and this increase was prevented by rapamycin, demonstrating a critical role for mTORC1 in this process. To determine if CaMKKα is essential for growth, muscles from CaMKKα knockout mice were stimulated to hypertrophy via unilateral ablation of synergist muscles (overload). Surprisingly, compared with wild-type mice, muscles from CaMKKα knockout mice exhibited greater growth (~15%) and phosphorylation of the mTORC1 substrate 70-kDa ribosomal protein S6 kinase (Thr³89; ~50%), demonstrating that CaMKKα is not essential for overload-induced mTORC1 activation or muscle growth. Collectively, these results demonstrate that activation of CaMKKα signaling is sufficient but not necessary for activation of mTORC1 signaling and growth in mouse skeletal muscle.