Calmodulin-dependent Protein Kinase Kinase 2 Research Articles

Abstract 1136: Regulation of AMPK by androgen receptor signaling and its role in promoting prostate cancer through the use of autophagy

Abstract While we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of prostate cancer, it is poorly understood what AR-regulated processes drive this pathology. Autophagy, an enigmatic cellular recycling process, has received increased attention as of late due to its potential oncogenic role in late-stage cancers. One of the master regulators of autophagy is the 5′-AMP-activated protein kinase (AMPK). Previous work from several independent laboratories has suggested AR signaling promotes cancer progression through an AMPK-dependent signaling mechanism. Further, it was demonstrated this enzymatic cascade was specifically regulated by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Given AMPK's known role as a regulator of autophagy, we hypothesized that AR-mediated CaMKK2-AMPK signaling could promote prostate cancer through increasing cellular rates of autophagy. To test this hypothesis, we initially assessed whether AR signaling could regulate autophagy and if so, what effect(s) this had on prostate cancer cell growth and tumor formation. We then used a variety of pharmacological and molecular approaches to determine whether CaMKK2-AMPK signaling was required and sufficient to regulate autophagy and subsequent prostate cancer cell growth in vitro and in vivo. Here, we have determined that 1) AR regulates cell metabolism and cell growth in part by increasing autophagy in prostate cancer cells, 2) functional autophagy was clinically detected in metastatic, castration-resistant cancers but not treatment-naïve, localized tumors and 3) autophagy is required for prostate cancer progression in preclinical animal models. Additionally, AR-mediated autophagy was mediated through the direct expression of CaMKK2 and subsequent phosphorylation/activation of AMPK. Correspondingly, levels of both CaMKK2 and phosphorylated/activated AMPK correlated with prostate cancer progression in genetic mouse models and patients. Mechanistically, AR-mediated autophagy appears to promote cell growth by augmenting intracellular lipid accumulation, a hallmark of prostate cancers. Taken together, our findings converge to demonstrate that AR signaling can co-opt the AMPK signaling cascade, a known homeostatic mechanism, to promote prostate cancer by increasing autophagy. The current study points to the potential utility of developing metabolic-targeted therapies directed towards the CaMKK2-AMPK signaling axis for the treatment of prostate cancer. Further, an inhibitor of this signaling cascade could serve as an alternative, more specific therapeutic compared to existing inhibitors of autophagy that, to date, have demonstrated limited efficacy in clinical trials due to their toxicity and poor pharmacokinetics. Citation Format: Yan Shi, Efrosini Tsouko, Alicia M. Blessing, Jayantha Tennakoon, Jenny J. Han, Michael M. Ittmann, Daniel E. Frigo. Regulation of AMPK by androgen receptor signaling and its role in promoting prostate cancer through the use of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1136. doi:10.1158/1538-7445.AM2015-1136

Mitochondrial biogenesis is rate limiting-factor for axonal growth

During early development neurons undergo complex morphological rearrangements. This active axo-dendritic growth must be supported by sufficient cellular energy, thus, cellular energy status could be a key factor in initiating this process. However, the precise mechanisms of how cells adapt their energetic status to physiological demand and upcoming energy needs and how they are coupled to adaptive energy generation, e.g., increased ATP production by mitochondria, remain unclear. Previous studies have shown that peroxisome-proliferator-activated receptor gamma co-activator 1 (PGC-1α) is a master regulator of mitochondrial biogenesis and cellular energy metabolism. Our aim was to examine whether neuronal growth depends on mitochondrial biogenesis and whether activation of cell growth pathways could promote mitochondrial biogenesis to support the energetic need of neuronal development. Over-expression of PGC-1α in cortical neurons increased mitochondrial density in the periphery of axonal tree and was two-fold higher in axonal tips compared to control group. Moreover, induction of mitochondrial biogenesis by PGC-1α facilitated the axonal growth and neuronal development. Activation of PGC-1α upstream kinases such as Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), transforming growth factor-β-activated kinase (TAK1) and STE-related adaptor (STRAD) increased PGC-1α transcriptional activity and mitochondrial density in axons. Most importantly, they promoted neuronal development through mitochondrial biogenesis. This study shows that mitochondrial biogenesis itself is limiting factor for axonal growth and AMPK-PGC-1α upstream pathways sensing cellular energy status could signal not only the energy deficit but also upcoming energy need to generate new mitochondria.

The camKK2/camKIV relay is an essential regulator of hepatic cancer.

Hepatic cancer is one of the most lethal cancers worldwide. Here, we report that the expression of Ca(2+) /calmodulin-dependent protein kinase kinase 2 (CaMKK2) is significantly up-regulated in hepatocellular carcinoma (HCC) and negatively correlated with HCC patient survival. The CaMKK2 protein is highly expressed in all eight hepatic cancer cell lines evaluated and is markedly up-regulated relative to normal primary hepatocytes. Loss of CaMKK2 function is sufficient to inhibit liver cancer cell growth, and the growth defect resulting from loss of CaMKK2 can be rescued by ectopic expression of wild-type CaMKK2 but not by kinase-inactive mutants. Cellular ablation of CaMKK2 using RNA interference yields a gene signature that correlates with improvement in HCC patient survival, and ablation or pharmacological inhibition of CaMKK2 with STO-609 impairs tumorigenicity of liver cancer cells in vivo. Moreover, CaMKK2 expression is up-regulated in a time-dependent manner in a carcinogen-induced HCC mouse model, and STO-609 treatment regresses hepatic tumor burden in this model. Mechanistically, CaMKK2 signals through Ca(2+) /calmodulin-dependent protein kinase 4 (CaMKIV) to control liver cancer cell growth. Further analysis revealed that CaMKK2 serves as a scaffold to assemble CaMKIV with key components of the mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway and thereby stimulate protein synthesis through protein phosphorylation. The CaMKK2/CaMKIV relay is an upstream regulator of the oncogenic mammalian target of rapamycin/ribosomal protein S6 kinase, 70 kDa, pathway, and the importance of this CaMKK2/CaMKIV axis in HCC growth is confirmed by the potent growth inhibitory effects of genetically or pharmacologically decreasing CaMKK2 activity; collectively, these findings suggest that CaMKK2 and CaMKIV may represent potential targets for hepatic cancer.

Inhibition of CaMKK2 reverses age-associated decline in bone mass

Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2−/− mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis.

Calcium calmodulin dependent kinase kinase 2 - a novel therapeutic target for gastric adenocarcinoma

Gastric cancer is one of the most common gastrointestinal malignancies and is associated with poor prognosis. Exploring alterations in the proteomic landscape of gastric cancer is likely to provide potential biomarkers for early detection and molecules for targeted therapeutic intervention. Using iTRAQ-based quantitative proteomic analysis, we identified 22 proteins that were overexpressed and 17 proteins that were downregulated in gastric tumor tissues as compared to the adjacent normal tissue. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was found to be 7-fold overexpressed in gastric tumor tissues. Immunohistochemical labeling of tumor tissue microarrays for validation of CAMKK2 overexpression revealed that it was indeed overexpressed in 94% (92 of 98) of gastric cancer cases. Silencing of CAMKK2 using siRNA significantly reduced cell proliferation, colony formation and invasion of gastric cancer cells. Our results demonstrate that CAMKK2 signals in gastric cancer through AMPK activation and suggest that CAMKK2 could be a novel therapeutic target in gastric cancer.

MicroRNA-224 and its target CAMKK2 synergistically influence tumor progression and patient prognosis in prostate cancer.

We previously demonstrated that microRNA (miR)-224 expression was significantly reduced in human prostate cancer (PCa) tissues and predicted unfavorable prognosis in patients. However, the underlying mechanisms of miR-224 have not been fully elucidated. In this study, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was identified as a target gene of miR-224. Then, we found that enforced expression of miR-224 could suppress PCa cell proliferation and cell cycle by regulating the expression of CAMKK2 in vitro. In addition, the expression levels of miR-224 in PCa tissues were negatively correlated with those of CAMKK2 mRNA significantly (Spearman's correlation: r = -0.66, P = 0.004). Moreover, combined low miR-224 expression and high CAMKK2 expression (miR-224-low/CAMKK2-high) was closely correlated with advanced clinical stage (P = 0.028). Furthermore, PCa patients with miR-224-low/CAMKK2-high expression more frequently had shorter overall survival than those in groups with other expression patterns of two molecules. In conclusion, our data offer the convincing evidence that miR-224 and its target gene CAMKK2 may synergistically contribute to the malignant progression of PCa. Combined detection of miR-224 and CAMKK2 expressions represents an efficient predictor of patient prognosis and may be a novel marker which can provide additional prognostic information in PCa.

AMPK activation by isorhamnetin protects hepatocytes against oxidative stress and mitochondrial dysfunction

Arachidonic acid (AA) is a ω−6 polyunsaturated fatty acid that is found in the phospholipids of membranes and released from the cellular membrane lipid bilayer by phospholipase A2. During this process, AA could produce excess reactive oxygen species and induce apoptosis and mitochondrial dysfunction by selectively inhibiting complexes I and III. Isorhamnetin, an O-methylated flavonol aglycone, has been shown to have cardio-protective, anti-adipogenic, anti-tumor, and anti-inflammatory effects. In the present study, we investigated the effects of isorhamnetin on hepatotoxicity and the underlying mechanisms involved. Our in vitro experiments showed that isorhamnetin dose-dependently blocked the hepatotoxicity induced by treatment with AA plus iron in HepG2 cells. Furthermore, isorhamnetin inhibited the AA+iron induced generation of reactive oxygen species and reduction of glutathione, and subsequently maintained mitochondria membrane potential in AA+iron treated HepG2 cells. In addition, isorhamnetin activated AMP-activated protein kinase (AMPK) by Thr-172 phosphorylation of AMPKα, and this was mediated with Ca(2+)/calmodulin-dependent protein kinase kinase-2 (CaMKK2), but not liver kinase B1. Experiments using CaMKK2 siRNA or its selective inhibitor, STO-609, revealed the role of CaMKK2 in the isorhamnetin-induced activation of AMPK in HepG2 cells. These results indicate isorhamnetin protects against the hepatotoxic effect of AA plus iron, and suggest that the AMPK pathway is involved in the mechanism underlying the beneficial effect of isorhamnetin in the liver.

Abstract A12: Regulation of ovarian cancer OVCAR-3 cell proliferation and viability by calcium/calmodulin-dependent protein kinase kinase 2

Abstract Protein kinases play crucial roles in cancer progression and are considered potential targets for novel anti-cancer therapeutics. Oncogenic kinases mediate a variety of events characteristic of the malignant phenotype including transcriptional activation, accelerated cell proliferation and enhanced cellular survival. Ca2+/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) has been implicated as a signaling mechanism in prostate and breast cancers, but has not been studied in ovarian cancer (OvCa). We report here the following. CaMKK2 expression is greatly increased in high grade serous papillary cystadenocarcinoma compared to benign tissue. Knockdown of CaMKK2 expression by RNA interference in OvCa OVCAR-3 cells resulted in slowing of cell growth and decreased number of cells entering S phase. At the molecular level, CaMKK2 knockdown led to significant decreases in cyclin D1 expression at both mRNA and protein levels and to a drop in phosphorylation of the retinoblastoma tumor suppressor (Rb) at Ser 807/811. These results indicate that CaMKK2 expression in OVCAR-3 OvCa cells is required for optimal G1/S phase cell cycle progression and proliferation. CaMKK2 knockdown also resulted in a significant decrease in OVCAR-3 cell survival and an increase in both Poly ADP Ribose Polymerase (PARP) cleavage (inactivation) and in the activity of executioner Caspases-3/7 indicating that CaMKK2 knockdown in OVCAR-3 cells results in apoptosis. Preliminary data showed a decrease in phosphorylation of the activation site of Akt (Thr 308) in CaMKK2 depleted cells suggesting that the G1/S phase cell cycle arrest and apoptosis in CaMKK2 silenced cells may occur either directly or indirectly through an Akt-dependent pathway. Altogether these data show that CaMKK2 is required for optimal growth and viability of OVCAR-3 cells and furthermore imply that CaMKK2 may be a promising and novel therapeutic target for OvCa. Citation Format: Angela M. Gocher, Gissou Azabdaftari, Arthur M. Edelman. Regulation of ovarian cancer OVCAR-3 cell proliferation and viability by calcium/calmodulin-dependent protein kinase kinase 2. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A12.

Convergent lines of evidence support CAMKK2 as a schizophrenia susceptibility gene

Genes that are differentially expressed between schizophrenia patients and healthy controls may have key roles in the pathogenesis of schizophrenia. We analyzed two large-scale genome-wide expression studies, which examined changes in gene expression in schizophrenia patients and their matched controls. We found calcium/calmodulin (CAM)-dependent protein kinase kinase 2 (CAMKK2) is significantly downregulated in individuals with schizophrenia in both studies. To seek the potential genetic variants that may regulate the expression of CAMKK2, we investigated the association between single-nucleotide polymorphisms (SNPs) within CAMKK2 and the expression level of CAMKK2. We found one SNP, rs1063843, which is located in intron 17 of CAMKK2, is strongly associated with the expression level of CAMKK2 in human brains (P=1.1 × 10(-6)) and lymphoblastoid cell lines (the lowest P=8.4 × 10(-6)). We further investigated the association between rs1063843 and schizophrenia in multiple independent populations (a total of 130 623 subjects) and found rs1063843 is significantly associated with schizophrenia (P=5.17 × 10(-5)). Interestingly, we found the T allele of rs1063843, which is associated with lower expression level of CAMKK2, has a higher frequency in individuals with schizophrenia in all of the tested samples, suggesting rs1063843 may be a causal variant. We also found that rs1063843 is associated with cognitive function and personality in humans. In addition, protein-protein interaction (PPI) analysis revealed that CAMKK2 participates in a highly interconnected PPI network formed by top schizophrenia genes, which further supports the potential role of CAMKK2 in the pathogenesis of schizophrenia. Taken together, these converging lines of evidence strongly suggest that CAMKK2 may have pivotal roles in schizophrenia susceptibility.

Inhibition of Ca2+/Calmodulin–Dependent Protein Kinase Kinase 2 Stimulates Osteoblast Formation and Inhibits Osteoclast Differentiation

ABSTRACT Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2−/− mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2−/− mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser133 phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis.

Down‐regulation of calcium/calmodulin‐dependent protein kinase kinase 2 by androgen deprivation induces castration‐resistant prostate cancer

Conversion into androgen-hypersensitive state and adaptation to the low concentration of androgen during ADT cause relapse of prostate cancer (PCa). It is important to identify differentially expressed genes between PCa and normal prostate tissues and to reveal the function of these genes that are involved in progression of PCa. We performed cDNA microarray analysis to identify differentially expressed genes, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2). Immunohistochemical analysis was conducted to investigate the relationship between the CAMKK2 expression level and prognosis. The function of CAMKK2 was assessed by generating CAMKK2 overexpressed LNCaP cells and by knockdown of CAMKK2. We identified CAMKK2 overexpressed six times in PCa more than normal prostate by cDNA microarray analysis. Immunohistochemical analysis of CAMKK2 protein showed that CAMKK2 protein was expressed more in PCa than normal tissue. However, the expression in the high-grade PCa diminished. Moreover, the narrowness of CAMKK2-positive area before ADT was a poor prognostic factor. Androgen-deprivation treatment from the medium in which LNCaP cells were cultured in the presence of 10 nM DHT repressed CAMKK2 expression. CAMKK2 overexpressed LNCaP cells (LNCaP/GFP-CAMKK2) attenuated androgen-sensitivity. Tumorigenesis of LNCaP/GFP-CAMKK2 cells in male SCID mice was decreased compared with control cells irrespective of castration. Finally, knockdown of CAMKK2 mRNA in LNCaP cells induced androgen-hypersensitivity and stimulated LNCaP cell proliferation. Induction of androgen-hypersensitivity after ADT may be involved in down-regulation of CAMKK2. This result may provide new therapeutic approach to keep androgen-sensitivity of PCa after ADT.

Abstract 944: Down-regulation of CAMKK2 by androgen deprivation induces castration-resistant prostate cancer

Abstract One of the mechanisms through which prostate cancer (PCa) relapses after androgen deprivation therapy (ADT) is to become androgen-hypersensitive and adapt to the low concentration of androgen during ADT. cDNA microarray analysis between PCa tissues before ADT and normal prostate tissues revealed that the level of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) mRNA was overexpressed 6 times in PCa more than in normal prostate. Immunohistochemical analysis of CAMKK2 protein using prostate tissue microarray showed that CAMKK2 protein was expressed well in PCa compared with normal tissue. However, the expression of CAMKK2 for the high grade PCa diminished compared with the low grade PCa. Moreover, we confirmed that the narrowness of a positive regional area of the CAMKK2 expression before ADT was a poor prognostic factor. Expression of CAMKK2 mRNA was up-regulated in the presence of DHT in LNCaP cells. Stable overexpression of CAMKK2 in LNCaP cells (LNCaP/GFP-CAMKK2), however, attenuated androgen-sensitivity. Tumorigenesis of LNCaP/GFP-CAMKK2 cells by subcutaneous injection in male SCID mice was also inhibited compared with control cells irrespective of castration. In contrast, knockdown of CAMKK2 mRNA in LNCaP cells induced androgen-hypersensitivity and stimulated LNCaP cell proliferation without DHT. Overexpression and knockdown of CAMKK2 in the presence of DHT were correlated with activation and inactivation of AMP-activated protein kinase (AMPK) that is a key kinase important for regulating cell proliferation, respectively. AMPK activation inhibited LNCaP cell proliferation. Taken together, induction of androgen-hypersensitivity after ADT may be involved in down-regulation of CAMKK2. This result may provide new therapeutic approach to keep androgen-sensitivity of PCa after ADT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 944. doi:1538-7445.AM2012-944

Methyl Cinnamate Inhibits Adipocyte Differentiation via Activation of the CaMKK2–AMPK Pathway in 3T3-L1 Preadipocytes

Methyl cinnamate, an active component of Zanthoxylum armatum , is a widely used natural flavor compound with antimicrobial and tyrosinase inhibitor activities. However, the underlying bioactivity and molecular mechanisms of methyl cinnamate on adipocyte function and metabolism remain unclear. The aim of this study was to investigate the inhibitory effect of methyl cinnamate on adipogenesis in 3T3-L1 preadipocytes. Methyl cinnamate markedly suppressed triglyceride accumulation associated with down-regulation of adipogenic transcription factor expression, including sterol regulatory element binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT/enhancer-binding protein α (C/EBPα). Additionally, methyl cinnamate-inhibited PPARγ activity and adipocyte differentiation were partially reversed by the PPARγ agonist troglitazone. Furthermore, methyl cinnamate stimulated Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and phospho-AMP-activated protein kinase (AMPK) expression during adipogenesis. This study first revealed methyl cinnamate has antiadipogenic activity through mechanisms mediated, in part, by the CaMKK2-AMPK signaling pathway in 3T3-L1 cells.

Deletion of CaMKK2 from the Liver Lowers Blood Glucose and Improves Whole-Body Glucose Tolerance in the Mouse

Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2(floxed) mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content.

Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation

Regulation between protein kinases is critical for the establishment of signaling pathways/networks to 'orchestrate' cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells has barely been explored. Here we examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca++/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKKβ1 variant proteins were found to be kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKKβ1 was found to be essential for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (-E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter one enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks.

The Ca2+/Calmodulin-Dependent Protein Kinase Kinase, CaMKK2, Inhibits Preadipocyte Differentiation

When fed a standard chow diet, CaMKK2 null mice have increased adiposity and larger adipocytes than do wild-type mice, whereas energy balance is unchanged. Here, we show that Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in preadipocytes, where it functions as an AMP-activated protein kinase (AMPK)α kinase. Acute inhibition or deletion of CaMKK2 in preadipocytes enhances their differentiation into mature adipocytes, which can be reversed by 5-aminoimidazole-4-carboxamide ribonucleotide-mediated activation of AMPK. During adipogenesis, CaMKK2 expression is markedly decreased and temporally accompanied by increases in mRNA encoding the early adipogenic genes CCAAT/enhancer binding protein (C/EBP) β and C/EBP δ. Preadipocyte factor 1 has been reported to inhibit adipogenesis by up-regulating sex determining region Y-box 9 (Sox9) expression in preadipocytes and Sox9 suppresses C/EBPβ and C/EBPδ transcription. We show that inhibition of the CaMKK2/AMPK signaling cascade in preadipocytes reduces preadipocyte factor 1 and Sox9 mRNA resulting in accelerated adipogenesis. We conclude that CaMKK2 and AMPK function in a signaling pathway that participates in the regulation of adiposity.

Abstract P2-07-06: Role of CaMKK2-AMPK Signaling in Breast Cancer Metabolic and Hypoxic Stress Responses

Abstract Background: Adenosine monophosphate-activated protein kinase (AMPK) is a major regulator of energy homeostasis at both the cellular and whole-body levels. AMPK is activated by elevated intracellular AMP level and by phosphorylation on threonine 172 (T172) in the activation loop of the α catalytic subunit by upstream kinases, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Once activated, AMPK switches on ATP-producing pathways while switching off ATP-consuming processes. Recent studies suggest that AMPK also controls processes relevant to tumor development, including protein synthesis, cell cycle progression, cell growth, survival and stress-induced autophagy. The purpose of our study is to determine the role of CaMKK2 as an AMPK upstream kinase in breast cancer cells, as well as its function in regulating cell proliferation, migration and survival. We chose the prototypical ERα + MCF-7 breast cancer cell line as our research model since this cell line does not express CaMKK1, which has similar functions to CaMKK2 but cannot activate AMPK. Materials and Methods: Illumina cDNA expression array was preformed on MCF-7, T-47D and MDA-MB-231 breast cancer cell lines, and normalized by rank-invariant method. MCF-7 cells with suppressed CaMKK2 or LKB1 expression were established using lentiviral gene specific shRNA interference. Stable knockdown cells (shNon-Target, shCaMKK2, shLKB1) were subject to acute and chronic stress conditions (i.e. ionomycin, 2-deoxy-D-glucose, hypoxia) and signaling events were evaluated using immunoblot analysis. Proliferation, anchorage-independent growth and motility of the knockdown cells were determined by MTT, soft agar formation and in vitro migration assay. Xenograft tumor studies were performed by inoculating estradiol-supplemented athymic nude mice with shNon-Target, shCaMKK2 and shLKB1 MCF-7 cells into the mammary fat pad. Tumor growth was determined by external caliper measurements twice a week. Forty-five days post-injection, mice were euthanized, tumors removed, weighed and prepared for histological, protein and RNA analyses. Results: CaMKK2 and LKB1 mRNAs are expressed at a similar level in the ERα + and ERα — breast cancer cell lines tested in the Illumina cDNA expression array. CaMKK2 activated AMPK phosphorylation (T172) under increased intracellular Ca2+ level induced by ionomycin. Under acute stress conditions, CaMKK2 functioned as the predominant AMPK upstream kinase. However, when cells are subject to long-term metabolic and hypoxic stress, CaMKK2 and LKB1 were both required for AMPK activation. Loss of CaMKK2 in MCF-7 cells promoted a moderate growth advantage under metabolic stress, increased soft agar colony formation and elevated cell migration capacity. CaMKK2 knockdown xenografts demonstrated less ductal structure formation, decreased E-Cadherin expression and reduced angiogenesis, as well as reduced VEGF and Glut1 mRNA levels in vivo. Conclusions: We have demonstrated that Ca2+-CaMKK2 signaling plays an important role in regulating breast cancer cell proliferation, migration and survival pathways. These findings suggest that Ca2+ activation through CaMKK2 promotes AMPK signaling, survival and angiogenesis in response to stress conditions in breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-06.

Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Transforming growth factor-beta-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain alphaG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic alphaG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of alphaG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.