Calcium Calmodulin Kinase Kinase 2
CaMKK2 was originally purified and characterized from rat brain as a CaMKI-activating kinase by Edelman et al. (1996). These authors demonstrated that CaMKK2 activated CaMKI and CaMKIV by phosphorylating an equivalent Thr residue within the “activation loop” region of both proteins. Later, rat and human CaMKK2 were cloned and characterized by two independent groups, which provided substantial information about the structure and function of CaMKK2 (Anderson et al. 1998; Kitani et al. 1997). The cAMP-activated protein kinase (AMPK) was subsequently identified as an additional substrate of CaMKK2 in 2005 (Hawley et al. 2005; Woods et al. 2005). The discovery of AMPK as a downstream target of CaMKK2 was an important milestone, which opened new research perspectives, providing mechanistic evidence for studying CaMKK2 regulation of general homeostasis, metabolism, and inflammatory responses (Racioppi and Means 2012; Marcelo et al. 2016).
CaMKK2 Structure and Function
CaMKK2 and Cell Development
The role of CaMKK2 in mediating cell development has been documented in neurons, adipose tissue, hematopoiesis, bone remodeling, and lineage reprogramming of pancreatic alpha and beta cells.
Neurons: CaMKK2 is highly expressed in different areas of the brain and is essential for neuronal development. It controls neuronal cytoskeleton remodeling in the hippocampus, the proliferation and migration of granule cells in cerebellar development, and the formation of spines and synaptic connections between neurons. Perhaps unsurprisingly, CaMKK2 deficiency impairs hippocampal long-term memory and spatial memory formation.
Adipocytes: CaMKK2 is expressed in preadipocytes but is not detectable in mature adipocytes. Genetic ablation or pharmacological inhibition of CaMKK2 accelerates differentiation of preadipocytes towards white adipocytes.
Blood cells: The expression of CaMKK2 is developmentally regulated in hematopoietic cells, and this kinase is selectively expressed in the myeloid lineage (Fig. 2). Genetic ablation of CaMKK2 facilitates the development of myeloid progenitors toward granulopoiesis.
Bone: CaMKK2 regulates bone remodeling by controlling the balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone matrix reabsorption. In in vitro culture, inhibiting CaMKK2 activity enhanced the differentiation of OB with higher alkaline phosphatase activity, and suppressed OC differentiation from mesenchymal stem cells (MSC).
Endocrine pancreas: The expression and phosphorylation of CaMKK2 are decreased in beta cells in comparison to alpha cells. Genetic ablation of CaMKK2 in alpha cells resulted in expression of beta-cell markers and increased insulin secretion.
CaMKK2 and the Nervous System
Isolated neurons: In cultured isolated neurons, CaMKK2 is required for the regulation of axonal growth cone morphology and outgrowth, dendritic arborization, and the formation of neuronal spines and synapses. Differential splicing of CaMKK2 transcripts and phosphorylation of critical Ser residues affect the ability of CaMKK2 to control dendrite/axon formation.
Hippocampal memory: Germ-line ablation of CaMKK2 impairs long-term memory formation. The absence of CaMKK2 is associated with selective loss of long-term potentiation at hippocampal CA1 synapses and a decrease in spatial training-induced cAMP response-element binding protein (CREB) activation in the hippocampus.
Cerebellar development: CaMKK2 is expressed in the cerebellum, as well as in isolated cerebellar granule cells (CGCs), which are the most abundant neurons in the cerebellum. Studies with mouse models revealed that loss of CaMKK2 impairs the ability of GCPs to cease proliferation and migrate to the internal granule layer.
Hypothalamus: In hypothalamic Neuropeptide Y/Agouti-related protein (NPY/AgRP) neurons, ghrelin determines the activation of the CaMKK2/AMPK pathway stimulating NPY synthesis. Accordingly, genetic ablation of CaMKK2 impairs hypothalamic AMPK activity and downregulates NPY and AgRP gene expression in NPY neurons, thus protecting mice from diet-induced obesity, hyperglycemia, and insulin resistance (Anderson et al. 2008). In hypothalamic ventromedial nuclei, CaMKK2 is coupled to 5-Hydroxytryptamine Receptor 2C (Htr2c) serotonin receptor signaling. Brain-derived serotonin stimulates CaMKK2 to phosphorylate CREB in response to signaling. Thus, CaMKK2 is required for regulating the expression of genes necessary for optimal sympathetic activity and, in turn, bone mass accrual, which is negatively correlated with sympathetic tone (Racioppi and Means 2012).
Brain and mental disorders: In human, CaMKK2 has been associated with bipolar affective disorders and has also been identified as a schizophrenia susceptibility gene (Barden et al. 2006; Luo et al. 2014). Mechanistically, it has been proposed that the T85S mutation in CaMKK2, which is linked to anxiety and bipolar disorder, disrupts the autophosphorylation and autonomous activity of CaMKK2 (Scott et al. 2015). CaMKK2 may also be involved in the progression of Alzheimer’s disease (AD). In this case, CaMKK2 is required for mediating the synaptotoxic effects of amyloid-β 1–42 (Aβ42) oligomers, as measured by dendritic spine loss on isolated hippocampal neurons. Interestingly, in vivo inhibition of CaMKK2 was observed to protect against the loss of dendritic spines in a mouse model of AD (Mairet-Coello et al. 2013).
CaMKK2 and Metabolism
CaMKK2 is functionally involved in regulating the whole-body energy homeostasis by controlling the satiety signal in the brain, metabolic conversion in the liver, energy storage in adipose tissue, and hormone secretion and sensitivity in the pancreas (Marcelo et al. 2016).
Hypothalamus: CaMKK2 plays a fundamental role in regulating systemic energy homeostasis by integrating the metabolic signals from peripheral organs. CaMKK2 is highly expressed in the hypothalamus. By phosphorylating AMPK and CaMKIV/CREB respectively, CaMKK2 regulates the orexigenic signals and serotonin signals in hypothalamus to modulate appetite and energy expenditure.
Liver: CaMKK2 is expressed in isolated cultured hepatocytes. Suppressing CaMKK2 in hepatocytes leads to decreased glucose utilization and increased lipogenesis and lipid oxidation, indicating a switch from glucose to fat metabolism, and improved whole-body glucose homeostasis.
Adipose tissue: CaMKK2 suppresses the differentiation of preadipocytes to mature adipocytes. While CaMKK2 is usually not expressed in mature adipocytes, its expression can be induced under thyroid or glucagon stimulation and regulates the adipose function.
Pancreas: CaMKK2 restrains beta-cell differentiation and insulin secretion. CaMKK2 regulates insulin-mediated metabolism by controlling leptin-induced insulin production under fasting conditions, or uridine diphosphate-induced insulin secretion under high glucose conditions.
Cardiovascular system: CaMKK2 mediates antiinflammatory and antioxidative responses in endothelial cells, protecting against atherosclerosis in the aorta.
CaMKK2 and Hematopoietic Cells
Granulopoiesis: The expression of CaMKK2 is developmentally regulated during granulocyte differentiation, with CaMKK2 being barely detectable in mature granulocytes. Deletion of CaMKK2 in engrafted hematopoietic stem cells (HSC) resulted in increased production of mature granulocytes in the BM and peripheral blood. Overexpression of CaMKK2 in the 32D myeloid cell line is sufficient to prevent granulocyte differentiation. These results identify CaMKK2 as a key regulator of granulocytic differentiation in early myeloid progenitors (Teng et al. 2011).
Macrophages: CaMKK2 is expressed selectively in monocytes/macrophages. Its ablation impairs the ability of macrophages to spread, phagocytize bacteria, and release cytokines/chemokines in response to bacterial lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncouples LPS-mediated signaling from the phosphorylation of protein tyrosine kinase 2 (PYK2/PTK2B), and activation of its downstream effectors, such as ERK1/2, NFκB, c-Jun, and AKT. In vivo, CaMKK2-null mice are protected from macrophage-mediated inflammation, as measured by endotoxin shock and fulminant hepatitis induced by bacterial LPS. Genetic ablation of CaMKK2 also protects mice from high fat diet-induced inflammation (Racioppi et al. 2012) which is partially mediated by macrophages.
CaMKK2 and Cancer
CaMKK2 is overexpressed in most malignant cells (Human Protein Atlas, http://www.proteinatlas.org/) where it regulates cellular survival, proliferation, migration, invasion, and metabolism (Racioppi and Means 2012; Marcelo et al. 2016).
Prostate cancer: CaMKK2 is overexpressed in human prostate cancer (PCa) and has been identified as a primary gene regulated by the androgen receptor (AR) signaling pathway. Accordingly, stimulation of AR signaling upregulates CaMKK2 expression in PCa cells, and promotes PCa progression by regulating gene expression that controls cell survival, proliferation, migration, and metabolism. In contrast, genetic ablation or pharmacological blockade of CaMKK2 restrains PCa growth in vivo and inhibits proliferation and migration of PCa cells in vitro.
Gastric cancer: CaMKK2 is overexpressed in gastric tumor tissues, and silencing of CaMKK2 expression using siRNA significantly inhibits proliferation, colony formation, and invasion of gastric cancer cells. It has been demonstrated that CaMKK2 signals in gastric cancer through the activation of AMPK. Thus, CaMKK2 could be a novel therapeutic target in gastric cancer.
Hepatic cancer: CaMKK2 is significantly upregulated in hepatocellular carcinoma (HCC) and is negatively correlated with prognosis. Genetic ablation of CaMKK2 by siRNA inhibits liver cancer cell growth in vitro and yields a gene expression signature that correlates with improvement in HCC patient survival. Ablation or pharmacological blockade of CaMKK2 has also been shown to impair the tumorigenicity of liver cancer cells in vivo.
CaMKK2 is one of the most versatile of the CaMKs, given its ability to phosphorylate and activate CaMKI, CaMKIV, and AMP-activated protein kinase. CaMKK2 is prevalently expressed in cells with stem/progenitor features, including neuronal progenitors, preadipocytes, hematopoietic stem/progenitor cells, and circulating monocytes. This kinase negatively regulates lineage fate, differentiation, and proliferation of stem/progenitor cells in cerebellum, adipose tissue, and hematopoietic system. CaMKK2 is also expressed at a detectable level in a small number of terminally differentiated cells, including neurons and macrophages. This kinase has an important function in hypothalamic nuclei, which regulate food intake, energy expenditure, and overall systemic homeostasis and metabolism. In addition, CaMKK2 regulates bone formation and the inflammatory immune response. In human diseases, CaMKK2 have been found associated with multiple brain and mental disorders. This kinase is also overexpressed in several types of tumors including prostate, gastric, and liver cancer. Importantly, the expression of CaMKK2 negatively correlates with malignancy prognosis, and the experimental pharmacological blockade of CaMKK2 has been shown to inhibit the growth of tumors. Taken together, the studies on the physiopathology of CaMKK2 identify this kinase as an important molecular hub that regulates key functions in normal and pathological cell types.
Pilot grant from the Opportunity Funds Management Core of the Centers for Medical Countermeasures against Radiation, National Institute of Allergy and Infectious Diseases; grant number U19AI067773 awarded to L.R
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