CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells

Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKα^Thr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKα^Thr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα ^Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase.     

DJ-1 regulates tyrosine hydroxylase expression through CaMKKβ/CaMKIV/CREB1 pathway in vitro and in vivo

Lack of dopamine production and neurodegeneration of dopaminergic neurons in the substantia nigra are considered as the major characteristics of Parkinson's disease, a prevalent movement disorder worldwide. DJ-1 mutation leading to loss of its protein functions is a genetic factor of PD. In this study, our results illustrated that DJ-1 can directly interact with Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) and modifies the cAMP-responsive element binding protein 1 (CREB1) activity, thus regulates tyrosine hydroxylase (TH) expression. In Dj-1 knockout mouse substantia nigra, the levels of TH and the phosphorylation of CREB1 Ser133 are significantly decreased. Moreover, Dj-1 deficiency suppresses the phosphorylation of CaMKIV (Thr196/200) and CREB1 (Ser133), subsequently inhibits TH expression in vitro. Furthermore, Knockdown of Creb1 abolishes the effects of DJ-1 on TH regulation. Our data reveal a novel pathway in which DJ-1 regulates CaMKKβ/CaMKIV/CREB1 activities to facilitate TH expression.     

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase

Activation of 5′-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5′-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase ce:sup>/ce:sup>/Mn²⁺-dependent (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the ce:sup>/ce:sup>/Mn²⁺-dependent protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggests that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

WSF-P-1, a novel AMPK activator,promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). The HMW adiponectin oligomers possess the most potent insulin-sensitizing activity. WSF-P-1(N-methyl-1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-7H-2,4α-methanonaphthalen-7-amine) is derived from natural sesquiterpene longifolene by chemical modifications. We found that WSF-P-1 activates AMPK in both 3T3-L1 adipocytes and 293T cells in this study. Activation of AMPK by WSF-P-1 promotes the assembly of HMW adiponectin and increases the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. We demonstrated that the Ca²⁺-dependent CaMKK signaling pathway is involved in WSF-P-1-induced AMPK activation and adiponectin multimerization. WSF-P-1 also activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes, making it a potential drug candidate for the treatment of type 2 diabetes, obesity, and other obesity-related metabolic diseases.     

Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

Hypothalamic lipotoxicity and the metabolic syndrome

Ectopic accumulation of lipids in peripheral tissues, such as pancreatic β cells, liver, heart and skeletal muscle, leads to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. Current evidence has demonstrated that hypothalamic sensing of circulating lipids and modulation of hypothalamic endogenous fatty acid and lipid metabolism are two bona fide mechanisms modulating energy homeostasis at the whole body level. Key enzymes, such as AMP-activated protein kinase (AMPK) and fatty acid synthase (FAS), as well as intermediate metabolites, such as malonyl-CoA and long-chain fatty acids-CoA (LCFAs-CoA), play a major role in this neuronal network, integrating peripheral signals with classical neuropeptide-based mechanisms. However, one key question to be addressed is whether impairment of lipid metabolism and accumulation of specific lipid species in the hypothalamus, leading to lipotoxicity, have deleterious effects on hypothalamic neurons. In this review, we summarize what is known about hypothalamic lipid metabolism with focus on the events associated to lipotoxicity, such as endoplasmic reticulum (ER) stress in the hypothalamus. A better understanding of these molecular mechanisms will help to identify new drug targets for the treatment of obesity and metabolic syndrome.     

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.     

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Background

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca²⁺/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.