Inactivation of MARK4, an AMP-activated Protein Kinase (AMPK)-related Kinase,Leads to Insulin Hypersensitivity and Resistance to Diet-induced Obesity

MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes.     

MARK2/4 promotes Warburg effect and cell growth in non-small cell lung carcinoma through the AMPKα1/mTOR/HIF-1α signaling pathway

MARKs kinase belongs to an AMPK-related family kinase plays a critical role in tumor progression, but its exact role and contribution of four different isoforms remain largely ambiguous. In this study, we used a clinical dataset compiled by The Cancer Genome Atlas (TCGA) and GEO revealed that MARK2 and MARK4 expressions were significantly upregulated in non-small cell lung cancer (NSCLC) compared with normal tissues. Furthermore, expressions of MARK2/4 were highly appeared in advanced stages and associated with the low survival rate of NSCLC patients. Functional assays demonstrated that MARK2/4 deletion or MARKs inhibition significantly suppressed aerobic glycolysis and cell growth in NSCLC cells. Mechanistically, MARK2/4 stimulates the mTOR/HIF-1α pathway and subsequently alleviates AMPK activity via physically associate with Raptor and AMPKα1, thereby facilitating aerobic glycolysis and cell growth in NSCLC cells. However, these effects were markedly reversed by MARKs inhibitor 39621, or MARK2/4 deletion, mTOR inhibitor rapamycin, or AMPK activator AICAR. Together, the data demonstrated that MARK2/4 exerts its oncogenic effects by facilitating metabolic reprogramming in NSCLC cells. Therefore, MARK2/4 might be a potential therapeutic target for lung cancer.     

Glycogen synthase kinase (GSK) 3beta directly phosphorylates Serine 212 in the regulatory loop and inhibits microtubule affinity-regulating kinase (MARK) 2

MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3beta serves the role of the inhibitory kinase. Because GSK3beta can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3beta, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.     

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.     

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.     

Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.     

Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway

5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)-->extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEK-->ERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease.     

Regulation of the AMPK-related protein kinases by ubiquitination

How can a constitutively active 'master' kinase with numerous downstream targets preferentially phosphorylate one or more of these without influencing all simultaneously? How might such a system be switched off? The characterization of the role of deubiquitination in regulating the phosphorylation and activation of AMPK (AMP-activated protein kinase)-related kinases by LKB1 suggests a novel and interesting mechanism for conferring signal transduction specificity and control at the kinase substrate level. In this issue of the Biochemical Journal, Al-Hakim et al. show that the AMPK-related kinases NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4) are polyubiquitinated in vivo and that they serve as substrates of the deubiquitinating enzyme USP9X; furthermore, the first evidence is provided for regulation of AMPK-related kinase family members mediated via unusual Lys(29)/Lys(33) polyubiquitin chains, rather than the more common Lys(48)/Lys(63) linkages.     

Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK->ERK1/2 pathway

5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEKERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease. 5'-AMP-activated protein kinase; nuclear transport     

Prostaglandin E2 signalling pathway in human T lymphocytes from healthy and conjunctiva basal cell carcinoma-bearing subjects

Prostaglandin E-induced signal transduction pathways in human T cells from healthy and uveal melanoma-bearing subjects were studied. Transfection experiments showed that PGE2 was able to phosphorylate and activate the fusion trans-activator of the cAMP responsive element-binding protein (CREB). Phosphorylation was at least partially mediated by protein kinase A, as evidenced by the effects of specific kinase inhibitors. Western blotting experiments, which were performed to identify the CREB/ATF2 family members involved in the response to PGE2, revealed a modulation of proteins CREB1, CREB2 and ATF2 and phosphorylation of the 43 kDa form of CREB. Experiments of immunoprecipitation with CREB-binding protein (CBP) demonstrated that, after PGE2 treatment, all of the CREB/ATF isoforms studied, as well as the phosphorylated form of CREB (p-CREB), interacted with CBP. In basal conditions, T cells from patients with conjunctiva basal cell carcinoma showed the presence of p-CREB, which coimmunoprecipitated with CBP. CREB phosphorylation did not modify after PGE2 treatment whereas the p-CREB fraction bound to CBP increased in a delayed manner compared to normal subjects.     

Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation and is highly conserved from yeast to mammals. The upstream kinases are also functionally conserved, and the AMPK kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase activate Snf1 in mutant yeast cells lacking the native Snf1-activating kinases, Sak1, Tos3, and Elm1. Here, we exploited the yeast genetic system to identify members of the mammalian AMPK kinase family by their function as Snf1-activating kinases. A mouse embryo cDNA library in a yeast expression vector was used to transform sak1Delta tos3Delta elm1Delta yeast cells. Selection for a Snf+ growth phenotype yielded cDNA plasmids expressing LKB1, Ca2+/calmodulin-dependent protein kinase kinase, and transforming growth factor-beta-activated kinase (TAK1), a member of the mitogen-activated protein kinase kinase kinase family. We present genetic and biochemical evidence that TAK1 activates Snf1 protein kinase in vivo and in vitro. We further show that recombinant TAK1, fused to the activation domain of its binding partner TAB1, phosphorylates Thr-172 in the activation loop of the AMPK catalytic domain. Finally, expression of TAK1 and TAB1 in HeLa cells or treatment of cells with cytokines stimulated phosphorylation of Thr-172 of AMPK. These findings indicate that TAK1 is a functional member of the Snf1/AMPK kinase family and support TAK1 as a candidate for an authentic AMPK kinase in mammalian cells.     

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

Hyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK (AMP-activated protein kinase)-related kinases MARK (microtubule-associated protein-regulating kinase/microtubule affinity-regulating kinase) and BRSK (brain-specific kinase) have been implicated in tau phosphorylation, but are insensitive to activation by cellular stress. In the present study, we show that AMPK itself phosphorylates tau on a number of sites, including Ser2?2 and Ser3??, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) activation of AMPK in response to Aβ (amyloid-β peptide)-(1-42) leads to increased phosphorylation of tau at Ser2?2/Ser3?? and Ser33??. Activation of AMPK by Aβ-(1-42) is inhibited by memantine, a partial antagonist of the NMDA (N-methyl-D-aspartate) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ-(1-42) activates CaMKKβ and AMPK via the NMDA receptor, suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau.     

PTEN inhibition attenuates endothelial cell apoptosis in coronary heart disease via modulating the AMPK–CREB–Mfn2-mitophagy signaling pathway

Atherosclerosis (AS) is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in the occurrence and development of AS. In our study, we attempted to evaluate the role of phosphatase and tensin homolog (PTEN) in endothelial cell apoptosis under oxidized low-density lipoprotein (ox-LDL) stimulation and identify the associated mechanisms. The results of our study demonstrated that ox-LDL induced human umbilical vein endothelial cell (HUVEC) death via mitochondrial apoptosis, as evidenced by reduced mitochondrial potential, increased mitochondria permeability transition pore opening, cellular calcium overload, and caspase-9/-3 activation. In addition, ox-LDL also suppressed cellular energy production via downregulating the mitochondrial respiratory complex. Moreover, ox-LDL impaired HUVECs migration. Western blot analysis showed that PTEN expression was upregulated after exposure to ox-LDL and knockdown of PTEN could attenuate ox-LDL-mediated endothelial cell damage. Furthermore, we found that ox-LDL impaired mitophagy activity, whereas PTEN deletion could improve mitophagic flux and this effect relied on the activity of the AMP-activated protein kinase (AMPK)–cAMP-response element-binding protein (CREB)–Mitofusin-2 (Mfn2) axis. When the AMPK–CREB–Mfn2 pathway was inhibited, PTEN deletion-associated HUVECs protection was significantly reduced, suggesting that the AMPK–CREB–Mfn2-mitophagy axis is required for PTEN deletion-mediated endothelial cell survival under ox-LDL. Taken together, our results indicate that ox-LDL-induced endothelial cell damage is associated with PTEN overexpression, and inhibition of PTEN could promote endothelial survival via activating the AMPK–CREB–Mfn2-mitophagy signaling pathway.