Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding

The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases

An AMPKa2-specific phospho-switch controls lysosomal targeting for activation

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy

The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body

The Spectrum of Neurological and White Matter Changes and Premutation Status Categories of Older Male Carriers of the FMR1 Alleles Are Linked to Genetic (CGG and FMR1 mRNA) and Cellular Stress (AMPK) Markers

The fragile X premutation (PM) allele contains a CGG expansion of 55–200 repeats in the FMR1 gene’s promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39–81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers – CGG repeat number and the levels of the FMR1 mRNA – were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85–90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings – suggestive of a role of this enzyme in the risk of FXTAS – need to be verified and expanded in future studies using larger samples and longitudinal assessment

Investigation of the structural and functional properties of the c-Jun N-terminal kinase (JNK)

Publications included in thesis:Ngoei, K. R. W., Catimel, B., Church, N., Lio, D. S., Dogovski, C., Perugini, M. A., Watt, P. M., Cheng, H., Ng, D. C. & Bogoyevitch, M. A. (2011). Characterization of a novel JNK (c-Jun N-terminal kinase) inhibitory peptide. Biochemical Journal, 434(3), 399-413. DOI: 10.1042/BJ20101244Ngoei, K. R. W., Ng, D. C., Gooley, P. R., Fairlie, D. P., Stoermer, M. J. & Bogoyevitch, M. A. (2013). Identification and characterization of bi-thiazole-2,2'-diamines as kinase inhibitory scaffolds. Biochimica et Biophysica Acta, 1834(6), 1077-1088. DOI: 10.1016/j.bbapap.2013.02.001Ngoei, K. R. W., Catimel, B., Milech, N., Watt, P. M. & Bogoyevitch, M. A. (2013). A novel retro-inverso peptide is a preferential JNK substrate-competitive inhibitor. International Journal of Biochemistry & Cell Biology, 45(8), 1939-1950. DOI: 10.1016/j.biocel.2013.06.006© 2013 Dr. Kevin Ronald NgoeiThe c-Jun N-terminal kinases (JNKs) are members of the larger group of mitogen-activated protein kinases (MAPKs), and are activated in response to various stimuli such as cellular stresses and cytokines. As the JNKs have been considered as therapeutic targets in the treatment of a number of pathologies such as stroke, neurodegenerative diseases (Alzheimer’s disease and Parkinson’s disease) and diabetes, an improved understanding of the biochemical regulation of the JNKs will likely impact on the development of a JNK-specific inhibitor for therapeutic purposes. In this PhD project, the aim has been to probe the biochemical properties of the JNKs with the development of new peptide and small molecule JNK inhibitors and by examining JNK interactions with a substrate that does not appear to conform to the canonical JNK recognition motif. In combination, the results from these studies will allow further elucidation of the requirements for JNK recognition of its substrates. In Chapter 2, a novel JNK-inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating transcription factor 2) and Elk1, and the microtubule-regulatory protein DCX (doublecortin) is described. Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11, within an F-X-F motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each alanine substitution decreased the interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1.Taken together, the results of this study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation. Chapter 3 capitalized on the current biochemical understanding of JNK in which an in silico screen of commercially available chemical databases to identify JNK1-interacting compounds was performed and the hits were subsequently tested for their in vitro JNK inhibitory activity. From in vitro and cell culture studies, a compound, 4'-methyl-N(2)-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123), but not the related compound (4'-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ was noted. The further screening of a broad panel of kinases using 10 μM JD123 identified inhibition of several other kinases, including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases such as PKBβ/Aktβ that are also potential therapeutic targets. In Chapter 4, a novel 18 amino acid peptide PYC98 was demonstrated to inhibit JNK1 activity toward c-Jun. A 5-fold increase in the potency of the retro-inverso form of the peptide, D-PYC98 (a D-amino acid peptide in the reverse sequence) was observed when compared with the inhibition achieved by L-PYC98, which prompted further evaluation of the D-PYC98 inhibitory mechanism. In vitro assays revealed that, in addition to the inhibition of c-Jun phosphorylation, D-PYC98 inhibited the JNK1-mediated phosphorylation of an EGFR-derived peptide, the ATF2 transcription factor, and the microtubule-regulatory protein DCX. Moreover, JNK2 and JNK3 activities toward c-Jun were also inhibited in vitro. Biophysical interaction studies showed direct D-PYC98 interaction with JNK1, but not towards JNK substrate, c-Jun and Elk-1. Further kinetics analyses revealed the non-ATP competitive mechanism of action of D-PYC98 as a JNK1 inhibitor. The targeting of the JNK1 common docking site by D-PYC98 was confirmed by the competition of binding by TIJIP. However, as mutations of JNK1 R127 and E329 within the common docking domain did not impact on the affinity of the interaction with D-PYC98 measured by surface plasmon resonance analysis, other residues in the common docking site appear to contribute to the JNK1 interaction with D-PYC98. Furthermore, it was observed that D-PYC98 inhibited the related kinase p38 MAPK, suggesting a broader interest in developing D-PYC98 for possible therapeutic applications. Lastly, in evaluating the efficacy of this peptide to act as a substrate competitive inhibitor in cells, the ability of cell-permeable D-PYC98-TAT to inhibit c-Jun Ser63 phosphorylation was highlighted during hyperosmotic stress. Thus, D-PYC98-TAT is a novel cell-permeable JNK inhibitor. Overall, the identification of D-PYC98 as a novel, retro-inverso peptide inhibitor of JNK provides a step towards further development of peptide inhibitors with improved stability and specificity to evaluate various JNK-mediated events in cellular context. In Chapter 5, the knowledge on JNK recognition was further expanded through studies to define the structural and functional properties of the microtubule-associated protein, doublecortin (DCX) as a JNK substrate. The evolutionary-conserved DC domains in DCX have been previously described to be important for JNK-DCX interaction. Both in vitro biochemical assays and biophysical studies showed that mutations that disrupt the JNK common docking region reduced the interaction of JNK with DCX. As naturally-occurring DCX pathogenic mutations clustering on the DC domains cause abnormal neuronal migration during brain development leading to X-linked lissencephaly in males and subcortical band heterotopia (SBH) in females, a number of mutations on the surface of N-terminal DC domain (NDC) previously reported to disrupt the DCX-tubulin interface were subsequently evaluated. Biochemical studies revealed DCX R78L or R89G mutants, but not the DCX R59L, R78H and R102S mutants, result in increased JNK phosphorylation. NMR 15N-HSQC experiments for the NDC R78L or R78H mutants suggested that the R78L mutation caused a broad perturbation on the JNK1 binding surface area whereas the R78H mutation caused minor changes mostly restricted around the site of mutation, with neither R78 mutant appearing to destabilize NDC. Interestingly, the increased JNK-mediated phosphorylation of DCX R78L led to the use of mass spectrometry analysis and the identification of a novel JNK phosphorylation site at DCX S28, within an N-terminal region thought to be unstructured. This site was confirmed through site-directed mutagenesis and phospho-specific antibody detection studies. Taken together, these structural insights on the putative JNK-DC domain interaction interface define a non-conventional mode of JNK substrate recognition, in addition to a new phosphorylation site at DCX S28 that may also contribute to DCX-mediated microtubule regulation. In summary, this project has identified and characterized several novel JNK inhibitors including JNK-inhibitory peptides PYC71N and D-PYC98, and a small-molecule inhibitor JD123. While additional studies to improve the potency and efficacy of these inhibitors are warranted, these inhibitors can be used as research tools for studying the JNKs. Furthermore, the links between the microtubule-associated protein, DCX and JNK signalling was further strengthened with new insights on the JNK-DCX interaction as well as identification of a novel JNK-mediated DCX phosphorylation site. Overall, these studies have enhanced our current understanding of molecular recognition by JNK and may inform future developments of new JNK inhibitors as novel therapeutics

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges

studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues

Identification and characterization of bi-thiazole-2,2'-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4'-methyl-N-2-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123, but not the related compound (4'-methyl-N-2--(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-gamma MAPK, but not ERK1, ERK2, or p38-alpha, p38-beta or p38-delta. Further screening of a broad panel of kinases using 10 mu M JD123, identified inhibition of kinases including protein kinase B beta (PKB beta/Akt beta). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-gamma MAPKs as well as other kinases that are also potential therapeutic targets such as PKB beta/Akt beta. (C) 2013 Elsevier B.V. All rights reserved

DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy

Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis