Woods, A. et al. Ca2+/calmodulin-dependent protein kinase kinase- acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2, 21-33

Columbia University, New York, New York, United States
Cell Metabolism (Impact Factor: 17.57). 08/2005; 2(1):21-33. DOI: 10.1016/j.cmet.2005.06.005
Source: PubMed


AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that plays a pivotal role in energy homeostasis. Activation of AMPK requires phosphorylation of threonine 172 (T172) within the T loop region of the catalytic alpha subunit. Recently, LKB1 was shown to activate AMPK. Here we show that AMPK is also activated by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). Overexpression of CaMKKbeta in mammalian cells increases AMPK activity, whereas pharmacological inhibition of CaMKK, or downregulation of CaMKKbeta using RNA interference, almost completely abolishes AMPK activation. CaMKKbeta isolated from rat brain or expressed in E. coli phosphorylates and activates AMPK in vitro. In yeast, CaMKKbeta expression rescues a mutant strain lacking the three kinases upstream of Snf1, the yeast homolog of AMPK. These results demonstrate that AMPK is regulated by at least two upstream kinases and suggest that AMPK may play a role in Ca(2+)-mediated signal transduction pathways.

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Available from: Richard Heath, Oct 02, 2015
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    • "In the absence of extracellular Ca 2+ , the phospho-Thr172-AMPK was not affected by STO-609 even at the highest concentration of 50μM, showing the dependence of CaMKKs towards extracellular Ca 2+ for AMPK phosphorylation. STO-609 is a selective inhibitor of both CaMKKα and CaMKK β[50], but it could inhibit other kinases such as AMPK itself when used at high concentrations in vitro1218]. Such action was ruled out here since AICAR-induced phosphorylation of AMPK was not affected by STO-609. "
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    ABSTRACT: Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells.
    Full-text · Article · Jan 2016 · PLoS ONE
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    • "Although there is no evidence of any Ca 2+ / CaM-dependent activity of Ssp1, it has recently been shown that Ssp1 has a conserved putative calmodulin binding domain (CBD) and a short stretch outside the kinase domain when compared to the amino acid sequences of human CaMKK1 and CaMKK2 [14]. In addition, Ssp1 shares a functional substrate with human CaMKKs, the AMP-activated protein kinase (Ssp2)1415161718 . Initially, Ssp1 kinase was reported to be required for growth polarity and actin localization at high temperature [19, 20]. "
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    ABSTRACT: Background: Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. Results: Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. Conclusions: These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role.
    Full-text · Article · Nov 2015 · PLoS ONE
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    • "We induced energy stress in HEK293 (expressing empty vector or FLAG SENP1) by subjecting the cells to low-glucose conditions, low glucose together with glycolysis inhibitor 2-DG, or treatment with phenformin (a biguanide compound that inhibits complex 1 of the mitochondria). CAMKK inhibitor STO-609 was included in the treatment regimen since CAMKKb can compensate for LKB1 by phosphorylating AMPK on the same site (Woods et al., 2005). Interestingly, immunoprecipitation of LKB1 revealed an increase in SUMO1 modification of LKB1 as the intracellular ATP levels declined (Figures 1C and S1D). "
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    ABSTRACT: SUMOylation has been implicated in cellular stress adaptation, but its role in regulating liver kinase B1 (LKB1), a major upstream kinase of the energy sensor AMP-activated protein kinase (AMPK), is unknown. Here, we show that energy stress triggers an increase in SUMO1 modification of LKB1, despite a global reduction in both SUMO1 and SUMO2/3 conjugates. During metabolic stress, SUMO1 modification of LKB1 lysine 178 is essential in promoting its interaction with AMPK via a SUMO-interacting motif (SIM) essential for AMPK activation. The LKB1 K178R SUMO mutant had defective AMPK signaling and mitochondrial function, inducing death in energy-deprived cells. These results provide additional insight into how LKB1-AMPK signaling is regulated during energy stress, and they highlight the critical role of SUMOylation in maintaining the cell's energy equilibrium. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Cell Reports
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