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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

ABSTRACT

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.
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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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    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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