title: Cellular energetic status supervises the synthesis of bis-diphosphoinositol tetrakisphosphate independently of AMP-activated protein kinase.
abstract: Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP](2)-InsP(4)), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP](2)-InsP(4) synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP](2)-InsP(4) synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP](2)-InsP(4) levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP](2)-InsP(4) synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2'-amino-3'-methoxyflavone (PD98059), had similar off-target effects. We conclude that the levels and hence the signaling strength of [PP](2)-InsP(4) is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.
authors.affiliation: Inositide Signaling Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Kuicheon
authors.initials: K
authors.lastname: Choi
authors.forename: Elahe
authors.initials: E
authors.lastname: Mollapour
authors.forename: Jae H
authors.initials: JH
authors.lastname: Choi
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D055372:AMP-Activated Protein Kinases; D000818:Animals; D002478:Cells, Cultured; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism* / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D051381:Rats
chemical_list: D007295:Inositol Phosphates; D009097:Multienzyme Complexes; D047428:Protein Kinase Inhibitors; C519007:inositol 1,2,3,4-tetraphosphate 5,6-dipyrophosphate; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases