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-InsP4), 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-InsP4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP]2-InsP4 synthesis without affecting other inositol phosphates. In addition, we report that the shortterm increases in [PP]2-InsP4 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-InsP4 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-InsP4 is supervised by cellular adenosine nucleotide balance, signifying a new link between signaling and bioenergetic networks.
Footnotes
-
This research was supported by the Intramural Research Program of the National Institutes of Health/National Institute of Environmental Health Sciences.
-
K.C. and E.M. contributed equally to this study.
-
ABBREVIATIONS: InsP7, PP-InsP5, diphosphoinositol pentakisphosphate; [PP]2-InsP4, InsP8, bis-diphosphoinositol tetrakisphosphate; InsP5, inositol pentakisphosphate; InsP6, inositol hexakisphosphate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; DN, dominant negative; AMPK, AMP-activated protein kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK, human embryonic kidney; MEF, mouse embryonic fibroblast; PP-InsP4, diphosphoinositol tetrakisphosphate; PPIP5K, PP-InsP5 kinase (E.C. 2.7.4.24), ZMP, 5-amino-4-imidazolecarboxamide riboside monophosphate; PD184352, 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide; MEK, mitogen-activated protein kinase kinase; siRNA, small interfering RNA; ERK, extracellular signal-regulated kinase; HPLC, high-performance liquid chromatography; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; PD98059, 2′-amino-3′-methoxyflavone.
-
↵1 Current affiliation: Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas
- Received December 21, 2007.
- Accepted May 6, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|