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PMID: 22621786 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636.

American journal of physiology. Cell physiology ·Vol. 303 ·No. 4 ·2012-08-15 ·Pages C436-46

Sbrissa D, Ikonomov OC, Filios C, Delvecchio K, Shisheva A

Abstract

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

MeSH Terms
3T3-L1 Cells Aminopyridines/pharmacology Animals CHO Cells Cricetinae DNA-Binding Proteins/genetics,metabolism HEK293 Cells Heterocyclic Compounds, 3-Ring/pharmacology Humans Insulin Mice Phosphatidylinositol 3-Kinases/genetics,metabolism Phosphatidylinositol Phosphates/biosynthesis Phosphoinositide-3 Kinase Inhibitors Phosphoproteins/genetics,metabolism RNA-Binding Proteins/genetics,metabolism
Chemicals
6-amino-N-(3-(4-(4-morpholinyl)pyrido(3',2'-4,5)furo(3,2-d)pyrimidin-2-yl)phenyl)-3-pyridinecarboxamide Aminopyridines DNA-Binding Proteins DOK1 protein, human Dok1 protein, mouse Heterocyclic Compounds, 3-Ring Insulin Phosphatidylinositol Phosphates Phosphoinositide-3 Kinase Inhibitors Phosphoproteins RNA-Binding Proteins phosphatidylinositol 3,5-diphosphate phosphatidylinositol 5-phosphate PIKFYVE protein, human Pikfyve protein, mouse
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Sbrissa Diego
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Ikonomov Ognian C
Filios Catherine
Delvecchio Khortnal
Shisheva Assia
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Article Info
Journal
American journal of physiology. Cell physiology
Abbr.
Am J Physiol Cell Physiol
ISSN
1522-1563
DOI
10.1152/ajpcell.00105.2012
Published
2012-08-15
Epub
2012-00-23
Pages
C436-46
Language
English
Region
United States
NLM ID
100901225
PMCID
PMC3422984
Subset
IM
Grants
NIDDK NIH HHS · P30 DK020572 · United States
NIDDK NIH HHS · DK-58058 · United States
External Links
PubMed source DOI: 10.1152/ajpcell.00105.2012 PMC Full Text View journal details
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