Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity

Ognian C. Ikonomov, Diego Sbrissa, Kristopher Mlak, Makoto Kanzaki, Jeffrey E. Pessin, Assia Shisheva

Research output: Contribution to journalArticle

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Abstract

PIKfyve enzymatic activity is required in maintaining late endocytic membrane integrity. PIKfyve is a dual specificity enzyme that phosphorylates phosphatidylinositol (PtdIns) and PtdIns 3-P at the 5-hydroxyl and unidentified endogenous protein substrate(s). To determine which of these activities (lipid versus protein kinase activity) is responsible for endomembrane homeostasis we analyzed a double mutant PIKfyveK1999E/K2000E. These substitutions in the putative lipid-substrate activation loop nearly completely abrogated the lipid kinase activity without any significant effect on the protein kinase activity of PIKfyveK1999E/K2000E. Expression of PIKfyveK1999E/K2000E in COS cells induced a dramatic dominant-negative effect in the form of endomembrane swelling and vacuolation. In addition, the lipid-substrate specificity of PIKfyve was modified by introducing single mutations in Lys-1999 or Lys-2000. This yielded proteins with preferentially abrogated synthesis of PtdIns 5-P (PIKfyveK2000E) or PtdIns 3,5-P2 (PIKfyveK1999E), of which only the PIKfyveK1999E mutant induced the characteristic endomembrane defects upon cell transfection. Furthermore, phosphoinositide microinjection into cells demonstrated a selective ability of PtdIns 3,5-P2 to correct the endomembrane defects induced by the dominant-negative PIKfyve lipid kinase. deficient mutants. Thus, PtdIns 3,5-P2 production by PIKfyve is crucial for endomembrane integrity, and Lys-1999 most likely directs the PIKfyve interactions with the 3-phosphate group in PtdIns 3-P.

Original languageEnglish (US)
Pages (from-to)9206-9211
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number11
DOIs
StatePublished - Mar 15 2002
Externally publishedYes

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Dissection
Phosphatidylinositols
Protein Kinases
Lipids
Phosphotransferases
Substrates
Defects
COS Cells
Microinjections
Substrate Specificity
Hydroxyl Radical
Transfection
Swelling
Proteins
Homeostasis
Substitution reactions
Chemical activation
Phosphates
Membranes
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity. / Ikonomov, Ognian C.; Sbrissa, Diego; Mlak, Kristopher; Kanzaki, Makoto; Pessin, Jeffrey E.; Shisheva, Assia.

In: Journal of Biological Chemistry, Vol. 277, No. 11, 15.03.2002, p. 9206-9211.

Research output: Contribution to journalArticle

Ikonomov, Ognian C. ; Sbrissa, Diego ; Mlak, Kristopher ; Kanzaki, Makoto ; Pessin, Jeffrey E. ; Shisheva, Assia. / Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 11. pp. 9206-9211.
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AU - Ikonomov, Ognian C.

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