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 Pessin, Assia Shisheva

研究成果: Article査読

91 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)9206-9211
ページ数6
ジャーナルJournal of Biological Chemistry
277
11
DOI
出版ステータスPublished - 2002 3月 15
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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