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

J Biol Chem. 2002 Mar 15;277(11):9206-11. doi: 10.1074/jbc.M108750200. Epub 2001 Nov 19.

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 PIKfyve(K1999E/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 PIKfyve(K1999E/K2000E). Expression of PIKfyve(K1999E/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 (PIKfyve(K2000E)) or PtdIns 3,5-P(2) (PIKfyve(K1999E)), of which only the PIKfyve(K1999E) mutant induced the characteristic endomembrane defects upon cell transfection. Furthermore, phosphoinositide microinjection into cells demonstrated a selective ability of PtdIns 3,5-P(2) to correct the endomembrane defects induced by the dominant-negative PIKfyve lipid kinase-deficient mutants. Thus, PtdIns 3,5-P(2) 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cell Membrane / metabolism*
  • Cricetinae
  • Endocytosis
  • Homeostasis
  • Molecular Sequence Data
  • Phosphatidylinositol 3-Kinases / chemistry
  • Phosphatidylinositol 3-Kinases / physiology*
  • Phosphatidylinositol Phosphates / biosynthesis*
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • Phosphatidylinositol Phosphates
  • phosphatidylinositol 3,5-diphosphate
  • phosphatidylinositol 5-phosphate
  • Phosphatidylinositol 3-Kinases