Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P2 production for endomembrane integrity.
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.
Ikonomov
Ognian C
OC
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Sbrissa
Diego
D
Mlak
Kristopher
K
Kanzaki
Makoto
M
Pessin
Jeffrey
J
Shisheva
Assia
A
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis; D006706:Homeostasis; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D018129:Phosphatidylinositol Phosphates; C106336:phosphatidylinositol 3,5-diphosphate; C113567:phosphatidylinositol 5-phosphate; D019869:Phosphatidylinositol 3-Kinases