Functional Dissection of Lipid and Protein Kinase Signals of PIKfyve Reveals the Role of PtdIns 3,5-P2 Production for Endomembrane Integrity

• Published in: The Journal of biological chemistry Vol. 277; no. 11; pp. 9206 - 9211
• Main Authors: Ikonomov, Ognian C., Sbrissa, Diego, Mlak, Kristopher, Kanzaki, Makoto, Pessin, Jeffrey, Shisheva, Assia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Cell Membrane - metabolism; CHO Cells; Cricetinae; Endocytosis; Homeostasis; Molecular Sequence Data; Phosphatidylinositol 3-Kinases - chemistry; Phosphatidylinositol 3-Kinases - physiology; Phosphatidylinositol Phosphates - biosynthesis; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108750200

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 (lipidversus 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.