PMID- 10419465
OWN - NLM
STAT- MEDLINE
DCOM- 19990819
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 31
DP  - 1999 Jul 30
TI  - PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes
      5-phosphoinositides. Effect of insulin.
PG  - 21589-97
AB  - One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group
      undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key
      functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and
      PtdIns 3,5-P(2), have now been identified in mammalian cells, but their
      biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235,
      whose exact substrate specificity remained to be determined (Shisheva, A.,
      Sbrissa, D., and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we
      report that recombinant p235 expressed in COS cells, like the authentic p235 in
      adipocytes, displays striking specificity for PtdIns over PI substrates and
      generates two products identified as PtdIns 5-P and PtdIns 3,5-P(2) by HPLC
      analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P(2) 
      but to a substantially lesser extent than PtdIns isolated from natural sources.
      Important properties of the p235 PI 5-kinase include high sensitivity to nonionic
      detergents and relative resistance to wortmannin and adenosine. By analyzing
      deletion mutants in a heterologous cell system, we determined that in addition to
      the predicted catalytic domain other regions of the molecule are critical for the
      p235 enzymatic activity. HPLC resolution of monophosphoinositide products,
      generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes
      acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels 
      as the corresponding p235 immune complexes of resting cells. However, the acute
      insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak,
      suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to
      p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro
      activity for PtdIns 5-P and PtdIns 3,5-P(2) generation, implying PIKfyve has a
      key role in their biosynthesis.
FAU - Sbrissa, D
AU  - Sbrissa D
AD  - Department of Physiology, Wayne State University School of Medicine, Detroit,
      Michigan 48201, USA.
FAU - Ikonomov, O C
AU  - Ikonomov OC
FAU - Shisheva, A
AU  - Shisheva A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Fungal Proteins)
RN  - 0 (Insulin)
RN  - 0 (Phosphatidylinositols)
RN  - 0 (Phospholipids)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Saccharomyces cerevisiae Proteins)
RN  - EC 2.7.1.- (FAB1 protein, S cerevisiae)
RN  - EC 2.7.1.- (Phosphatidylinositol 3-Kinases)
RN  - EC 2.7.1.- (Phosphotransferases (Alcohol Group Acceptor))
RN  - EC 2.7.1.137 (Pikfyve protein, mouse)
SB  - IM
MH  - 3T3 Cells
MH  - Adipocytes/*enzymology
MH  - Animals
MH  - COS Cells
MH  - Catalytic Domain
MH  - Chromatography, High Pressure Liquid
MH  - Fungal Proteins/metabolism
MH  - Insulin/*pharmacology
MH  - Mammals
MH  - Mice
MH  - Phosphatidylinositol 3-Kinases/chemistry/*metabolism
MH  - Phosphatidylinositols/*biosynthesis/chemical synthesis
MH  - Phospholipids/*metabolism
MH  - Phosphotransferases (Alcohol Group Acceptor)/*metabolism
MH  - Recombinant Proteins/metabolism
MH  - Saccharomyces cerevisiae/enzymology
MH  - *Saccharomyces cerevisiae Proteins
MH  - Substrate Specificity
MH  - Transfection
EDAT- 1999/07/27 00:00
MHDA- 1999/07/27 00:01
CRDT- 1999/07/27 00:00
PHST- 1999/07/27 00:00 [pubmed]
PHST- 1999/07/27 00:01 [medline]
PHST- 1999/07/27 00:00 [entrez]
AID - 10.1074/jbc.274.31.21589 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Jul 30;274(31):21589-97. doi: 10.1074/jbc.274.31.21589.