ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion.
Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins. ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions. PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and here we report that PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K). The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming. Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway. The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.
Hay
J C
JC
Program in Cell and Molecular Biology, University of Wisconsin, Madison 53706.
Fisette
P L
PL
Jenkins
G H
GH
Fukami
K
K
Takenawa
T
T
Anderson
R A
RA
Martin
T F
TF
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis; D008565:Membrane Proteins; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010716:Phosphatidylinositols / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010738:Type C Phospholipases; D002118:Calcium; D009638:Norepinephrine