ABSTRACT
Ins(1,4,5)P(3) 3-kinase (IP3K) phosphorylates the Ca(2+)-mobilizing second messenger Ins(1,4,5)P(3) to yield the putative second messenger Ins(1,3,4,5)P(4). A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5)P(3) and increased levels of Ins(1,3,4,5)P(4) after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca(2+) responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca(2+), compared with 3% of uninduced cells. The quantity of Ca(2+) within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca(2+) entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5)P(3) levels, and consequently in Ca(2+) responses following stimulation of cells with Ins(1,4,5)P(3)-elevating agonists.
Subject(s)
Calcium Signaling , Calcium/metabolism , Homeostasis , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcium/agonists , Calcium Signaling/drug effects , Chromatography, High Pressure Liquid , Electric Conductivity , Enzyme Induction/drug effects , Gene Expression/drug effects , HeLa Cells , Histamine/pharmacology , Homeostasis/drug effects , Humans , Inositol/metabolism , Phosphotransferases (Alcohol Group Acceptor)/biosynthesis , Phosphotransferases (Alcohol Group Acceptor)/genetics , Promoter Regions, Genetic/genetics , Rats , Recombinant Proteins/drug effects , Tetracycline/pharmacology , Thapsigargin/pharmacology , TransfectionABSTRACT
The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).
