title: G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway.
abstract: Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.
authors: Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors: N E
authors: NE
authors: Chatah
authors: C S
authors: CS
authors: Abrams
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005799:Genes, Dominant; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D011956:Receptors, Cell Surface; D006160:Guanosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D013917:Thrombin; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins