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.
Highlights
The synthesis of the membrane-bound phospholipid, phosphatidylinositol 4,5-bisphosphate (PI4,5P2)[1] is a critical step in signal transduction
phosphatidylinositol-4-phosphate 5-kinase type I (PIP5K I) can associate, in a nucleotide-independent fashion, with the small GTP-binding proteins Rho and Rac (26, 27). These GTPases can lead to the activation of PIP5K I, but this effect on lipid kinase activity may be limited to the GTP-bound form of the nucleotide-binding protein (28)
Stimulation of a G-protein-coupled Receptor Leads to the Membrane Recruitment of PIP5K I␣—Previous reports demonstrated that stimulation of the predominant thrombin receptor on human platelet, PAR1, leads to an increase in PI4,5P2 synthesis (41, 42)
Summary
The synthesis of the membrane-bound phospholipid, phosphatidylinositol 4,5-bisphosphate (PI4,5P2)[1] is a critical step in signal transduction. The second pathway involves phosphorylation at the D-4 position of phosphatidylinositol 5-phosphate (PI5P) by a enzyme currently referred to as phosphatidylinositol-5-phosphate 4-kinase (PIP4K), but previously named phosphatidylinositol4-phosphate 5-kinase type II (PIP5K II) or type C (PIP5K C) (16, 18) It is currently thought the pathway utilizing PIP5K I (␣, , or ␥ isoform) is the predominant mechanism of PI4,5P2 synthesis in mammalian cells and, is a major regulator of actin organization, secretion, and endocytosis (19 –22). We demonstrate that stimulation of the PAR1 receptor induces a translocation of PIP5K I␣ from the Golgi to the cell membrane This process involves heterotrimeric G-proteins as well as both Rac and Rho, but not Arf. Coincident with the intracellular translocation of PIP5K I␣, we found that its enzymatic activity increased 5- to 7-fold when co-expressed with either large or small GTP-binding proteins. These results delineate a signaling pathway that is initiated by a G-protein-coupled receptor and leads to the production of PI4,5P2
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