Trafficking of phosphatidylinositol by phosphatidylinositol transfer proteins

2007 ◽  
Vol 74 (1) ◽  
pp. 259 ◽  
Author(s):  
Shamshad Cockcroft
Keyword(s):  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells

2001 ◽  
Vol 356 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Jef A. PINXTEREN ◽  
Bastien D. GOMPERTS ◽  
Danise ROGERS ◽  
Scott E. PHILLIPS ◽  
Peter E. R. TATHAM ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Mast Cells ◽  
Protein Kinase ◽  
Aminoglycoside Antibiotic ◽  
Pleckstrin Homology Domain ◽  
Kinase C ◽  
Streptolysin O ◽  
Regulatory Machinery ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca2+ and guanosine 5′-[γ-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20–30min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25μM ATP can extend responsiveness significantly; this effect is maximal at 50μM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPα and PITPβ have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C16), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C16 suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.

Functional Analysis of Phosphatidylinositol Transfer Proteins

1996 ◽  
pp. 327-338 ◽  
Author(s):  
Brian G. Kearns ◽  
James G. Alb ◽  
Robert T. Cartee ◽  
Vytas A. Bankaitis
Keyword(s):  
Functional Analysis ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

scholarly journals Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

2000 ◽  
Vol 347 (3) ◽  
pp. 837-843 ◽  
Author(s):  
Philip SWIGART ◽  
Robert INSALL ◽  
Andrew WILKINS ◽  
Shamshad COCKCROFT
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phospholipase C ◽  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Hl60 Cells ◽  
Single Organism ◽  
Membrane Compartment ◽  
Phosphatidylinositol Transfer ◽  
Cloned Cdna ◽  
Phosphatidylinositol Transfer Proteins

Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (α and β) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPα and PITPβ. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

Class I Phosphatidylinositol Transfer Proteins (PITPs) Promote PIP2 Synthesis and IP3 Production in Mouse Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 363-363
Author(s):  
Liang Zhao ◽  
Lurong Liang ◽  
Aae Suzuki ◽  
Sang Min ◽  
Charles S. Abrams
Keyword(s):  
Conflicts Of Interest ◽  
Second Messengers ◽  
Yeast Cells ◽  
Class I ◽  
Null Mutation ◽  
Phosphoinositide Metabolism ◽  
Double Knockout ◽  
Akt Phosphorylation ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Abstract Abstract 363 Phosphoinositides are the phosphorylated forms of phosphatidylinositol. Although they constitute only 1% of platelet membrane phospholipids, phosphoinositides are important contributors to platelet signaling. As an example the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PIP2) has been shown to regulate integrin activation, actin assembly, and secretion in platelets. PtdIns(4,5)P2 contributes to these events by directly binding to signaling proteins, and also by serving as a substrate for PLC and PI3K to generate the second messengers Inositol(3,4,5)P3 (Ins(3,4,5)P3) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). The phosphoinositide concentration in different cellular compartments is tightly regulated both in time and space, and their relative amounts of various phosphoinositides change within seconds of agonist stimulation. Given their importance in platelet biology, we sought to better understand the mechanism of phosphoinositide synthesis and function. Class I PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that bind to and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner. Although there are no studies on the function of PITPs in hematopoietic cells, the proteins are essential in yeast cells for the biosynthesis and metabolism of phosphoinositides. This suggests that PITPs may also be essential for phosphoinositide metabolism in platelets. There are two dominant Class I PITP family members, PITPα and PITPβ in murine platelets. We introduced conditional loss-of-function mutations into the murine PITPα and PITPβ genes These mice were crossed with transgenic mice expressing Cre recombinase under the control of the platelet specific promoter PF4, thereby generating mice lacking individual PITP isoforms only in platelets and megakaryocytes. Mice lacking either platelet PITP isoform have approximately one-third lower platelet counts than littermate controls, but otherwise have normal blood counts and appear phenotypically normal. We have also generated a few viable double knockout (PITPαfl/fl PITPβfl/fl PF4Cre+) mice and found that these mice produce some platelets even in the absence of both PITP isoforms. Next, we analyzed the concentration of individual phosphoinositides in platelets of the knockout mice by thin layer chromatography. Although loss of either PITP isoform did not affect PtdIns(4)P synthesis in resting or thrombin stimulated platelets, PtdIns(4,5)P2 synthesis was significantly inhibited in platelets lacking either PITPα (∼40% of normal) or PITPβ (∼50% of normal). This suggests that both PITPα and PITPβ are required for normal PtdIns(4,5)P2 synthesis in platelets. To test whether PITP isoforms are critical for second messenger formation, we analyzed the formation of Ins(3,4,5)P3 in response to thrombin stimulation. We found that the PITPα-null mutation causes an almost absence of Ins(3,4,5)P3 formation. The PITPβ-null mutation also caused a profound defect in Ins(3,4,5)P3 formation, albeit less than that seen following PITPα deletion. Nonetheless, both PITPα and PITPβ knockout platelets were able to increase their intracellular calcium concentration following thrombin stimulation to approximately 50% of normal. These data imply that a significant portion of the calcium response in platelets results from the influx of extracellular calcium and not by the Ins(3,4,5)P3–dependent release of calcium from intracellular stores. Further, platelets lacking either PITP isoform exhibited only a moderate (∼50%) defect in Akt phosphorylation – a PtdIns(3,4,5)P3 synthesis-dependent event. Although both Ins(3,4,5)P3 and PtdIns(3,4,5)P3 are derived from PtdIns(4,5)P2, it is remarkable that PITP enzymes are more critical for Ins(3,4,5)P3-synthesis than for PtdIns(3,4,5)P3-synthesis (as suggested by Akt phosphorylation). Finally, we performed pilot experiments of the ex vivo function of platelets lacking both PITP isoforms and found that double knockout platelets had a profound loss of agonist-induced platelet aggregation. Together, our data suggest that PITPα and PITPβ are both critical for the synthesis of essential platelet phosphoinositides and second messengers. Thus, these enzymes may represent novel therapeutic targets for the prevention and treatment of arterial occlusion. Disclosures: No relevant conflicts of interest to declare.

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