The Derivatives Of Phosphatidylinositol In Cell Signalling Biology Essay

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For over past few decades, phosphatidylinositol and its derivatives have thought to be involved in various cell signaling and membrane trafficking events in eukaryotes. Different phosphatidylinositol shows different sub cellular localization. Golgi membranes are found to be rich in phosphatidylinositol -4 phosphate [PI(4)P] and in higher phosphorylated derivatives such as phosphatidylinositol 4,5 bisphosphate. A higher derivative, phosphatidylinositol 3,4,5 triphosphate are found in the inner leaflet of plasma membrane. After activation by growth factor, phosphotidylinositol 3,4,5 triphosphate may also accumulate on endomembranes. Endosomal organelles are characterized by the presence of phospatidylinositol 3 phosphate ( Micheal kraub et al). Among the phospholipids, the head group of the phosphotidylinositol is found to be unique as 3 out of the 6 hydroxyl group found on the inositol ring can be subjected to reversible covalent modification by phosphorylation. Phosphotidylinositol being a substrate of various lipid kinases and phosphatases, it is converted to a biologically active derivative phosphoinositides. Since these phosphoinositides are also substrates of various phospholipases, they end up producing various products which acts as secondary messenger molecule with biological function of their own. Phosphoinositides can bind to different types of proteins which include cytoplasmic factors and membrane integral channels and transporters. This binding takes place by electrostatic interactions between the negatively charged phosphoinositides head group and the positive charges present in the protein to be targeted.(Michael Krauß )(Thomas Strahl )

Folich , in 1940, concluded that inositol was found to be a natural constituent of brain phospholipids who`s structure remained unknown for few year. Mabel and Lowell Hokin found a new discipline of lipid signaling by demonstrating that certain phospholipids, especially, PtdIns are rapidly metabolized by cells in response to external stimuli( Micheal kraub et al).The field gained a boom after Brockerhoff et al in 1980 suggested that PtdIns[4,5]P2 are produced after the phosphorylation of PtdIns and might act as a secondary messenger for Phospholipase C isoform(PLC) (fig 1). Two secondary messenger molecules, 1,2diacylglycerol (DAG) and inositol 1,4,5 triphosphate(Ins[1,4,5]P3, were produced after the hydrolytic degradation of PtdIns[4,5]P2 by receptor stimulated phospholipase C (PLC) in response to external stimuli. (Thomas Strahl ). Berridge and Nishizuka found that DAG in animal cells activates certain forms of protein kinase C (PKC) isoforms which results in changes in gene expression. Also Berridge et al in different studies and Joseph et al demonstrated that after binding of Ins[1,4,5]P3 to group of intracellular receptors causes release of Ca+2 from intracellular reserves.

Saccharomyces cerevisiae was used as the model organism for the study of vesicle mediated membrane trafficking caused due to PtdIns and its derivatives. Schekman and his colleagues isolated a gene, sec 14 which is responsible for the production of PtdIns transfer protein (PITP) required for functioning of the Golgi apparatus (Bankaitis et al). Emr et al found a gene product, Vps 34, which is responsible for protein trafficking between the vacuoles and the Golgi apparatus which encodes an enzyme PtdIns 3 Kinase. In two different studies it was found that PtdIns 4 Kinase was required for the generation of exocystic vesicles from the golgi apparatus.

The role of kinases and phospholipids second messengers has been established in regulation of chemotaxis, cell differentiation, rearrangement of actin cytoskeleton, adaptations of the lower eukaryotes under conditions of stress (Furuya et al. 2000; De la Roche & Cote 2001; De la Torre-Ruiz et al. 2002; DeWald 2002).The phosphorylation of PI and its metabolites is catalyzed by multiplex phosphoinositide kinases, each of which phosphorylates at specific positions of hydroxyl groups of the inositol ring ( Yasunori Kanaho et al.) which are characterized into two families: phosphoinositide(PI) kinases and phosphoinositide(PIP) phosphate kinases. These characterizations are based on thesubstrate specificity and similarity in their sequences. The PI kinases are sub divided into PI 3 kinase and PI 4 kinases. The PI 3 kinases phosphorylated at the D3 position and are further divided in 3 classes of isozymes . PI4 kinases phosphorylates at D4 hydroxyl position and are further divided into 2 classes of isozymes-type II and type III. The phosphoinositide phosphate kinases are made up PI 4 phosphate 5 kinase[PI(4)P5- Kinases] PI 5-phosphate 4-kinases [PI(5)P 4-kinases], and PI 3-phosphate [PI(3)PI 5-kinase [PI(3)P 5-kinasel, which phosphorylate PI(4)P at the D-5 position, PI(5)P at the D-4 position, and PI(3)P at the D-5 position, respectively (Yasunori Kanaho et al).

VPS 34 was the first ever Phosphoinositide 3 kinase that was cloned from S cerevisiae. Rothaman et al, in 1993, came to a conclusion that VPS 34 encoded PtdIns 3 Kinase only after finding a stricking similarity in the COOH terminal with the catalytic subunit of mammalian Type I PtdIns 3 kinase. In the same year, Flanagan et al showed that PIK1 was responsible for encoding PtdIns 4 Kinase Type IIIβ. It is know till now that S cerevisiae contains PtdIns[3]P, PtdIns[4]P, PtdIns[3,5]P2 and in S Pombe, the fission yeast has PtdIns[3,4,5]P3. It was also found that in addition to the COOH terminal Vps 34also share stricking similarity with other PtdIns Kinases. These are called Lipid kinase unique(LKU) whos function is unknow and the C2 domain which is responsible for protein-lipid interactions. It was also seen that Vps 34 was found to be insensitive to the inhibitors (wortmannin and LY294002) used to block the mammalian TypeI PtdIns 3 Kinase activity both in vivo and in vitro(Stack et al 1992, Yano et al 1993, Okada et al 1994). Early studies on vps34 mutants, utilizing both light and

electron microscopy, revealed morphologically intact vacuoles (the yeast vacuole is functionally equivalent to the mammalian lysosome), which receive protein traffic from both the secretory and endocytic pathways, but also a number of different abnormal membranous structures in the cytoplasm of these

cells.(Thomas Strahl ). Different studies on analysis of various Vps mutants revealed there is similarity in phenotypes of Vps 34 and Vps 15. From these studies, it was suggested that the gene products are involved in the vacuolar protein sorting at the same step. Stack et al in 1995 found that the activity of PtdIns 3 kinase of Vps 34 is essential for proper targeting of certain soluble vacuolar protein, but this is not the case with Vps 15.

Auger et al in 1989, suggested the existence of PtdIns(3,5)P2 but it was officially identified by Dove et al in 1997. PtdIns(3,5)P2 are present everywhere in the eukaryotes and is one of the seven polyphosphoinositides that controls membrane trafficking in endosomal/lysosomal system. It is also involved in the regulation of size, shape and acidity of at least one compartment of endo-lysosomal compartment.(Dove et al,2009). The PtdIns(3,5)P2 has the ability to carry out this control by using multiple effector proteins whereby each effectors that is specific for a subset of PtdIns(3,5)P2 dependent process is used. Synthesis of PtdIns(3,5)P2 takes place after the phosphorylation of Phosphoinositide 3 phosphate. Both PI3P and PtdIns(3,5)P2 are generated in the endomembrane system, a heterogeneous system of membranes involved in traffic to and from the plasma membrane, the lysosome, and the Golgi (Di Paolo and De Camilli, 2006). PtdIns(3,5)P2 is a minor protein in comparison to Phosphoinositide 3 phosphate. The values of PtdIns(3,5)P2 in yeast is found to be <0.01% of the total lipids and in mammalian cells it was found to be <0.05% of the total lipids whereas Phosphoinositide 3 phosphate in yeast was 2.5% of total lipids and 0.2% in mammalian cells. White et al in 1997 labeled mouse fibroblast by using non-equilibrium [32 P]Pi and Dove et al in 1997 using hyperosmotically stressed S. cerevisiae showed that by the action of 5-OH Kinase activity, PtdIns(3,5)P2 is synthesized from PtdIns 3P. (Cooke 2002). PtdIns(3,5)P2 Is found in low quantity but Dove et al in 1997 showed that the amount of PtdIns(3,5)P2 increases in yeast with hyperosmotic stress. He also showed that PtdIns(3,5)P2 is also present in Schizosaccharomyces pombe and plant cells. Various studies indicated that there are minimum of five proteins - the lipid kinase, Fab1p, lipid phosphatase Fig4p, the Fab1p activator Vac7p,the Fab1p inhibitor Atg18p, and Vac14p, a protein required for the activity of both Fab1p and Fig4p-are responsible for interconversion of PI3P and PtdIns(3,5)P2. In yeast, Vac14p, Fab1p and Fig4p are localized on the vacuole membrane and form a complex.(Cooke 2002). In different studies it was found that, the protein product of yeast FAB1 gene was necessary for membrane efflux from vacuole and was found to encode a PtdIns3P 5-kinase. Gary et al in 1998 found that PtdIns(3,5)P2 was absent in fab1 mutant but a moderate level of PtdIns(4,5)P2 was found. Yamamoto et al in 1995, found a phenotypic mutation in FAB1 gene which showed enlarged vacuoles and defective vacuolar acidification. He also showed that fab1Δ, a mutant, was not able to grow at 37 ̊C and Cacl2 dependent. McEwan et al in 1999 found a Fab1p homologue gene in fission yeast Schizosaccharomyces pombe , designated as SpFab1p, restores the PtdIns(3,5)P2 deficency in S. cerevisiae mutant but did not recover the temperature sentive growth and vacuolar morphology. It was observed by Bidlingmaier et al in 1992 that strains with this defect also showed different mating projection in response to pheromones secreted formatting. It found by various studies that the pheromone signaling in yeast was dependent on the vesicle transport system. Morishita et al in 2002 investigated ste12 gene, having an enlarged vacuole like the S. cerevisiae fab1mutant, found to be defective in mating which indicated that PtdIns(3,5)P2 is involved in signaling pathway related to mating competence. This could be due to ste12 mutant which was not able to traffick the necessary factors to the plasma membrane.

Phenotypic analysis of two yeast mutants that cannot synthesize PtdIns(3,5)P2, fab1and vac7 , suggested that this phospholipid is required for selected vacuole membrane functions. Genetic analysis in yeast shows that Vac14p is required for Fab1p and Fig4p function (Bonangelino et al., 1997; Gary et al., 1998).(Cecilia J. Bonangelino, Johnathan J. Nau, Jason E. Duex, Mikala Brinkman, Andrew E. Wurmser, Jonathan D. Gary, Scott D. Emr, and Lois S. Weisman ). Different studies showed that most of the fab1 mutation effects are deleted when the mutation from VAC7 or VAC14 are deleted. On individual deletion, it was seen that there was a significant drop in the levels of PtdIns(3,5)P2 although vac14 retains a low amount of activity permitting the strain to grown at high temperature. Turnover of this phospholipid is predominantly mediated by Fig4p, a Sac related lipid phosphatase specific for PtdIns(3,5)P2.(Efe, Botelho and Emr 2005) . 2002). Erdman et al in 1998 identified FIG4 as a pheromone induced gene. He also found that the expression of FIG4 is upregulated more than 40 folds upon treatment ofcellswith α factors. In a study conducted by Gary et al in 2002 suggested a functional relationship of Fig4 in regulating Fab1 lipid kinase and Vac7. Fig4 possesses

polyphosphoinositide phosphatase activity and PtdIns[3,5]P2 functions as its major substrate both in vitro and in vivo.(Thomas Strahl ). The sac domain is composed of seven conserved motifs that define a phosphoinositide phosphatase enzymatic activity (Guo et al., 1999; Hughes et al., 2000). In vitro, the sac domains of Sac1, Sjl2 and Sjl3, dephosphorylate PtdIns(3)P, PtdIns(4)P, and PtdIns(3,5)P2 (Guo et al., 1999; Hughes et al., 2000).Gary et al in 2002 and Guo et al in 1999 showed that there is no significant change in PI levels when FIG4 is deleted. This study suggested that the amount of lipid that Fig4 metabolize is not significant or the other sac domains take over the fig4 function and maintain the levels of phosphoinositides. Fig4 is also involved in the up -regulation of cellular PtdIns[3,5]P2 levels upon hyperosmotic shock.

CECILIA J. BONANGELINO, JOHNATHAN J. NAU, JASON E. DUEX, MIKALA BRINKMAN, ANDREW E. WURMSER, JONATHAN D. GARY, SCOTT D. EMR, AND LOIS S. WEISMAN, Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p. .

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