PtdIns 4,5 P

Dressman, M.A., Olivos-Glander, I.M., Nussbaum, R.L., and Suchy, S.F, 2000, Ocrll, a PtdIns(4,5)P(2) 5-phosphatase, is localized to the trans-Golgi network of fibroblasts and epithelial cells. J. Histochem. Cytochem. 48 179-190. 

Godi, A., Fertile, P., Meyers, R., Marra, P., Di Tullio, G., lurisci, C., Luini, A., Corda, D., and De Matteis, M. A. (1999). ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P-2 on the Golgi complex. Nat. Cell Biol 1, 280-287. 

In the plant kingdom, the pathway of synthesis of 3- and 4-phosphorylated phosphoinositides has been described only in Spirodela polyrhiza L. [1]. If animal models of phosphoinositide metabolism are to be applied to plants, it is imperative to determine the route of synthesis of phosphoinositides in other members of the plant kingdom in which signaling functions for phosphoinositides and inositol phosphates have been implicated. Recently, we have rigorously identified 3- and 4-phosphorylated phosphoinositides in stomatal guard cells of Commelina communis L. [2]. In the present work we have set out to determine the metabolic route by which PtdIns(3,4)P and PtdIns(4,5)P synthesis occurs and to examine the metabolic relationship between 3- and 4-phosphoryrated lipids in stomatal guard cells. 

Since the 3 phosphate of PtdIns(3,4)P is less stron y labelled than the 4 phosphate, synthesis of PtdIns(3,4)P in Conmmina communis L. occurs by successive 3- and 4-phosphorylation of Ptdms. In contrast to all animal and plants cell systems described to date, in stomatal guard cells PtcUns(3,4)P predominates, in labelling terms at least, over PuiIns(4,5)P PtdIns(4,5)P is synthesis in Commelina communis L. by successive 4- and3-phosphorylation of Ptdins. 

All class I PI3Ks are heterodimeric enzymes composed of a 110 kDa catalytic subunit (with the isoforms pi 10 a,(3,5 or y) that associates with a regulatory subunit. Although the class I PI3Ks are capable of phosphorylating Ptdlns, PtdIns(4)P and PtdIns(4,5)P2 in vitro, it appears that they only use PtdIns(4,5)P2 as a substrate in vivo. Receptor-induced formation of Ptdlns... 

Polymerization and depolymerization of actin, the main component of microfilaments, is controlled by a series of proteins, the activity of which is controlled by Ca and/or Ptdlns(4,5)P2. The Ca regulated proteins (see 6.7) are chiefly involved in processes of depolymerization of actin. Many of the proteins involved in the opposite process, actin polymerization, have specific binding sites for PtdIns(4,5)P2 and Ptlns(4)P and are regulated by the availability of phosphoinositides. Examples of such proteins are profilin, gelsolin, villin and talin (review Janmey, 1994). 

Hawkins, P.T. Jackson, T.R. Stephens, L.R. Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature, 358, 157-159 (1992)... 

Di Paolo G, Moskowitz HS, Gipson K, Wenk MR, Voronov S, Obayashi M, Flavell R, Fitzsimonds RM, Ryan TA, De Camilli P (2004) Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature 431 415-22 Dulubova I, Khvotchev M, Liu S, Huryeva I, Siidhof TC, Rizo J (2007) Muncl8-1 binds directly to the neuronal SNARE complex. Proc Natl Acad Sci USA. 2007 Feb 14 104, 2697-702 Edeling MA, Smith C, Owen D (2006) Life of a clathrin coat insights from clathrin and AP structures. Nat Rev Mol Cell Biol 7 32 14... 

In the second procedure, it is possible to assay specific immunoprecipitated proteins (e.g., PI 3-kinase subunits, receptors, receptor component chains, or even cellular proteins) for associated lipid kinase activity under in vitro assay conditions using distinct substrates such as Ptdlns (6-8). It is also possible to use PtdIns(4)P or PtdIns(4,5)P2 as in vitro substrates for the prototypical class IA PI 3-kinase, but generally these are more expensive to buy. 

Resuspend dried lipids in 50 pL chloroform and spot onto a dry TLC plate previously impregnated with 1% potassium oxalate (see Note 10). Spot 5-10 pL drops at a time and dry each drop using a low-power hair dryer. It is also advisable to spot on 10 pL of standard PtdIns(4)P and PtdIns(4,5)P2 solutions as standards. 

Phosphatidylinositols and their phosphorylated deri tives are substrates for several Ptdins kinases. For example, Ptdins is converted by Ptdins 3-kinase to PtdIns-3-P, by Ptdins 4-kinase to PtdIns-4-P, and by Ptdins 5-kinase to PtdIns-5-P. Moreover, PtdIns-3-P is converted by phosphatidylinositol phosphate 4-kinase, (PIP 4 kinase), to PtdIns-3,4-P2, and PtdIns-4-P is converted by PIP 3-kinase to PtdIns-3,4-P2 and to PtdIns-4,5-P2 by PIP 5-kinase. Finally, PtdIns-4,5-P2 is hydrolysed by phospholipase C to diacylglycerol (DAG) and IP3, and the Ptdins bisphosphates (PtdIns-3,4-P2 and... 

M., Madden, D.T. et al.. Interaction of Slap2p s ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytotic internalization, Mol. Biol. Cell 16, 717-730, 2005 Yao, P.J., Bushlin, I., and Petralia, R.S., Partially overlapping distribution of epsinl and HIPl at the synapse analysis by immunoelectron microscopy, J. Comp. Neurol. 494, 368-379, 2006. 

Figure 2. The so-called canonical phosphoinositide pathway . The continuous phosphorylation/dephosphorylation reactions allow a steady-state level of Ptdins, PtdIns(4)P and PtdIns(4,5)P2 in the plasma membrane (PM). Cleavage of PtdIns(4,5)P2 by phospholipase C (PLC) generates the two well-known second messengers, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and diacylglycerol (DAG). Besides its role as a protein kinase C (PKC) activator, DAG can be phosphorylated to phosphatidic acid (PA). The resynthesis of Ptdins from inositol and PA occurs mainly in the endoplasmic reticulum (ER). PPi, inorganic phosphate. PA-Pase, phosphatidic acid phosphatase. PA-TP, phosphatidic acid transport protein. PtdIns-TP, phosphatidylinositol transport protein. CDP-DAG, cytidine diphosphate-diacylglycerol. CMP, CDP and CTP, cytidine mono-, di- and triphosphate, respectively.

Another method exists to quantify the intracellular PtdIns(3,4,5)P3 level (Van der Kaay et al., 1997). Briefly, lipids are extracted from cells and hydrolysed by KOH to obtain Ins(l,3,4,5)P from PtdIns(3,4,5)P3. A competition with radiolabelled Ins(l,3,4,5)P versus an Ins(l,3,4,5)P -binding protein from cerebellum is then performed. 

A mass assay for PtdIns(5)P has been developed based on the use of recombinant type II PtdInsP-kinase a, a kinase that selectively phosphorylates this lipid to PtdIns(4,5)P2 (Morris et al, 2000 Niebuhr et al, 2002). 

As discussed above, PtdIns(4)P is the major precursor of PtdIns(4,5)P2, but it can also be phosphorylated by type II PI 3-kinases to produce PtdIns(3,4)P2. 

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