Ptdins 4 kinase

For Ptdins 4 kinase and Ptdins (4) P5 kinase activities, the assay can be modified with assay buffer (50 mM Tris-HCl, pH 7.2 10mM MgCh 1 mM DTT 0.4% Triton X-100), 0.5 mM of substrate Ptdins or PtdIns4P, and 1 (xCi [y- P]-ATP. Terminate the reaction with 0.6 mL chloroformimethanol (1 1, v/v). After addition of 0.5mL 12N HCl, phosphoinosi-tides are extracted into the lower chloroform phase, which are washed with 1 mL methanoLlM HCl (1 1, v/v) followed by 1 mL methanoLO.l mol/L HCl (1 1, v/v). The radioactive reaction product can be isolated by TLC and quantified by liquid scintillation counting. 

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... 

Verghese, M. Fernandis, A.Z. Subrahmanyam, G. Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a Ptdins 4-kinase from lymphocytes. Indian J. Biochem. Biophys., 36, 1-9 (1999)... 

The chemotactic response to cAMP of the slime mould Dictyostelium discoideum is presented as an example. When these cells are starving, they sense cAMP signals, and in response to the hunger signal, cAMP, the cells differentiate (reviewed in ref. 67). In Dictyostelium discoideum the response to cAMP is mediated by G-protein-coupled hepta-helical receptors and is transmitted by the Py subunits of a heterotrimeric G protein. In response to the chemoattractant, cAMP, a homologue of PKB (protein kinase B) is rapidly activated by phosphorylation througji a Ptdins kinase specific for the 3-OH position (see also Chapter 4). 

Family of enzymes phosphorylating phosphatidylinositol (Ptdlns), PtdIns(4)phosphate, and PtdIns(4,5)phosphate in the 3-position. The Ptdlns(3 phospholipids are second messengers in processes like cell growth, cytoskeletal rearrangement, and vesicular transport. PI 3-kinases are heterodimers composed of a catalytic and a regulatory subunit. The enzymes are activated by insulin, many growth factors, and by a variety of cytokines. Their activity can be inhibited by wortmannin and LY294002. 

P2 is generated from PtdIns(4)P by the enzymatic activity of phosphatidylinositol 4-phosphate 5-kinase (PDP5K) (Fig. 1). Additional pathways are likely to be discovered. 

Domin J, Harper L, Aubyn D, et al. The class II phosphoinositide 3-kinase PI3K-C2beta regulates cell migration by a PtdIns(3)P dependent mechanism. J Cell Physiol 2005 205(3) 452 162. 

Phosphatidyl inositol (Ptdins) is first phosphorylated by specific kinases at the 4 and 5 positions of the inositol residue, leading to formation of phosphatidyl inositol-4,5-bisphosphate (PtdIns(4,5)P2). 

Fig. 6.3. Formation of diacylglycerol, Ins(l,4,5)P3 and PtdIns(3,4,5)P3. PL-C phospholipase of type C PI3-kinase phosphatidyl inositol-3 -kinase.

Fig. 6.4. Formation and function of diacylglycerol and Ins(l,4,5)P3. Formation of diacylglycerol (DAG) and Ins(l,4,5)P3 is subject to regulation by two central signaling pathways, which start from transmembrane receptors with intrinsic or associated tyrosine kinase activity (see Chapters 8 11) or from G-protein-coupled receptors. DAG activates protein kinase C (PKC, see Chapter 7), which has a regulatory effect on ceU proliferation, via phosphorylation of substrate proteins. Ins(l,4,5)P3 binds to corresponding receptors (InsPs-R) and induces release of Ca from internal stores. The membrane association of DAG, PtdIns(3,4)P2 and PL-C is not shown here, for clarity.

Several metabohc pathways lead from phosphatidyl inositol to compounds with second messenger" character (review Liscovitch and Cantley, 1994 Divecha and Irvine, 1995). One main pathway, the formation of diacylglycerol and Ins(l,4,5)P3 from PtdIns(4,5)P2, has already been described in 6.4 and Fig. 6.3. Other compounds of regulatory importance can be formed by phosphorylation at the 3 position of the inositol part of Ptdins (Fig. 6.9). The reaction is catalyzed by a class of enzymes known as phosphatidylinositide 3-kinases (P13-kinases). The P13-kinases phosphorylate various phosphatidyl inositol compoimds at the 3 position. For example, PtdIns(3,4,5)P3, produced by 3 phosphorylation of Ptdlns(4,5)P2, has an important function as an intracellular messenger (see 6.6.2). 

Fig. 6.9. A) Metabolism of phosphatidyl inositol lipids. The figure shows different membrane-associated phosphatidyl inositol phosphate compounds and their mutual conversion. PI3-kinase (PI3K) is responsible, in particular, for the formation of PtdIns(3,4,5)P3 from PtdIns(3,4)P2. For the reactions marked with a question mark, involvement of PI(3) kinase is not certain.

The PI3 kinase (PI3-K) is translocated to the membrane by interaction of the SH2 domain of its p85 subunit with phosphotyrosine residues of the activated receptor. There it converts PtdIns(3,4)P2.into PtdIns(3,4,5)P3 which binds to PH domains of various effector molecules and recruits them into the signaling chain. The effector molecules can stimulate cell division or can induce the programmed cell death. The tumor suppressor PTEN hydrolyses phosphates from PtdIns(3,4,5)P3 and thus inhibits the growth promoting effect of the PI3 kinase signaling. An important effector of PI3 kinase is the protein kinase Akt which is also termed protein kinase B (PKB). GF growth factor GFR growth factor receptor. 

An important function in growth regulation is attributed to the P13-kinase. PtdIns(3,4,5)P3 is not detectable in resting cells. On stimulation of the cells with a growth factor, a rapid increase in Ptdlns(3,4,5)P3 occurs. An associated translocation of PI3-kinase to the membrane is observed. 

The products of the PI3-kinase reaction are different phosphoinositide derivatives phosphorylated at the 3 position, of which PtdIns(3,4,5)P3 has the greatest regulatory importance. PtIns(3,4,5)P3, like cAMP, has the function of a messenger substance that activates effector molecules in the sequence for further signal conduction. In contrast to cAMP, Ptdlns(3,4,5)P3 is localized in the cell membrane and performs its function in close association with processes at the cell membrane. 

In the Akt signaling pathway (review Downward, 1998), first an extracellular growth factor activates the corresponding transmembrane receptor (e.g., PGDF receptor, see 8.1). Consequently, tyrosine phosphorylation takes place on the cytoplasmic domain of the receptor. The tyrosine residues serve as docking sites for the SH2 domain of the p85 subimit of the PI3-kinase. The associated translocation of PI3-kinase is synonymous with its activation. The PtdIns(3,4,5)P3 formed binds to the PH domain of the signal protein next in sequence, the Akt kinase, which recruits the latter to the membrane. 

The great importance of PtdIns(3,4,5)P3 metabolism for growth regulation is illustrated by the observation that an enzyme of PtdIns(3,4,5)P3 metabolism has been identified as a tumor suppressor protein (Wu et al., 1998). PTEN tumor suppressor protein has lipid phosphatase activity that is specific for hydrolysis of PtdIns(3,4,5)P3. It is assumed that PTEN lipid phosphatase is a negative regulator of the Akt pathway by lowering the concentration of PtdIns(3,4,5)P3 and counteracting stimulation of Akt kinase. 

Fig. 6.11. Formation and function of diacylglycerol. The figure schematically shows two main pathways for formation of diacylglycerol (DAG). DAG can be formed from PtdInsP2 by the action of phospholipase C (PL-C). Another pathway starts from phosphatidyl chohne. Phospholipase D (PL-D) converts phosphatidyl choline to phosphatidic add (Ptd), and the action of phosphatases results in DAG. Arachidonic add, the starting point of biosynthesis of prostaglandins and other intracellular and extracellular messenger substances, can be cleaved from DAG. PKC protein kinase C Ptdins phosphatidyl inositol.

Additional information <1, 2, 3, 4, 38> (<2>, in cells loaded with protein-tyrosine phosphatase antibody, phosphatidylinositol 3 -kinase activity is increased by 38%, respectively, compared with control cells loaded with preim-mune IgG [11] <4>, b2-integrins activate the tyrosine kinases p58c-fgr and p59/61hck and causes them to associate with the p85 subunit of Ptdins 3-... 

T. Clustering of b2-integrins on human neutrophils activates dual signaling pathways to Ptdins 3-kinase. Exp. Cell Res., 256, 257-263 (2000)... 

PtdIns(4)P-5-kinase B isoform PtdIns(4)P-5-kinase C isoform type II PIP kinase... 

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)... 

PtdIns-3-kinase C2a PtdIns-3-kinase C2/I PtdIns-3-kinase C2y class II phosphoinositide 3-kinase C2a class II phosphoinositide 3-kinase C2/I... 

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