Other Phosphatidylinositol Kinases

The patent literature contains several additional applications. Exelixis has claimed methods for modulating the IGFR pathway by controlling Itpk activity and described screens for the identification of modulators of Itpk.162 IRM LLC has published three patent applications for ItpkB selective 

PIP5K produces the versatile phospholipid phosphatidylinositol 4,5-bispho-sphate (PI4,5P(2)). The physiological functions of the various PIP5K isoforms are not yet fully characterized. In an abstract presented at the AACR in 2009, Treinies reported on a novel PIP5K assay developed by CRT Discovery 

Laboratories.172 A high throughput screen led to the identification of two distinct chemical series that have since entered a hit to lead program. The compounds inhibited PIP5K with nanomolar potencies and were selective against a panel of protein kinases including PI3K. 

PIKfyve is a mammalian class III phosphatidylinositol phosphate kinase which synthesizes phosphatidylinositol 3,5-bisphosphate. The physiological role of this pathway is not yet clear. The PI3K inhibitor PI-103 (85) also inhibits PIKfyve. A structural analog, YM201636 (86), was shown to be 

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Other enzymes present in myelin include those involved in phosphoinositide metabolism phosphatidylinositol kinase, diphosphoinositide kinase, the corresponding phosphatases and diglyceride kinases. These are of interest because of the high concentration of polyphosphoinositides of myelin and the rapid turnover of their phosphate groups. This area of research has expanded towards characterization of signal transduction system(s), with evidence of G proteins and phospholipases C and D in myelin. 

Apart from PKA, some other protein-kinases were found to be controlled by forskolin, such as cytosolic sphingosine kinase in rat periosteal cells [186] and protein kinase B (PKB) [187]. The latter was found to be stimulated by the activation of PKA through a PI3 (phosphatidylinositol 3)-kinase-independent pathway. Furthermore, a distinct activation mechanism was suspected, other than that normally observed by growth factors such as insulin, since substitution of the serine at the S473 position of PKB with alanine could not prevent activation by forskolin. The JAK family of protein kinases in T lymphocytes can also be regulated by forskolin through the activation of PKA [188]. Thus it seems obvious that many other enzymes could be susceptible to control by forskolin. 

Of the different classes of non-protein kinases, the development of inhibitors of lipid kinases has received by far the most attention. A majority of this work centers on inhibitors of phosphatidylinositol kinases, which reflects the important role that phosphatidylinositols play as second messengers. In this section, we review inhibitors of PI3K and related kinases, as well as inhibitors of other phosphatidyl inositol kinases. We also discuss sphingosine kinase inhibitors and we end this section with a brief summary of inhibitors of other lipid kinases. 

Figure 1, Phosphoinositide Pathway. Enzymes a. phosphatidyl-inositol synthetase (PI synthetase) h. phosphatidylinositol kinase (PI kinase) c. phosphatidylinositol-4-phosphate kinase (pip kinase) d. phospholipase C (R = palmitoyl or other long-chain fatty acid chain).

Figure 1. Structure of phosphatidylinositol. Phosphatidylinositol (Ptdins) constitutes about 10% of the total phospholipids in eukaryotic cells and is the precursor of the other phosphoinositides (polyphosphoinositides) through sequential phosphorylations by specific kinases. As indicated, its inositol head group can be phosphorylated at three positions (D-3, D-4 and D-5) by specific kinases in vivo. The cleavage by phosphoinositide-specific phospholipase C (PLC), which has as its preferred substrate PtdIns(4,5)P2, is also shown. PI3K, phosphoinositide 3-kinase. PI-K II and III, phosphatidylinositol kinase types II and III. PIP-K I, phosphatidylinositol monophosphate kinase type I. PIP-K II, phosphatidylinositol monophosphate kinase type II.

The other response to G-protein activation involves phosphatidylinositol, one of the phospholipids in cell membranes (section 4.3.1.2). As shown in Figure 10.9, phosphatidylinositol can undergo two phosphorylations, catalysed by phosphatidylinositol kinase, to yield phosphatidylinositol bisphosphate (PIPj). PIPj is a substrate for phospholipase C, which is activated by the binding of (G-protein a-subunit)—GTE The products of phospholipase C action are inositol trisphosphate (IPj) and diacylglycerol, both of which act as intracellular second messengers. 

FIGURE 14-6 Main signaling pathways for histamine receptors. Histamine can couple to a variety of G-protein-linked signal transduction pathways via its four different receptors. The Hj receptor activates the phosphatidylinositol turnover via Gq/11 proteins. The other receptors either positively (H2 receptor) or negatively (H3 and H4 receptor) regulate adenylyl cyclase activity via Gs and GUo protein activation respectively. Several additional signaling pathways have been described, which are not shown. Abbreviations PfP2, phosphatidylinositol 4,5-bisphosphate PIC, phospholipase C AC, adenylyl cyclase ATP, adenosine triphosphate cAMP, cyclic AMP PKC, protein kinase C PICA, protein kinase A. 

The family of heterotrimeric G proteins is involved in transmembrane signaling in the nervous system, with certain exceptions. The exceptions are instances of synaptic transmission mediated via receptors that contain intrinsic enzymatic activity, such as tyrosine kinase or guanylyl cyclase, or via receptors that form ion channels (see Ch. 10). Heterotrimeric G proteins were first identified, named and characterized by Alfred Gilman, Martin Rodbell and others close to 20 years ago. They consist of three distinct subunits, a, (3 and y. These proteins couple the activation of diverse types of plasmalemma receptor to a variety of intracellular processes. In fact, most types of neurotransmitter and peptide hormone receptor, as well as many cytokine and chemokine receptors, fall into a superfamily of structurally related molecules, termed G-protein-coupled receptors. These receptors are named for the role of G proteins in mediating the varied biological effects of the receptors (see Ch. 10). Consequently, numerous effector proteins are influenced by these heterotrimeric G proteins ion channels adenylyl cyclase phosphodiesterase (PDE) phosphoinositide-specific phospholipase C (PI-PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phospholipase A2 (PLA2), which catalyzes the hydrolysis of membrane phospholipids to yield arachidonic acid. In addition, these G proteins have been implicated in... 

More recently, the importance of a group of highly polar inositol lipids, present in neutrophils and many other cell types, has been recognised. Activation of neutrophils by fMet-Leu-Phe results in the transient accumulation of phosphatidylinositol 3-phosphate (Ptdlns 3-P), phosphatidylinositol 3,4-bisphosphate (Ptdlns 3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (Ptdlns 3,4,5-P3). Apparently, the enzyme phosphatidylinositol 3-hydroxy (3-OH) kinase plays a key role in the formation of these novel lipids. This enzyme can catalyse the formation of these lipids from phosphatidylinositol, phosphatidylinositol 4-phosphate (Ptdlns 4-P) and phosphatidylinositol 4,5 bisphosphate (Ptdlns 4,5-P2) in vitro (Fig. 6.10). Alternatively, it is possible that Ptdlns 3,4-P2 and Ptdlns 3-P are derived from the sequential dephosphorylation of Ptdlns 3,4,5-P3. 

The binding of the sperm to a receptor on the membrane of the oocyte either activates a membrane-bound phospholipase or releases a phospholipase into the oocyte. The phospholipase hydrolyses phosphatidylinositol bisphos-phate to produce the two intracellular signals, inositol tris-phosphate (IP3) and diacylglycerol within the ovum. As in other cells, the IP3 signal increases the level of cytosolic Ca + ions and the diacylglycerol (DAG) signal activates protein kinase C. 

FIGURE 12-19 Hormone-activated phospholipase C and IP3. Two intracellular second messengers are produced in the hormone-sensitive phosphatidylinositol system inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Both contribute to the activation of protein kinase C. By raising cytosolic [Ca2+], IP3 also activates other Ca2+-dependent enzymes thus Ca2+ also acts as a second messenger. 

The phosphoinositides constitute 2-8% of the lipid of eukaryotic cell membranes but are metabolized more rapidly than are other lipids.265 278 279 283 - 285 A simplified picture of this metabolism is presented in Fig. 11-9. Phosphatidylinositol is converted by the consecutive action of two kinases into phosphatidylinositol 4,5-bisphosphate.286287 The InsP3 released from this precursor molecule by receptor-stimulated phospholipase C is thought to mobilize calcium ions by... 

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