Phosphoinositide kinases, phosphate

Adenylate cyclase Na+, id -ATPase phosphoinositide kinase Glyceraldehyde-3-Phosphate dehydrogenase... 

Figure 11.2 Structure of the insulin receptor (a). Binding of insulin promotes autophosphorylation of the (3-subunits, where each (3-subunit phosphorylates the other (3-subunit. Phosphate groups are attached to three specific tyrosine residues (tyrosines 1158, 1162 and 1163), as indicated in (b). Activation of the (3-subunit s tyrosine kinase activity in turn results in the phosphorylation of various intracellular (protein) substrates which trigger the mitogen-activated protein kinase and/or the phosphoinositide (PI-3) kinase pathway responsible for inducing insulin s mitogenic and metabolic effects. The underlying molecular events occurring in these pathways are complex (e.g. refer to Combettes-Souverain, M. and Issad, T. 1998. Molecular basis of insulin action. Diabetes and Metabolism, 24, 477-489)...

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. 

The inositol polyphosphate 5-phosphatases belong to a family of enzymes that terminate the signals generated by inositol lipid kinases and PLC. To date, two major types of 5-phosphatase have been identified, both of which share a common 5-phosphatase domain of approximately 300 amino acids, with several highly conserved motifs. Type-I enzymes are 43-65 kDa and preferentially hydrolyze 1(1,4,5)P3 and 1(1,3,4,5)P4, with the attendant formation of I(1,4)P2 and 1(1,3,4)P3, but have little or no activity towards membrane-bound phosphoinositides. The pro-totypic form of a type-15-phosphatase is a 43 kDa protein that is post-translationally modified by farnesylation of the carboxyl terminus CAAX motif this modification juxtaposes the enzyme with the membrane. Type-II enzymes are larger (75-160 kDa) and will hydrolyze both water-soluble inositol phosphates and lipids that... 

Kozawa, O., Kawamura, H, and Uematsu, T., 2000a, Sphingosine 1-phosphate amplifies phosphoinositide hydrolysis stimulated by prostaglandin F2a in osteoblasts involvement ofp38 MAP kinase. Prostaglandins Leukot. Essent. Fatty Acids 62 355-359. 

Kozawa, O., Yamamoto, T., tanabe, K., Akamatsu, S., Dohi, S. and Uematsu, T., 2000, Enhancement by sphingosine 1 -phosphate in vasopressin-induced phosphoinositide hydrolysis in aortic smooth muscle cell involvement ofp38 MAP kinase, J. Cell. Biochem. 18 46-52. 

Rakhit, S., Conway, A-M., Tate, R., Bower, T, Pyne, N.J. and Pyne, S., 1999, Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle role of endothelial differentiation gene-1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem. J. 338 643-649. 

The probes of this type were shown to selectively label at least 75% of human kinases in crude cell lysates, thus demonstrating their selectivity and promiscuity for kinases [101]. As a follow up, the labeled kinases were subjected to proteolytic digestion, and the biotinylated peptides purified on avidin beads and analyzed by LC-MS/MS. This analysis demonstrated that the site of probe labeling was indeed the conserved active-site lysine as predicted. In contrast to the promiscuity demonstrated by the acyl phosphate probes, several selective covalent inhibitors of protein kinases have been used as ABPP probes. Wortmannin is a natural product derived from the fungus Penicillium funiculosum. It is a potent and specific covalent inhibitor of phosphoinositide 3-kinase (PI3K) and the PI3K-related kinase (PIKK) families [102, 103]. The use of natural products in relation to ABPP is covered by Breinbauer et al. [104]. 

Wyman MP, Bulgarelli-Leva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield MD, Panayotou G (1996) Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 16 1722-1733... 

There are two main procedures for measuring PI 3-kinase activity which measure lipid kinase activity in intact cells or broken cell lysates respectively, and both rely on detecting the transfer of the y- phosphate of ATP to the D-3 position of the inositol head group of phosphoinositide lipids. The first method relies on metabolic labeling of intact cellular pools of ATP with [32P]Pi followed by lipid extraction (3,4) and separation of the phosphorylated lipids by high-performance liquid chromatography (HPLC) analysis (5). The advantages of this procedure are ... 

The detection of PI in bacterial and archaeal systems implies that PI synthase, which is an integral membrane protein, is present. Once PI is available, kinases could add phosphates to generate various phosphoinositides, although to date this chemistry has only been demonstrated to occur in eukaryotes. Bacterial catabolic activities specific to PI have also been identified. In most cases these are secreted soluble proteins that can cleave PI and in some cases GPI-anchored... 

Rho proteins are involved in regulation of the actin cytoskeleton and induce formation of stress fibers and focal adhesions (Paterson et al., 1990 Ridley and Hall, 1992 Tominaga et al., 1993 Laudanna et al., 1996). Most probably independently of their role in actin regulation, Rho proteins have been proposed to participate as molecular switches in the control of phosphoinositide-3-kinase (Zhang et al., 1993), phosphatidylinositol-4-phosphate-5-kinase (Chong et al.. 

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, PtdIns(3,4,5)P3 is localized in the cell membrane and performs its function in close association with processes at the cell membrane. The concentration of PtdIns(3,4,5)P3 in the cell depends both on the rates of synthesis by PI3-kinases and the rates of hydrolysis of its phosphate residues. Several inositol polyphosphate phosphatases have been identified that remove the phosphates at position 3 or 5 of the inositol moiety. Among the inositol polyphosphate phosphatases with specificity for the 3-position, the PTEN phosphatase has been identified as a tumor supressor protein (see below). 

A FIGURE 13-28 Synthesis of DAG and IP3 from membrane-bound phosphatidylinositol (PI). Each membrane-bound PI kinase places a phosphate (yellow circles) on a specific hydroxyl group on the inositol ring, producing the phosphoinositides... 

---