Lipid kinases

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. 

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A subfamily of Rho proteins, the Rnd family of small GTPases, are always GTP-bound and seem to be regulated by expression and localization rather than by nucleotide exchange and hydrolysis. Many Rho GTPase effectors have been identified, including protein and lipid kinases, phospholipase D and numerous adaptor proteins. One of the best characterized effector of RhoA is Rho kinase, which phosphorylates and inactivates myosin phosphatase thereby RhoA causes activation of actomyosin complexes. Rho proteins are preferred targets of bacterial protein toxins ( bacterial toxins). 

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... 

Topham, M. K. and Prescott, S. M. Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions./. Biol. Chem. 274 11447-11450,1999. 

Other members of class I of the PI3-kinases, such as PI3-kinase of the y subtype, are stimulated by interaction with Pycomplexes (see Chapter 5.5.7) and have their own regulatory subunit. It is interesting that both a lipid kinase activity and a protein kinase activity have been identified in the catalytic domain of the P13-kinase y subtype in brain (Bondeva et al., 1998). Activation of the MAPK pathway (see Chapter 10) may take place via the protein kinase activity, so that this enzyme can produce a bifurcated signal the lipid kinase activity stimulates the Akt kinase (see below), the protein kinase the MAPK pathway. Proliferation promoting signals are transmitted via both pathways. 

Fiunter, T, When is a hpid kinase not a lipid kinase When it is a protein kinase (1995) CeU 83, 1-4... 

Baynes, K.C.R. Beeton, C.A. Panayotou, G. Stein, R. Soos, M. Hansen, T Simpson, H. O Rahilly, S. Shepherd, P.R. Whitehead, J.P. Natural variants of human p85a phosphoinositide 3-kinase in severe insulin resistance a novel variant with impaired insulin-stimulated lipid kinase activity. Diabetologia, 43, 321-331 (2000)... 

Bajjalieh, S.M. Martin, T.F.J. Floor, E. Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles. J. Biol. Chem., 264, 14354-14360 (1989)... 

Sugiura, M. Kono, K. Liu, H. Shimizugawa, T. Minekura, H. Spiegel, S. Kohama, T. Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization. J. Biol. Chem., 277, 23294-23300 (2002)... 

Yee M-C, Fas CF, Stohlmeyer MM, Wandless TJ, Cimprich KA (2005) A cell-permeable, activity base probe for protein and lipid kinases. J Biol Chem 280 29053-29059... 

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 ... 

Does not necessarily distinguish between the lipid kinase activity of various different isoforms and subtypes of the PI 3-kinase family. 

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. 

May detect other lipid kinases such as PI 4-kinases that coassociate with immunoprecipitated proteins or nonspecifically associate with protein A-Sepharose beads. 

Wash immunoprecipitates three times in lysis buffer containing nonionic detergent such as Nonidet P-40 (see Note 8), twice in buffer without detergent, twice in phosphate buffered saline, twice in 0.5M LiCI, 100 mM Tris, pH 7.2, once in water and once in lipid kinase assay buffer. Resuspend immunoprecipitates in 40 pL of lipid kinase buffer and place at room temperature (see Note 8). 

Fig. 3. TLC separation of D-3 phosphoinositide lipids derived from in vitro lipid kinase assay of p85 immunoprecipitates using Ptdlns as a substrate. Immuno-precipitates are derived from resting (lane 1) or RANTES-stimulated (lane 2) human T lymphocytes.

In summary, the KinaTor technology appears to be extremely potent for potentially revealing all binding partners of biologically active kinase inhibitors. Transmembrane domain proteins bind, as well as lipid kinases and other nucleotidebinding proteins, such as heat-shock proteins and oxidoreductases (unpublished data). So far, there are no limitations. 

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