Lipid Kinase Assay

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. 

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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 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. 

Figure 2. Membranes isolated from Pyrococcus furiosus will phosphorylate Ptdlns to form PtdIns3P. Microsomal membranes were isolated from Daucus carota (wild carrot cells) grown in cell culture and P. furiosus. Lipid kinase activity was assayed as previously described using Ptdlns as a substrate (Bunney et al., 2000). The lipids were extracted and separated by thin layer chromatography as described by Walsh et al. (1991) to separate Ptdlns3P and Ptdlns4P. The migrations of the lipid standards are indicated (Brglez and Boss, unpublished results).

The sizes of these panels vary from about 200 kinases at Caliper to over 400 at Ambit. The assay counts include the lipid kinases, mutant kinases and kinases that are available in more than one assay condition (the number of wild type kinases in any of the panels is less than the total). These assay panels enable assessment of broad selectivity and liability information needed for lead and candidate characterization. They also enable broad screening for chemical starting points for kinases across the kinome in parallel. Indeed, we reported doing this in 2008 against the 203 kinases available in the Ambit panel at that time, which will be discussed elsewhere in this review. 

By combining assays from the nine providers in Appendix 1.1, inhibitor binding for up to 407 wild type protein or lipid kinases can be assessed. The data in Appendix 1.1 shows that the kinase families can be well covered by a combination of the commercial assay panels. Where one panel may be deficient, other panels provide coverage. 

The Millipore human kinase panel consists of 236 wt kinases (including some lipid kinases fluorescence energy transfer technology is used for lipid kinases). They also provide assays for 40 mutant kinases and for 11 kinases with special substrates or in activated states. As with most catalytic assay suppliers, whole panel screens are typically done at one ATP concentration (10 pM ATP for Millipore), but assays at the ATP Km can be requested. For more information on the Millipore panel, see http //www.millipore.com/drugdiscovery/dd3/ kinaseprofiler. 

The RBC kinase panel consists of 325 wt kinases (including some lipid kinases), 35 mutants and 6 kinases with special substrates or in activated states. A review of the technology as well as most of the other technologies discussed here was recently published by authors from RBC.23 This paper provides a nice overview of many of the standard catalytic assays used by RBC and other kinome assay providers. For more information on the RBC panel, see http // www.reactionbiology.com/pages/kinase.htm. 

Caliper Life Sciences also provides a CE based kinase panel. The Caliper Life Sciences panel consists of 177 wt kinases (including some lipid kinases) and 12 mutant kinases. This company provides kinases and substrates on assay plates and is not strictly a screening service, but it could be a good way forward for labs that have access to a Caliper CE instrument. The company does also provide a smaller panel as a screening service. For more information about both of these, see http //www.caliperls.com/products/reagents/kinase-profiling/ profilerpro-kinase-selectivity-assay-kits.htm. 

The Carna human kinase panel consists of 275 wt kinases (including some lipid kinases), 29 mutants and 2 kinases with alternate binding partners. The standard ATP concentration for the Carna panel is the ATP apparent Km, and half of the panel can also be run at up to 1 mM ATP. As indicated above, the Carna assay technology is primarily CE, but they also offer some assays that are not amenable to CE, and these are run primarily in ELISA format. Similar to Caliper Life Sciences, Carna also offers large subset of their kinase panel as kits for Caliper CE instruments. Details and specific kinase can be found at https //www.carnabio.com/english/product/assay-msa.html. 

Hampton, R.Y. Raetz, C.R.H. Lipid A 4-kinase from Escherichia coli enzyme assay and preparation of 4- 2p-labeled probes of high specific radioactivity. Methods Enzymol., 209, 466-475 (1992)... 

Regulatory properties and precise substrate specificities of the PI 3-kinase(s) may be remarkably distorted by the assay conditions and form of lipid presentation used. 

Furthermore, the expression and function of the Pgp can be modulated by indirect mechanisms, such as interactions with membrane lipids [67] or inhibition of protein kinase C. The reversal of MDR is established using tumor cells lines that are made resistant by the exposure to an anticancer agent or by transfection of the mdrl or mrpl genes. The parameter most widely used to show the activity of MDR reversal agents is reversal factor (RF). This type of assay assumes that the reversal agent does not show inherent cytotoxicity at the concentrations tested. 

All other panels of isolated kinases use catalytic assays and measure the conversion of a substrate (either ATP or peptide/lipid) to product. Because these are catalytic assays, they are all run in the presence of ATP plus a substrate. The ability to assay inhibitors at multiple ATP concentrations provides the scientist with the ability to obtain some information on whether the inhibitor is competitive with ATP or not and to rapidly eliminate the... 

For the assay of PI kinase, A431 cell membrane and [y-32p]ATP were mixed in 20 mM HEPES buffer (pH 7.2). The reaction mixture was incubated for 20 minutes at 20"C and stopped by the addition of CHCI3, MeOH, and IN HCl (4 1 2). After vigorous vortexing, the lower phase was applied to a silica gel column for the separation of phospholipid and unreacted [y-32p]ATP (31). The phosphorylated lipid was eluted with CHCI3, MeOH, and 4N NH4OH (9 7 2) and the radioactivity was quantified by liquid scintillation counting. 

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). 

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