Microarrays for the Functional Analysis of the Chemical-Kinase Interactome

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals formonitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (K m ATP = 3.3 µ M) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 µ M adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coatedmicroarrayswas inhibited in the nanoliter reactions by GW5074 at the expected IC 50of 9 nM. Common kinase inhibitors were printed onmicroarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRβ, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC 50 determinations, and selectivity profiling.

Effect of tannins in the rumen microbial growth measured by 32P incorporation

The phenolic compounds are substances which reduce feed intake, protein digestibility and reduce ruminal activity in sheep and goats. To reduce the effects of tannins a lot of synthetic materials, for example PEG (polyethylene glycol) is tested as tannin-binding agent, and let protein free for the digestibility. The aim of this work was to test the effect of tannins in the rumen microbial growth using the in vitro 32P incorporation technique.

UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor

We have compared the activation and trafficking of epidermal growth factor receptor (EGFR) induced by UV light and EGF. Tyrosine phosphorylation of EGFR was not detected in UV-exposed cells by immunoblotting of whole cell lysates or EGFR immunoprecipitates with antibodies specific for each of the five activated autophosphorylation sites of EGFR. In addition, EGFR of UV-irradiated cells did not demonstrate increased 32P-incorporation. However, UV-exposed cells demonstrated a gel mobility shift of EGFR, which was not abolished by alkaline phosphatase treatment. UV-exposure did not induce dimerisation of EGFR. Furthermore, UV induced internalisation of EGFR without polyubiquitination or degradation. UV-exposed EGFR was transferred to early endosomes and arrested in transferrin-accessible endosomes close to the cell surface. Whereas inhibition of the EGFR tyrosine kinase effectively inhibited tyrosine phosphorylation and internalisation of EGF-activated EGFR, internalisation of UV-exposed EGFR was unaffected. UV induced neither relocalisation of Shc and Grb2 nor activation of Raf, but activation of MEK and MAPK was observed. Our work indicates that UV induces internalisation of EGFR independent of its phosphorylation or receptor tyrosine kinase activation, and altered EGFR trafficking compared with ligand-activated receptor. In addition, MAPK activation by UV does not appear to be mediated by EGFR activation.

Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl

The effect of cytoplasmic Cl concentration ([Cl]i) on the activation state ([3H]benzmetanide binding rate) and phosphorylation state (32P incorporation) of the Na-K-Cl cotransporter was evaluated in secretory tubules isolated from the dogfish shark rectal gland. Reduction of [Cl]i at relatively constant cell volume (by removal of extracellular Cl or Na or by addition of bumetanide) increased cotransporter activation and phosphorylation. Raising extracellular K concentration ([K]o) from 4 to 80 mM, a maneuver that elevated [Cl]i above normal, reduced basal cotransport activity and rendered it entirely refractory to forskolin. High [K]o also blocked activation and phosphorylation in response to cell shrinkage, despite the fact that [Cl]i was already greatly elevated as a consequence of osmotic water loss. The phosphatase inhibitor calyculin A also promoted activation, but not in cells preexposed briefly to high [K]o. In summary, maneuvers than lower [Cl]i activate the cotransporter, whereas those that elevate [Cl]i (or prevent it from decreasing) block activation in response to secretory stimuli. Cell Cl appears to govern its own rate of entry via Na-K-Cl cotransport by impeding regulatory phosphorylation of the Na-K-Cl cotransport protein.

Dietary medium-chain triglycerides can prevent changes in myosin and SR due to CPT-1 inhibition by etomoxir

To define determinants of subcellular structures of heart, Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were treated for 5 wk with 15 mg.kg-1.day-1 etomoxir [reduces mitochondrial carnitine palmitoyltransferase-1 (CPT-1) activity and fatty acid synthesis]. To bypass CPT-1 inhibition, etomoxir-treated rats were fed a medium-chain fatty acid (MCFA) diet. Etomoxir induced a proportionate growth of heart, which could partially (WKY, P < 0.05) or completely (SHR, P < 0.05) be prevented by the MCFA diet. Also the etomoxir-induced increase in myosin V1 was partially prevented (P < 0.05). Etomoxir increased (P < 0.05) rate of sarcoplasmic reticulum (SR) Ca2+ uptake of WKY and SHR ventricular homogenates in the presence or absence of the SR Ca2+ release inhibitor ruthenium red. The MCFA diet resulted in SR Ca2+ uptake rates that were in between those of etomoxir-treated and untreated rats. The in vitro 32P incorporation into phospholamban and troponin I did not differ significantly in WKY. Etomoxir induced, however, an increase (P < 0.05) in the phosphorylated intermediate of the Ca2+ adenosinetriphosphatase in WKY that was prevented by the MCFA diet. In SHR, etomoxir increased the in vitro phospholamban phosphorylation, which was reduced compared with WKY. The data show that myosin and SR are affected by a chronically altered substrate utilization of heart.

Modulation of the phosphorylation state of tau in situ: the roles of calcium and cyclic AMP

Alterations in situ in the phosphorylation state of the microtubule-associated protein tau were examined in response to increasing intracellular levels of Ca2+ through N-methyl-D-aspartate (NMDA)-receptor activation, or activating cyclic AMP (cAMP)-dependent protein kinase (cAMP-PK), in rat cerebral-cortical slices. Increasing intracellular concentrations of Ca2+ by treatment of the brain slices with the glutamate analogue NMDA in depolarizing conditions (55 mM KCl) resulted in dephosphorylation of tau. Addition of KCl+NMDA to the slices resulted in a 40% decrease in 32P incorporation into tau, whereas addition of KCl or NMDA alone had no effect on tau phosphorylation. The KCl+NMDA-induced dephosphorylation of tau was blocked by the non-competitive NMDA-receptor antagonist MK801. Determine the involvement of the Ca2+/calmodulin-dependent phosphatase, calcineurin, in the KCl+NMDA-induced dephosphorylation of tau, slices were pretreated with the calcineurin inhibitor Cyclosporin A. Pretreatment of the rat brain slices with Cyclosporin A completely abolished the dephosphorylation of tau induced by the addition of KCl+NMDA. The dephosphorylation of tau in situ was site-selective, as indicated by the loss of 32P label from only a few select peptides. Activation of cAMP-PK by stimulating adenylate cyclase in rat cerebral-cortical slices with forskolin resulted in a 73% increase over control levels in 32P incorporation into immunoprecipitated tau. Two-dimensional phosphopeptide mapping revealed that most of the sites on tau phosphorylated in brain slices in response to increased cAMP levels were the same as those phosphorylated on isolated tau by purified cAMP-PK. Although the state of tau phosphorylation is certainly regulated by many protein phosphatases and kinases in vivo, to our knowledge this study provides the first direct evidence of a specific protein phosphatase and kinase that modulate the phosphorylation state of tau in situ.