Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis

Bacillus subtilis has three active tyrosine kinases, PtkA, PtkB and McsB, which play an important role in the physiology of the bacterium. Genome sequence analysis and biochemical experiments indicated that the ortholog of McsB, BAS0080, is the only active tyrosine kinase present in Bacillus anthracis. The autophosphorylation of McsB of B. anthracis was enhanced in the presence of an activator protein McsA (BAS0079), a property similar to that reported for B. subtilis. However, the process of enhanced phosphorylation of McsB in the presence of McsA remains elusive. To understand the activation mechanism of McsB, we carried out spectroscopic and calorimetric experiments with McsB and McsA. The spectroscopic results suggest that the binding affinity of Mg-ATP for McsB increased by one order from 103 to 104 in the presence of McsA. The calorimetric experiments revealed that the interaction between McsB and McsA is endothermic in nature, with unfavourable positive enthalpy (Δ H) and favourable entropy (Δ S) changes leading to an overall favourable free energy change (Δ G). Kinetics of binding of both ATP and McsA with McsB showed low association rates (ka) and fast dissociation rates (kd). These results suggest that enhanced phosphorylation of McsB in the presence of McsA is due to increased affinity of ATP for McsB

Combination Treatments with the PKC Inhibitor, Enzastaurin, Enhance the Cytotoxicity of the Anti-Mesothelin Immunotoxin, SS1P

<div><p>Activated protein kinase C (PKC) contributes to tumor survival and proliferation, provoking the development of inhibitory agents as potential cancer therapeutics. Immunotoxins are antibody-based recombinant proteins that employ antibody fragments for cancer targeting and bacterial toxins as the cytotoxic agent. Pseudomonas exotoxin-based immunotoxins act via the ADP-ribosylation of EF2 leading to the enzymatic inhibition of protein synthesis. Combining PKC inhibitors with the immunotoxin SS1P, targeted to surface mesothelin, was undertaken to explore possible therapeutic strategies. Enzastaurin but not two other PKC inhibitors combined with SS1P to produce synergistic cell death via apoptosis. Mechanistic insights of the synergistic killing centered on the complete loss of the prosurvival Bcl2 protein, Mcl-1, the loss of AKT and the activation of caspase 3/7. Synergy was most evident when cells exhibited resistance to the immunotoxin alone. Further, because PKC inhibition by itself was not sufficient to enhance SS1P action, enzastaurin must target other kinases that are involved in the immunotoxin pathway.</p></div

Cytotoxic activity of the anti-mesothelin immunotoxin, SS1P, in combination with PKC inhibitors on KB cells.

<p>PKC inhibitors (Enzastaurin, Sortastaurin or Go6976) at 10 uM were added in combination with SS1P (50 ng/ml) to KB cells and viability assessed using the CellTiter-Glo® Viability Assay after 48 hrs. Enz = enzastaurin, Sotras = sotrastaurin.</p

IC50 of combination (SS1P & Enzastaurin) treatment relative to SS1P alone.

<p>IC50 of combination (SS1P & Enzastaurin) treatment relative to SS1P alone.</p

Enzastaurin in combination with agents that inhibit protein synthesis.

<p>KB cells were treated with CHX (A), DT (B) or HB21-PE40 (C) alone or in combination with enzastaurin at the concentrations indicated for 24 hrs (Caspase Assay) or 48 hrs (CellTiter-Glo® Assay). Both ATP and Caspase levels are represented in relative luminescence units. Panel D shows a replot of data where HB21-PE40 was added at 1.25 ng/ml without or with enzastaurin at the indicated concentrations. Data are presented as % control compared to untreated cells (for SS1P) or to enzastaurin alone (for the combination).</p

SS1P in combination with enzastaurin results in loss of Akt.

<p>KB cells were treated with SS1P, enzastaurin, SS1P+Enzastaurin, HB21-PE40 - overnight. Akt was detected via immunoblot as indicated in materials and methods. β-actin was used as a loading control. Control (Lane 1), SS1P 500 ng/ml (Lane 2), Enzastaurin 1 uM (Lane 3), SS1P 500 ng/ml+Enzastaurin 1 uM (Lane 4), Enzastaurin 10 uM (Lane 5), SS1P 500 ng/ml+Enzastaurin 10 uM (Lane 6) and HB21-PE40 200 ng/ml (Lane 7).</p

Cell viability following incubation with SS1P, enzastaurin and combinations of both in KB and KLM1 cells.

<p>KB and KLM1 cells were seeded in 6 well plates and treated with the indicated concentration of SS1P, Enzastaurin or both for 48<b>.</b></p

Protein synthesis levels in KB cells treated with SS1P,enzastaurin alone or a combination of both.

<p>KB cells were treated with SS1P and enzastaurin alone or in combination at the concentrations indicated in the figure. Protein synthesis was determined by measuring the incorporation of 3H-leucine into cells at 24 h post treatment. Data is represented in cpm/well. Error bars show one standard deviation of triplicate samples.</p

Caspase 3/7 activity following SS1P, enzastaurin or combinations of both agents.

<p>SS1P (100 ng/ml) or enzastaurin (10 uM) were added to KB, KLM1 or HAY cell lines individually or in combination. Caspase 3/7 activity is represented as relative luminescence units and was measured after cells were treated with an overnight incubation (approximately 20 hr).</p