e separated from the inhibition of Tyr 177 phosphorylation, we performed kinase assays with immune complexes harvested from Bcr Ablt 32D cells with anti Jak2 antibodies, and determined whether Jak2 inhibition would decrease levels of pTyr177 and whether levels of Bcr Abl in the immune complexes would also be reduced by Jak2 inhibition. The Bcr Abl Protein was not decreased MLN518 FLT-3 inhibitor in these immune complexes by Jak2 inhibition but importantly levels of pTyr177 were strongly decreased. Similarly, levels of pTyr Bcr Abl were not reduced by treatment of the kinase reaction mixture with 5 and 10 mM TG. Importantly, Jak2 and Bcr Abl coprecipitated in immune complexes.
These results indicate that the events leading to the decrease of Bcr Abl occurred within intact cells but not in immune complexes Bosutinib from these same cells and, more importantly, the inhibition of phosphorylation of Tyr177 by TG can readily occur in these subcellular fractions under conditions wherein Bcr Abl levels were stable. Importantly, we found that Jak2 inhibition caused only background levels of apoptosis during the first 4 h of treatment wherein Bcr Abl levels were strongly reduced. Jak2 inhibitor TG101209 rapidly decreased the levels of pTyr177 Bcr Abl We tested the effects of Jak2 inhibition in various Bcr Ablt cell lines and CML cell lines. Jak2 inhibition reduced levels of active Jak2 in a dose dependent manner.22 The 50% point of Jak2 inhibition as measured by pTyr1007 phosphorylation was estimated to be 5 mM as estimated by quantitation of the intensity values.
Similarly, the level of Bcr Abl pTyr177 was inhibited in a dose dependent manner and the 50% inhibitory point was estimated to be 5 mM. The 50% inhibitory point for Bcr Abl reduction was estimated to be B7.5 mM. Interestingly, the selective Jak2 inhibitor TG rapidly decreased levels of phosphorylation of Tyr177 of Bcr Abl but also decreased levels of Bcr Abl during this time period. The CML cell line K562 R showed rapid loss of pTyr177 Bcr Abl and the Bcr Abl protein after 3 h, as did KBM7 CML cells. Reductions of pTyr177 Bcr Abl and Bcr Abl were seen in CML cell line K562. Cells from a blast crisis CML patient treated with TG101209 also had a reduction of pTyr177 Bcr Abl and Bcr Abl protein. We also showed that Jak2 inhibition rapidly reduced the levels of total pTyr proteins. The Jak2 consensus sites include YxxV/L/I31 and there are a number of these Jak2 consensus sites in Bcr Abl.
We wondered whether YxxF would also be a site that is phosphorylated by Jak2, as F like V/L/I is a hydrophobic amino acid. There are three such sites in Bcr Abl including Tyr 360. We had made a mouse monoclonal antibody against the pTyr360 sequence of Bcr. This sequence specific pTyr monoclonal antibody detected a signal in Bcr Abl, and Jak2 inhibition by a new Jak2 inhibitor WP1193 dramatically reduced the level of pTyr360 Bcr Abl within 60 min, suggesting that Jak2 also phosphorylates Tyr360 of Bcr Abl. pTyr177 within P160 BCR is also rapidly reduced by Jak2 inhibition Similar to Bcr Abl, phosphorylation of Tyr177 within P160 BCR was also rapidly reduced by treatment with 10 mM TG. As Bcr is believed to form heterotetramers with Bcr Abl, these results suggest that Jak2 would phosphorylate Tyr177 within Bcr Abl and Bcr in these heterotetrame