Jak2 inhibition rapidly decreased pTyr levels of STAT5, which is consistent with downregulation of STAT5 transcriptional activity. It remains to be determined whether inactivation of Bcr Abl by Jak2 interferes with Bcr Abl,s ability to activate STAT5. Jak2 knockdown reduced levels of tyrosine phosphorylation of Shc and Gab2 The reduction of Grb2 binding to Bcr Abl and the resultant Trichostatin A 58880-19-6 decrease in Ras activation prompted an examination of Jak2 effects on phosphorylation of Shc. It is known that pTyr Shc also binds Grb2. Knockdown of Jak2 strongly decreased levels of pTyr Shc in CML cell line K562 R and BV173 cells. Thus, Jak2 inhibition not only reduced levels of Grb2 binding to Bcr Abl, but Jak2 knockdown also reduced the alternate pathway for activating Ras, namely the tyrosine phosphorylation of Shc.
Both of the effects of Jak2 inhibition would lead to reduced levels of Ras activation. Jak2 knockdown also reduced levels of pTyr Gab2, which binds to Grb2,6 thereby reducing the activation of the PI 3 kinase pathway in K562 R cells. The levels of pMEK1 and 2, pSer 9 of GSK3 and pTyr STAT5 BMS-540215 were also reduced by Jak2 knockdown in BV173 cells. Jak2 controls the Gab2/PI 3 kinase, Akt and GSK3 through its ability to induce phosphorylation of Gab220 and would control Ras activation and downstream MEK activation by Jak2,s ability to phosphorylate Tyr177 of Bcr Abl and Tyr 239/240 of Shc. We note that Jak2 knockdown did not decrease either Lyn kinase or total Grb2 levels. Our findings indicate that Lyn kinase is not part of the Bcr Abl/Jak2/HSP90 network complex.
22 Thus, prolonged Jak2 inhibition would seriously depress levels of activated Ras and PI 3 kinase activation in Bcr Ablt leukemia cells. A new pan Jak kinase inhibitor, inhibits Jak2 effects on Bcr Abl and Tyr177 phosphorylation We tested the effects of a more potent analog of the Jak2 inhibitor AG490 for its ability to reduce Bcr Abl and to inhibit phosphorylation of Tyr177 of Bcr Abl. WP1193 inhibited the phosphorylation of Tyr177 housed within the Bcr peptide with 50% inhibition point of o2.5 mM. Like AG490, WP1193 inhibited tyrosine phosphorylation of Jak2 in cells and also inhibited tyrosine phosphorylation of Jak1 and Jak3, WP1193 also inhibited the autophosphorylation of Jak2 in vitro. It has been reported that Jak1 kinase interacts with Jak2 leading to the strengthening of the downstream effects of cytokine signaling through Jak2.
30 WP1193 rapidly reduced levels of Bcr Abl and pTyr177 Bcr Abl within several Bcr Ablt cell lines including T315I cells and cells from blast crisis CML patients. WP1193 appeared to be more potent than TG. The estimated point of 50% inhibition of phosphorylation of Tyr177, and Bcr Abl reduction forWP1193 was between 2.0 and 3.0 mM in whole cells, respectively. Overall, the pan Jak inhibitor, although much less potent in Jak2 kinase assays than TG101209, WP1193 was similar if not more potent at reducing levels of Bcr Abl and pTyr177 compared with TG101209. Like TG, WP1193 was able to reduce binding of Grb2 to Bcr Abl complexes while reducing levels of pTyr177 Bcr Abl. WP1193 rapidly reduced RAS GTP levels and pTyr Gab2, and STAT5 levels. WP1193 was a potent inhibitor of the Jak2 kinase in a test tube kinase assay but did not in