Materials and methods See Supplementary Material and Methods. Results Jak2 knockdown reduced levels of Bcr Abl protein and Tyr 177 phosphorylated form STF-62247 inhibitor of Bcr Abl in mouse hematopoietic and CML cell lines Our findings with Bcr Ablt cell lines including cells expressing imatinib mesylate resistant forms of Bcr Abl indicate that Jak2 is a critical player in CML.20,22 We began exploring the mechanisms behind this critical role of Jak2 in CML. We found that Jak2 controls Bcr Abl protein levels, as Jak2 knockdown causes a rapid disappearance of Bcr Abl. We used a specific mouse form of Jak2 small interfering RNA to knockdown Jak2 in Bcr Ablt 32D mouse myeloid cells.
Jak2 knockdown BMS 794833 dramatically reduced levels of the pTyr Bcr Abl protein. To offset the possible nonspecific effects of Jak2 knockdown, we rescued Jak2 knockdown by transducing Jak2 cDNA into cells at the time of Jak2 knockdown. The rescued cells had restored levels of pTyr Bcr Abl in cells corresponding to the restored expression of Jak2, suggesting that Jak2 controls the expression of Bcr Abl. We also used an inducible form of specific human Jak2 short hairpin RNA to knockdown Jak2 in three CML cell lines, namely BV173, KBM 7 and K562 R. Levels of Jak2 were reduced and also Bcr Abl protein levels were drastically reduced following Jak2 knockdown in K562 R cells. Reversal of Jak2 knockdown restored the expression of the Bcr Abl protein. In Jak2 knockdown experiments, levels of Tyr177 phosphorylation within Bcr Abl were also strongly inhibited as expected as Bcr Abl had also disappeared.
Importantly, rescue experiments partially restored levels of pTyr177. Similar results were observed by Jak2 knockdown in CML cell lines BV173 and KBM7. Jak2 phosphorylated the tyrosine 177 Bcr sequence found in Bcr Abl An analysis of the Bcr Abl sequence revealed various tyrosine residues that are Jak2 consensus phosphorylating sites.31 We observed that tyrosine 177 within Bcr Abl fits the consensus Jak2 phosphorylation motif. To test whether Jak2 would phosphorylate Tyr177, we made a Bcr peptide that contains sequences surrounding the tyrosine 177 sequence of Bcr Abl, and used that peptide as a target for Jak2. Purified recombinant Jak2 readily phosphorylated this peptide and this phosphorylation was strongly inhibited by the selective Jak2 inhibitor TG101209 but not by IM.
We note that TG is a potent inhibitor of Jak2,s ability to phosphorylate Tyr 177 in kinase assays with an BIC 50 of less than 0.01 mM 35. Jak2 immune complexes isolated from Bcr Ablt 32D cells also phosphorylated the Tyr 177 Bcr peptide and phosphorylation was inhibited by TG but not by IM. These Jak2 immune complexes contain Bcr Abl, Jak2 and HSP90 and other signaling members such as Akt and STAT3.20,21 Thus, although Bcr Abl is present in the Jak2 immune complex, it does not phosphorylate Tyr 177 as IM does not inhibit tyrosine phosphorylation of the peptide. We note that purified near full length recombinant c Abl kinase only poorly phosphorylated the Tyr 177 site in the Bcr peptide. Jak2 inhibition of phosphorylation of Tyr177 is a separate event from disappearance of Bcr Abl To determine whether the disappearance of Bcr Abl could