In all cases, BCR ABL phosphorylation CI-1040 MEK inhibitor was not completely suppressed after 24 h in culture with addition of Dox. These results were consistently observed in two individual cotransduced cell lines. Similarly, BCR ABL protein expression was also suppressed to a lesser degree in cotransduced cells than in cells transduced with BCR ABL only, and Ahi 1 protein expression was found to be higher in cotransduced cells than in cells transduced with BCR ABL only. We next evaluated the eff ect of increased BCR ABL expression and tyrosine phosphorylation of cotransduced cells on the activity of candidate signaling mechanisms. We observed increased levels of phosphorylation of JAK2, STAT5, NF B p65, and Src in BCR ABL inducible cells and Ahi 1 cotransduced BCRABL inducible cells compared with control BaF3 cells.
Interestingly, phosphorylation of most downstream proteins was down regulated when BCR ABL expression was inhibited by Dox, but sustained phosphorylation Bergenin of JAK2 and STAT5 was consistently observed in cotransduced cells in the presence of IL 3 and Dox. In the absence of IL 3, phosphorylation of JAK2 and STAT5 was reduced in cotransduced cells when BCR ABL expression was suppressed. A similar, albeit less pronounced, fi nding of sustained phosphorylation of Src was also observed, particularly in Ahi 1 BCRABL 1 cells in the presence of IL 3. These results suggest that Ahi 1 may play a regulatory role in mediation of BCRABL activity associated with enhanced activation of JAK2 and STAT5 through the IL 3 signaling pathway.
AHI 1 physically interacts with BCR ABL To detect a physical interaction between AHI 1 and BCRABL in CML cells, coimmunoprecipitation experiments were performed. We demonstrated a direct interaction between AHI 1 and BCR ABL at endogenous levels by detection of BCR ABL in human CML cells after IP with a human AHI 1 antibody. This interaction was not found in a BCR ABL T cell line or in control antibody coimmunoprecipitated K562 cells. The result was confi rmed by detection of AHI 1 in the same cells after IP with a specifi c antibody to ABL. Importantly, tyrosine phosphory lated p210 BCR ABL could be detected in K562 cells using an antiphosphotyrosine antibody after IP with the AHI 1 antibody. Interestingly, this protein interaction complex is also associated with a 120 kD tyrosine phosphorylated protein.
To determine whether this 120 kD protein could be an AHI 1 isoform, the same membrane was washed and reprobed with an AHI 1 antibody. The full length and a 100 kD isoform of AHI 1 protein were identifi ed by Western blot analyses. Using co IP, we examined several candidate 120 kD tyrosine phosphorylated proteins known to interact with BCRABL, including CBL and JAK2. We determined that JAK2 was associated with this protein interaction complex, as AHI 1 could be detected by an anti AHI 1 antibody in K562 cells after IP with a specifi c antibody to JAK2, this interaction was further confi rmed by reverse detection of JAK2 after IP with the anti AHI 1 antibody. In addition, the antigenic peptide derived from the sequence of AHI 1 specifi cally blocked the ability of the AHI 1 antibody to precipitate both tyrosine phosphorylated BCR ABL and the 120 kD protein, whereas an unrelated peptide had no eff ect. Interestingly.