Figure 2

Jak2 phosphorylates Bcr–Abl on Tyr177. (a) Jak2 inhibitor TG101209 (TG, TargeGen Inc., San Diego, CA, USA) but not IM inhibited phosphorylation of a Bcr Tyr177 peptide (custom synthesized by Bachem Co., Torrance, CA, USA) in Jak2 immune complexes from the cell lysates of 32Dp210 cells immunoprecipitated with anti-Jak2 antibody coupled to agarose beads. The target peptide has the Bcr sequence H-Ala-Glu-Lys-Pro-Phe-Tyr(177)-Val-Asn-Val-Glu-Phe-His-His-Glu-(Lys-Lys-Lys). The bolded amino acids are the Grb2 binding site in Bcr–Abl (YVNV) and are the Jak2 consensus phosphorylation site. The three Lys residues allow binding to Whatman 3 mm filters. Anti-Jak2 immune complexes were incubated in a kinase assay with γ p32 ATP in the presence of various concentrations of either Jak2 inhibitor TG or IM. (b) Jak2 inhibition of recombinant Jak2 (JH1 domain, 50 ng; Invitrogen, Carlsbad, CA, USA) by TG but not IM inhibited phosphorylation of a Tyr177 Bcr peptide. The Jak2 kinase assay with γ p32 ATP was performed using recombinant Jak2 kinase (JH1 domain, GST-Jak2 aa808–1132 in presence of either Jak2 inhibitor TG or IM using the Bcr synthetic target peptide containing Tyr177 sequence. (c) Jak2 efficiently phosphorylates the Tyr177 Bcr whereas c-Abl prefers to phosphorylate the YxxP sequence. Recombinant JH1 of Jak2 was used to phosphorylate either the Bcr Tyr177 peptide or the Abltide peptide (BIOMOL International, Plymouth Meeting, PA, USA). (d) Jak2 inhibition reduced levels of pTyr177 Bcr–Abl in kinase assays performed with anti-Jak2 immune complexes but did not reduce levels of Bcr–Abl. Mouse 32Dp210 cells were lysed and anti-Jak2 immune complexes were harvested as described.² Lysates were western blotted with either pTyr 177 Bcr antibody or anti-Abl 8e9 antibody. (e) Jak2 inhibition reduced levels of pTyr 177 Bcr–Abl in kinase assays performed with anti-Abl p6D monoclonal antibody² but had no effect on levels of the Bcr–Abl protein.