Fig. 3: JAK2 phosphorylates CHK2 at Y156 to maintain the spindle assembly checkpoint.

A JAK2 activity was increased by nocodazole treatment, as evidenced by enhanced JAK2 Y1007/8 phosphorylation (pY1007/8). CHK2 KO cells stably re-expressing myc-tagged WT CHK2 were treated with nocodazole overnight. Lysates were prepared for western blotting using the indicated antibodies. B Co-expressed full-length JAK2 (JAK2-FL) promoted tyrosine phosphorylation in WT but not Y156F CHK2 in HEK293T cells. C, D In vitro kinase assays showing that immunoprecipitated FLAG-JAK2-C (amino acids 747–1132) (C) or recombinant His-JAK2-C (amino acids 843–1122) phosphorylated recombinant His-CHK2 at Y156. E Interaction of endogenous JAK2 with CHK2 in HEK293T cells as assessed by co-immunoprecipitation. Cells were treated with nocodazole overnight. F, G Co-immunoprecipitation of JAK2 and CHK2 from HeLa CHK2 KO cells stably expressing myc-CHK2 WT with anti-JAK2 (F) or anti-myc (G) antibody. Cells were treated with nocodazole overnight. (H) Nocodazole treatment can moderately increase JAK2-CHK2 interaction. Lysates as described in (A) were used for immunoprecipitation. I–K Inhibition of JAK2 and CHK2 led to abnormal metaphase. Nocodazole-arrested cells were released into the medium containing either CHK2 inhibitor II (5 μM) or JAK2 inhibitor IV (2.5 μM) for 1 h. Representative images (I) were captured after 1 h following release. White arrows indicate misaligned chromosomes. Percent cells in metaphase were determined and shown in (J), of which those with abnormal metaphase were counted and shown in (K). Scale bar, 10 μM. L, M The phospho-mimicking CHK2 Y156E mutant restored normal metaphase even in the presence of JAK2 inhibitor. Wild type or mutant CHK2 was transiently expressed in CHK2 KO cells, followed by overnight Noc treatment. Images were taken 1 h following release in the presence or absence of JAK2 inhibitor IV. mCherry was coexpressed to mark the transfected cells.