RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition.
The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1 which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK; and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear relocalization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and relocalization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.
Reiterer
Gudrun
G
Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA.
Chen
Lihua
L
Tassef
Ryan
R
Varner
Jeffery D
JD
Chen
Chang-Yan
CY
Yen
Andrew
A
D002467:Cell Nucleus / Q000378:metabolism; D042822:Genomic Instability; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D053612:Janus Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D008938:Mitosis; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D007211:Indoles; D010636:Phenols; D047428:Protein Kinase Inhibitors; D053612:Janus Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone