Stress-induced inactivation of the c-Myb transcription factor through conjugation of SUMO-2/3 proteins.
Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys(523) and Lys(499). These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress.
Sramko
Marek
M
Center of Molecular Medicine, Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia.
Markus
Ján
J
Kabát
Juraj
J
Wolff
Linda
L
Bies
Juraj
J
D000818:Animals; D019556:COS Cells; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D015536:Down-Regulation / Q000502:physiology*; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009997:Osmotic Pressure; D011499:Protein Processing, Post-Translational / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism* / Q000502:physiology; D013312:Stress, Physiological / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism* / Q000502:physiology
D020598:Proto-Oncogene Proteins c-myb; C515972:SUMO2 protein, mouse; D025841:Small Ubiquitin-Related Modifier Proteins; C117908:Sumo3 protein, mouse; D015534:Trans-Activators; D014452:Ubiquitins; D008239:Lysine