A GTPase-activating protein binds STAT3 and is required for IL-6-induced STAT3 activation and for differentiation of a leukemic cell line.
We previously identified a guanosine triphosphatase (GTPase)-activating protein (GAP) male germ cell Rac GAP (MgcRacGAP) that enhanced interleukin-6 (IL-6)-induced macrophage differentiation of murine M1 leukemia cells. Later, MgcRacGAP was found to play crucial roles in cell division. However, how MgcRacGAP enhanced IL-6-induced differentiation remained elusive. Here we show that MgcRacGAP enhances IL-6-induced differentiation through enhancement of signal transducer and activator of transcription-3 (STAT3) activation. MgcRacGAP, Rac, and STAT3 formed a complex in IL-6-stimulated M1 cells, where MgcRacGAP interacted with Rac1 and STAT3 through its cysteine-rich domain and GAP domain. In reporter assays, the wild-type MgcRacGAP enhanced transcriptional activation of STAT3 while a GAP-domain deletion mutant (DeltaGAP) did not significantly enhance it, suggesting that the GAP domain was required for enhancement of STAT3-dependent transcription. Intriguingly, M1 cells expressing DeltaGAP had no effect on the differentiation signal of IL-6, while forced expression of MgcRacGAP rendered M1 cells hyperresponsive to the IL-6-induced differentiation. Moreover, knockdown of MgcRacGAP by RNA interference profoundly suppressed STAT3 activation, implicating MgcRacGAP in the STAT3-dependent transcription. All together, our data not only reveal an important role for MgcRacGAP in STAT3 activation, but also demonstrate that MgcRacGAP regulates IL-6-induced cellular differentiation in which STAT3 plays a pivotal role.
Tonozuka
Yukio
Y
Division of Cellular Therapy, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Minoshima
Yukinori
Y
Bao
Ying Chun
YC
Moon
Yuseok
Y
Tsubono
Yohei
Y
Hatori
Tomonori
T
Nakajima
Hideaki
H
Nosaka
Tetsuya
T
Kawashima
Toshiyuki
T
Kitamura
Toshio
T
D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D007938:Leukemia / Q000473:pathology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D015850:Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C115995:mgcRacGAP; D020830:rac1 GTP-Binding Protein