Interleukin-3 Stimulation of mcl-1 Gene Transcription Involves Activation of the PU.1 Transcription Factor through a p38 Mitogen-Activated Protein Kinase-Dependent Pathway

Abstract

We have previously demonstrated that the antiapoptotic gene mcl-1 is activated by interleukin-3 (IL-3) in Ba/F3 pro-B cells through two promoter elements designated the CRE-2 and SIE motifs. While the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through the phosphatidylinositol 3-kinase/Akt-dependent pathway, the identity and cytokine activation pathway of the SIE-binding complex remains unclear. In this report, we demonstrated that PU.1 is one component of the SIE-binding complex. A chromatin immunoprecipitation assay further confirmed that PU.1 binds to the mcl-1 promoter region containing the SIE motif in vivo. While IL-3 stimulation does not significantly alter the SIE-binding activity of PU.1, it markedly increases PU.1's transactivation activity. The latter effect coincides with the increased phosphorylation of PU.1 following IL-3 activation of a p38 mitogen-activated protein kinase (p38^MAPK)-dependent pathway. A serine-to-alanine substitution at position 142 significantly weakens PU.1's ability to be phosphorylated by the p38^MAPK immunocomplex. Furthermore, this S142A mutant is impaired in the ability to be further stimulated by IL-3 to transactivate the mcl-1 reporter through the SIE motif. Taken together, our results demonstrate that IL-3 stimulation of mcl-1 gene transcription through the SIE motif involves phosphorylation of PU.1 at serine 142 by a p38^MAPK-dependent pathway.

ACKNOWLEDGMENTS

We thank Jiahuai Han for the p38 antibody used in the in vitro kinase assay and the plasmids expressing DN-p38α and DN-p38β; Koichi Nakajima and Toshio Hirano for the plasmid expressing DN-STAT3; Masato Kasuga for the Δp85 (DN-p85) expression vector; and Tzu-Hao Wang for the DN-JNK, DN-ERK1, and DN-ERK2 expression vectors.

This work was supported by intramural funds from Academia Sinica. J.-M. Wang was supported by a postdoctoral fellowship awarded by Academia Sinica.