c-Myb transcription is activated by protein kinase B (PKB) following interleukin 2 stimulation of Tcells and is required for PKB-mediated protection from apoptosis

Abstract

During T-cell activation, c-Myb is induced upon interleukin 2 (IL-2) stimulation and is required for correct proliferation of cells. In this paper, we provide evidence that IL-2-mediated induction of the c-myb gene occurs via the phosphoinositide 3-kinase (PI3K) signaling pathway, that protein kinase B (PKB) is the principal transducer of this signal, and that activation of the c-myb promoter can be abolished by deletion of conserved E2F and NF-kappaB binding sites. We show that Myb is required to protect activated peripheral T cells from bcl-2-independent apoptosis and that overexpression of oncogenic v-Myb is antiapoptotic. Overexpression of a Myb dominant-negative transgene abrogates PKB-mediated protection from apoptosis. Taken together, these results suggest that induction of c-myb transcription is an important downstream event for PKB-mediated protection of T cells from programmed cell death.

FIG. 1

c-myb is activated by PI3K following IL-2 stimulation. T cells were starved for 16 h and stimulated with 10 ng of IL-2/ml in the presence or absence of various inhibitors. After the times indicated (in hours), total RNA was obtained from cells and c-myb, c-jun, and GAPDH expression was analyzed in RNase protection assays. (A) ConA-activated splenocytes were starved and stimulated with IL-2 ± 50 μM LY294002 or 50 μM PD98059. (B) ConA-activated splenocytes were starved and then stimulated with IL-2 for 5 h. Extracts were blotted and probed with anti-c-Myb antibody (upper lanes) or anti-PKB antibody as a loading control (lower lanes). (C) B6.1 cells were starved and stimulated with IL-2 ± 50 μM LY294002 or 50 μM PD98059. (D) B6.1 cells were starved and stimulated with IL-2 ± 25 μM PDTC.

FIG. 2

The c-myb promoter is activated by PI3K and PKB. (A) Schematic of the murine c-myb promoter. E2F and NF-κB sites are underlined and shown in bold, and the putative SP1 site is overlined. (B) NIH 3T3 cells were transfected with the c-myb-βG reporter either alone or in concert with effector plasmids encoding activated PI3K, activated PKB, or dominant-negative PKB. Transcription from c-myb-βG and endogenous GAPDH was analyzed in RNase protection assays. (C) NIH 3T3 cells were transfected with c-myb-βG either alone or with the Ras effector mutants shown (see text). RNA analysis was as described above. (D) NIH 3T3 cells were transfected with c-myb-αG either alone or together with effector plasmids encoding activated PI3K or activated PKB.

FIG. 3

E2F and NF-κB proteins bind to the c-myb promoter. (A) Bandshifts with an E2F probe from the c-myb promoter or a mutant thereof. Lanes 1 and 2, in vitro-translated E2F1 and DP1; lanes 3 and 4, activated T-cell extracts. (B) Bandshifts with an NF-κB probe from the c-myb promoter. Purified NF-κB proteins and IκBα were added as detailed in Materials and Methods. (C) Bandshifts with the c-myb NF-κB probe and activated T-cell extracts. Unlabeled competitor oligonucleotides and antibodies were added as shown.

FIG. 4

The E2F and NF-κB sites are required for basal and PI3K-activated transcription from the c-myb promoter. Transfections into NIH 3T3 cells and RNase protection analyses were performed as described above. (A) Cotransfection of c-myb-βG and E1A or E2F expression vectors results in up-regulation of c-myb-βG. (B) Individual or double mutation of the E2F and NF-κB sites results in loss of activity of the c-myb-βG reporter. (C) Lanes 1 to 3, mutation of the E2F site to a GAL4 site causes loss of PI3K responsiveness. Lanes 4 to 6, mutation of the E2F site to a GAL4 site and inhibition of NF-κB activity causes complete loss of PI3K inducibility.

FIG. 5

Myb is a survival factor during T-cell activation. Splenocytes were activated with ConA and assessed for bcl-2 levels and apoptosis as described in the text. Activated T cells were gated for expression of αβTCR and CD25. (A) Activated MEnT splenocytes have reduced bcl-2 expression. Peak 1, ConA-stimulated MEnT splenocytes; peak 2, ConA-stimulated nontransgenic splenocytes. (B) Left panel: MEnT causes enhanced apoptosis during T-cell activation. Right panel: v-Myb protects activated cells from apoptosis. The percentages of dead and dying cells in the cultures were quantitated by annexin V and 7-AAD staining. Error bars indicate the standard deviation from the mean of at least three separate experiments.

FIG. 6

Myb lies downstream of PKB. Splenocytes from 5-week-old littermates derived from a MEnT × B6/PKB cross were activated with ConA for 2 days. Activated T cells were gated for expression of αβTCR and CD25 and assessed for apoptosis by staining with annexin V-FITC and 7-AAD.