IL-3 activation of mcl-1
gene transcription is mediated through two promoter elements designated the CRE-2 and SIE motifs. In our previous reports, we stated that the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through a PI3-K/Akt-dependent pathway (49
). In the present study, we demonstrated that the Ets family of transcription factor PU.1 is one component of the SIE-binding complex. Unlike the case with the CREB protein, which binds to the CRE-2 motif in an IL-3-inducible manner, PU.1 binds to the SIE element constitutively but its transactivation activity is increased upon IL-3 stimulation of cells. Furthermore, we showed that IL-3 stimulation of mcl-1
gene transcription through the SIE motif involves phosphorylation of PU.1 at serine 142 by a p38MAPK
-dependent pathway. These results, together with those in our previous report (49
), indicate that the PI3-K/Akt/CREB and p38MAPK
/PU.1 pathways cross talk at the mcl-1
gene locus (Fig. ). Considering the fact that the core sequences of the CRE-2 and SIE motifs are only separated by 9 bp, there may be some protein-protein interactions between CREB- and PU.1-containing complexes that, via an unknown mechanism, stimulate transcription of the mcl-1
gene. More experiments are required to investigate this possibility.
Schematic representation of molecules involved in the IL-3 signaling pathways that lead to mcl-1 gene transcription through the SIE and CRE-2 elements (see text for details).
PU.1 is a hematopoietic, lineage-specific transcription factor with some characterized functional domains. These include multiple N-terminal acidic and glutamine-rich transactivation domains (11
), a central proline-, glutamic-acid-, serine-, and threonine-rich domain that is important for protein-protein interactions (34
), and a C-terminal Ets DNA-binding domain (19
). The activity of PU.1 is primarily regulated posttranscriptionally by phosphorylation (reviewed in reference 26
). While phosphorylation of two serine residues (positions 41 and 45) located at one of the acidic transactivation domains is necessary for macrophage colony-stimulating factor-dependent proliferation of bone marrow macrophages (4
), a serine phosphorylation at position 148 in the proline-, glutamic-acid-, serine-, and threonine-rich domain mediates the interaction between PU.1 and the B-cell enhancer factor (NF-EM5). The latter interaction is required for optimal transactivation of the 3′ enhancer elements located in the immunoglobulin κ and λ light-chain genes (35
). The serine 148 phosphorylation was also found to be required for the lipopolysaccharide-induced transactivation function of PU.1 (27
). In contrast, in this study, we demonstrated that serine phosphorylation of PU.1 at position 142 instead of position 148 mediates, at least partially, the SIE-dependent IL-3 stimulation of mcl-1
reporter activity. While casein kinase II has been suggested to be responsible for phosphorylation of PU.1 at serine 148 (27
), our data indicate that the serine 142 phosphorylation is induced via activation of a p38MAPK
-dependent pathway. The IL-3-induced in vivo phosphorylation of PU.1 is inhibited by SB203580, suggesting that p38α, p38β, or both mediate this phosphorylation event. However, the fact that IL-3-stimulated transactivation activity of PU.1 is inhibited by the dominant negative mutant form of p38α but not that of p38β (Fig. ) suggests that serine 142 phosphorylation is induced via a p38α-dependent pathway. On the other hand, while our results presented in Fig. cannot distinguish whether PU.1 is directly phosphorylated by p38MAPK
or by another associated kinase in the p38MAPK
immunocomplex, the lack of phosphorylation of PU.1 at Ser-142 in an in vitro kinase assay using bacterium-produced p38α (data not shown) suggests that PU.1 is an indirect substrate of p38α. More experiments are required to reveal the identity of the putative p38α-associated kinase that catalyzes the phosphorylation of PU.1 at Ser-142 in response to IL-3 stimulation.
PU.1 binds to a cis
element with a 5′-GGAA/T-3′ core sequence. Its binding to some gene promoters is constitutive (44
), whereas in some other cases, its binding activity is stimulated by extracellular stimuli or cytokines (18
). PU.1 binds to the SIE motif of the mcl-1
promoter irrespective of IL-3 stimulation, and IL-3 stimulates its phosphorylation-and-transactivation function. These results suggest that IL-3 stimulates the phosphorylation of PU.1 at serine 142, which either directly increases the transactivation activity of PU.1 or increases PU.1's ability to recruit another transcriptional activator and activates mcl-1
gene transcription in an indirect manner. PU.1 was shown to interact with many transcription factors or coactivators, including NF-EM5, Ets-1, NF-IL-6, HMG-I(Y), c-Jun, IRF-1, ICSBP, AML1, and CBP (2
). It remains to be determined what other factors are associated with PU.1 and mediate the SIE-dependent IL-3 stimulation of mcl-1
gene transcription. In the gel shift assay with the SIE probe, in addition to the B2 complex that contains PU.1, another specific B1 complex was consistently detected in the Ba/F3 cell extracts (Fig. ). The formation of this B1 complex, like that of B2, is not influenced by the presence or absence of IL-3. It is not clear whether the B1 complex plays any role in the SIE-mediated IL-3 induction of mcl-1
gene transcription. If it does play a role in this process, the inability of the PU.1 antibody to supershift this complex suggests either that B1 does not contain PU.1 or that the PU.1 protein present in the B1 complex exists in a conformation that cannot be recognized by the antibody used in the assay. More experiments are required to address this issue.
An SIE-like element (termed SRE) is present in the human mcl-1
gene promoter (between nucleotides −105 and −92) (47
). This DNA element was shown to be recognized by a protein complex containing serum response factor and Elk-1. Both serum response factor and Elk-1 act coordinately to affect both the basal activity and tetradecanoyl phorbol acetate inducibility of the human mcl-1
gene in human K-562 cells. Unlike the SIE motif reported in this study, the tetradecanoyl phorbol acetate inducibility of SRE in the human mcl-1
promoter was shown to be mediated through the extracellular signal-regulated kinase pathway (47
). On the other hand, the STAT3 protein in the peripheral blood mononuclear cell extracts isolated from patients with large granular lymphocyte leukemia has been reported to bind to the murine SIE motif (9
). In contrast, although STAT3 is activated by IL-3 in Ba/F3 cells (30
; our unpublished data), no evidence suggests that it binds to SIE or plays a role in the IL-3 regulation of murine mcl-1
gene transcription (our unpublished results). Furthermore, Mcl-1 has been found to be expressed in many other cell types of nonhematopoietic origin (23
). Taken together, these results suggest that SIE-dependent mcl-1
gene transcription is likely to be regulated by different transcriptional factors in a cell type- and stimulation signal-dependent manner.
The function of PU.1 is pivotal to myeloid and lymphoid development, as many genes essential for these two processes are regulated by this transcription factor. Our identification of PU.1 as being involved in IL-3 regulation of mcl-1
gene expression suggests that Mcl-1 may also contribute to the development of myeloid and/or lymphoid cells. Mcl-1-deficient embryos did not survive beyond the peri-implantation stage of mouse development (39
). This early embryo lethality precludes a direct assessment of Mcl-1 function during myeloid and lymphoid development. It would be interesting to investigate how severe mcl-1
gene expression is affected in PU.1-null embryos and whether overexpression of Mcl-1 would rescue any defects of these mutant phenotypes.