ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain

Abstract

Previous studies have shown that upregulation of the orphan steroid receptor Nur77 is required for the apoptosis of immature T cells in response to antigen receptor signals. Transcriptional upregulation of Nur77 in response to antigen receptor signaling involves two binding sites for the MEF2 family of transcription factors located in the Nur77 promoter. Calcium signals greatly increase the activity of MEF2D in T cells via a posttranslational mechanism. The mitogen-activated protein (MAP) kinase ERK5 was isolated in a yeast two-hybrid screen using the MADS-MEF2 domain of MEF2D as bait. ERK5 resembles the other MAP kinase family members in its N-terminal half, but it also contains a 400-amino-acid C-terminal domain of previously uncharacterized function. We report here that the C-terminal region of ERK5 contains a MEF2-interacting domain and, surprisingly, also a potent transcriptional activation domain. These domains are both required for coactivation of MEF2D by ERK5. The MEF2-ERK5 interaction was found to be activation dependent in vivo and inhibitable in vitro by the calcium-sensitive MEF2 repressor Cabin 1. The transcriptional activation domain of ERK5 is required for maximal MEF2 activity in response to calcium flux in T cells, and it can activate the endogenous Nur77 gene when constitutively recruited to the Nur77 promoter via MEF2 sites. These studies provide insights into a mechanism whereby MEF2 activity can respond to calcium signaling and suggest a novel, unexpected mechanism of MAP kinase function.

FIG. 1

ERK5 stimulation of the MEF2-dependent reporter construct in three different cells lines. DO11.10 (A), NIH 3T3 (C), and S194 (D) cells were transfected with a MEF2-luc (35) and full-length wild-type, full-length kinase-deficient [ERK5(AEF), ERK5 D183A, and ERK5 D203A], or truncated ERK5. Activated MEK5 [MEK5(D)] was added where indicated. (D) Mutant MEF2-luc (35) was used to confirm MEF2 specificity. Open bars indicate activity in unstimulated cells. Hatched or filled bars indicate activity in cells stimulated with PMA and ionomycin. Expression of transfected ERK5 constructs in DO11.10 and NIH 3T3 cells was determined by Western blot analysis using anti-ERK5 (B) or anti-HA (C) antibodies.

FIG. 2

Mapping of the ERK5 transcriptional activation domain. DO11.10 cells were transfected with either GAL4-dependent (GAL4-luc) or MEF2-dependent (MEF2-luc) reporter constructs. Luciferase activity was measured in unstimulated cells (shaded bars in panel A and open bars in panels C and D) or cells treated with PMA and ionomycin (open bars in panel A, filled bars in panel C, and hatched bars in panel D). (A) Activation of Gal4-luc by GAL4-ERK5 fusions. (B) Anti-GAL4 Western blot analysis on nuclear extracts from transfected DO11.10 cells. (C) Activation of MEF2-luc by MEK5(D) plus either full-length ERK5 or ERK5 lacking the C-terminal transactivation domain [ERK5(1-740)]. Expression of full-length and truncated ERK5 were demonstrated by Western blotting using anti-ERK5 antibody (inset). (D) Suppression of activation-dependent MEF2-luc activity by ERK5(1-740) compared to empty vector (pCI) or full-length ERK5.

FIG. 3

Interaction of ERK5 with MEF2D. (A) HA epitope-tagged deletions of ERK5 were transfected into DO11.10 hybridomas. Cell lysates were incubated with either plain nickel-agarose beads (beads) or beads bearing bacterially expressed MEF2D(1-92) (His-MEF2D). Bead-bound proteins and whole-cell lysates were resolved by SDS-PAGE, blotted, and probed with anti-HA antibody. GST-Cabin 1 (2037-2220) or GST-Cabin 1 (2037-2179) proteins were added to the binding reactions as indicated. (B) Mammalian two-hybrid assay for activation-dependent MEF2-ERK5 interaction. Gal4-luc and either Gal4 DNA binding domain (positions 1 to 142) or Gal4(1-142) fused to the ERK5(400-739) [Gal4-ERK5ΔTAD] were transfected into DO11.10 cells with or without MEF2(1-92)–VP16. Ionomycin (iono) or ionomycin plus cyclosporine (iono/CsA) were added as indicated. The values shown for Gal4-VP16 are 10% of their actual light units. (C) Overexpression of both MEF2D and ERK5(400-806) in DO11.10. Neither molecule alone can overcome the requirement for PMA and ionomycin stimulation for maximal MEF2-luc activity. (D) DO11.10 cells were transfected with a MEF2-dependent reporter construct together with MEF2D, ERK5, and MEK5 expression constructs as indicated. Luciferase activity was measured in untreated cells and cells treated with PMA plus ionomycin (PMA/ionos).

FIG. 4

Effect of ERK5, calcineurin, or MEF2 on expression of endogenous Nur77 family members in DO11.10 cells. (A) Either the wild-type or mutant NBRE-luc reporter construct (5) was transfected into DO11.10 cells along with MEF2, calcineurin, or ERK5 expression vectors as indicated. PMA and ionomycin were added to only one set of samples (+PMA iono), showing stimulus-dependent upregulation of Nur77 family members by endogenous factors. (B) Cells were transfected as described above, except a CD8 expression plasmid was used instead of an NBRE-luc plasmid to mark transfected cells, which were then magnetically isolated using anti-CD8 antibodies. Whole-cell extracts were made and Western blot analysis with anti-Nur77 monoclonal antibody was performed.

FIG. 5

Dependence of ERK5 coactivation of MEF2D on amino acids 1 to 92 of MEF2D and amino acids 440 to 806 of ERK5. (A) Constructs containing fusions of either full-length MEF2D or amino acids 93 to 514 of MEF2D to the Gal4 DNA binding domain were transfected into DO11.10 cells together with a Gal4-dependent reporter construct and either empty vector, ERK5 (440-806), or a combination of full-length ERK5 and MEK5(D) expression constructs. Activity was measured in untreated cells or cells treated with PMA plus ionomycin (PMA/iono). (B) Transfection and measurement were performed as for panel A, except that a fusion of the HSV VP16 transcriptional activation domain to the Gal4 DNA binding domain was used. (C) DO11.10 cells were transfected with a MEF2-dependent reporter construct together with ERK5 and MEK5 expression constructs as indicated. Luciferase activity was measured in untreated cells and cells treated with PMA plus ionomycin (PMA/ionos).