Mitogen-activated protein kinase (MAPK)-docking sites in MAPK kinases function as tethers that are crucial for MAPK regulation in vivo

Abstract

Docking sites on targets of mitogen-activated protein kinases (MAPKs) facilitate accurate and efficient substrate phosphorylation. MAPK/ERK kinases (MEKs, or MKKs), the upstream regulators of MAPKs, also contain N-terminal MAPK-docking sites, or 'D-sites'; however, the in vivo functions of MEK D-sites are incompletely understood. Here we found that the ability of constitutively-active human MEK1 and MEK2 to stimulate ERK phosphorylation and to induce the neoplastic transformation of NIH 3T3 cells required the integrity of the D-site. In addition, D-site mutants of otherwise wild-type MEK1/2 were unable to anchor unphosphorylated ERK2 in the cytoplasm. ERK activation, cytoplasmic anchoring and release were completely retained in 'swap' mutants in which MEK2's D-site was replaced with the D-site of MEK1 or yeast Ste7. Furthermore, these abilities were significantly retained when MEK2's D-site was moved to its C-terminus, or replaced by an unrelated MAPK-binding domain taken from the Ets-1 transcription factor. We conclude that the D-sites in MEKs are crucial for the activation of their cognate MAPKs in vivo, and that their primary function is to tether their cognate MAPKs near the MEK's kinase domain. This proximity effect is sufficient to explain the contribution that the D-site interaction makes to several biologically important signaling events.

Fig. 1

Deletion of the docking site from active MEK proteins inhibits their ability to phosphorylate their target MAPKs. (A) Sequence of MEK1, MEK2, MKK3 and MKK6 N-termini, with the core of their MAPK-docking sites highlighted in grey. The most N-terminal residue shown is indicated on the left. (B) HEK293 cells were transfected with plasmids encoding full-length constitutively active MEK1 or MEK2 (MEK1-ED and MEK2-DD, respectively), docking site deficient constitutively active MEK1 or MEK2 (MEK1-EDΔ and MEK2-DDΔ, respectively) or control empty vector (vector control). Twenty-four hours post-transfection cells were transferred to low-serum (0.5%) medium and grown for a further 24 h. Cells were then harvested and lysed and ERK1/2 phosphorylation was analyzed by Western blotting. For serum activation, cells were incubated with complete medium (10% serum) for 15 min prior to harvesting. (C) Cells were transfected with plasmids encoding full-length constitutively active MKK3 or MKK6 (MKK3-EE and MKK6-EE), docking site deficient active MKK3 or MKK6 (MKK3-EEΔ and MKK6-EEΔ) or control empty vector (vector control). Cells were lysed 48 h post-transfection, and p38 phosphorylation was assessed by Western blotting. For stimulation, cells were incubated for 30 min with 100 ng/ml anisomycin.

Fig. 2

Docking-site mutants of MEK1 and MEK2 are unable to induce the morphological transformation of NIH3T3 cells. NIH3T3 cells were transfected with plasmids encoding full-length constitutively active MEK1 or MEK2 (MEK1-ED and MEK2-DD, respectively), docking site deficient constitutively active MEK1 or MEK2 (MEK1-EDΔ and MEK2-DDΔ, respectively), activated V12-Ras (H-Ras V12) or empty vector (control). (A) Twenty-one days post-transfection cells were viewed using an IX70 Olympus light microscope and morphological transformation was assessed. (B) Cells were fixed and permeabilized and then stained with 0.1% methylene blue to visualize foci. (C) Quantification of the number of foci formed per 10 cm dish (±standard deviation, n =3).

Fig. 3

The MEK-mediated cytosolic retention of ERK2 is dependent on docking sites. HeLa cells were grown on coverslips and were co-transfected with plasmids encoding ERK2 and either full-length or docking-site deficient MEK1 or MEK2 (V5-tagged). Forty-eight hours post-transfection cells were fixed and permeabilised and incubated with monoclonal anti-V5 and rabbit polyclonal anti-ERK1/2 antibodies followed by anti-mouse AlexeFluor 488 and anti-rabbit AlexaFluor 564 secondary antibodies. Scale bar=10 μm.

Fig. 4

Replacement of MEK1/2 docking sites with other MKK docking sites can restore ERK activation. (A) Alignment of N-terminal sequences of MEK2, MEK1 and the yeast MEK protein, Ste7, with conserved docking site regions highlighted in grey. The most N-terminal residue shown is indicated on the left. (B) HEK293 cells were transfected with full-length constitutively active (V5-tagged) MEK1 or MEK2, MEK1/MEK2 docking sites swaps, or with empty vector (control). Twenty-four hours post-transfection, cells were transferred to low-serum (0.5%) medium and grown for a further 24 h. Cells were then harvested and ERK1/2 phosphorylation was analyzed by Western blotting. (C) Schematic representation of a MEK2-Ste7 docking fusion protein. Residues 3–14 of MEK2 were replaced with residues 9–17 of Ste7. (D) HEK293 cells were transfected with plasmid constructs encoding constitutively active full-length MEK2-DD, MEK2-DDΔ, MEK2-DDΔ-Ste7, MEK2_EEAA or MEK2-Ala. Cells were processed as above and lysates were analyzed by Western blotting.

Fig. 5

N-terminal location of the MEK2 docking site is not critical for its function. (A) Schematic of the MEK2-DDΔ-Cdock fusion protein. Residues 1–14 of MEK2 were moved to its C-terminus. (B) HEK293 cells were transfected with vectors encoding MEK2-DD, MEK2-DDΔ, MEK2-DDΔ-Cdock or empty vector. Lysates were prepared as described above and were analyzed by Western blotting. (C) Quantification of the levels of phosphorylated ERK1/2. Data represent mean values±standard deviation (n =5).

Fig. 6

Replacement of the MEK2 docking site with the ERK-binding domain of the Ets-1 transcription factor is able to restore MEK-mediated ERK activation. (A) Schematic diagram of the Ets-1-MEK2-DDΔ fusion protein. Residues 1–14 of MEK2 were replaced with residues 54–138 of Ets-1. (B) HEK293 cells were transfected with MEK2-DD, MEK2-DDΔ, Ets-1-MEK2-DDΔ or empty vector. Lysates were prepared and analyzed by Western blotting. (C) Quantification of the levels of phosphorylated ERK1/2. Data represent mean values±standard deviation (n =5). (D) HeLa cells were transfected with Ets-1-MEK2-DDΔ, MEK2-DD, MEK2-DDΔ or empty vector (control) and their effects on the distribution of endogenous ERK1/2 was examined. Twenty-four hours post-transfection, cells were transferred to low-serum (0.5%) medium and grown for a further 24 h. Cells were then processed as described in Fig. 3. For serum activation (control+), cells were incubated with complete medium (10% serum) for 15 min prior to fixation. (E) HeLa cells were co-transfected with vector encoding ERK2 and either Ets-1-MEK2 or MEK2Δ-encoding vectors. Cells were processed as described above. Scale bar=10 μm

Fig. 7

Replacement of the MEK2 docking site with other docking sites restores MEK –MAPK complex formation in vitro. (A) ³⁵S-labelled MEK2-DD, MEK2-DDΔ, Ets-1-MEK2-DDΔ, MEK2-DDΔ-cdock or MEK2-EEAA proteins were tested for their ability to bind GST-ERK2 (10 μg). Co-sedimented proteins, along with 10% of the original input, were separated by SDS-PAGE and visualized by autoradiography. (B) Relative levels of binding exhibited for each of the MEK-fusion proteins. Results were normalized by setting the amount of full-length MEK2-DD sedimented to 100%. Data shown is the average of three independent experiments with error bars indicating the standard error.