Biochemical and biological functions of the N-terminal, noncatalytic domain of extracellular signal-regulated kinase 2

Abstract

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are important components in signal transduction pathways involved in many cellular processes, including cell differentiation and proliferation. These proteins consist of a central kinase domain flanked by short N- and C-terminal noncatalytic domains. While the regulation of ERK2 by sequences within the kinase domain has been extensively studied, little is known about the small regions outside of the kinase domain. We performed mutational analysis on the N-terminal, noncatalytic domain of ERK2 in an attempt to determine its role in ERK2 function and regulation. Deleting or mutating amino acids 19 to 25 (ERK2-Delta19-25) created an ERK2 molecule that could be phosphorylated in response to growth factor and serum stimulation in a MEK (mitogen-activated protein kinase kinase or ERK kinase)-dependent manner but had little kinase activity and was unable to bind to MEK in vivo. Since MEK acts as a cytoplasmic anchor for the ERKs, the lack of a MEK interaction resulted in the aberrant nuclear localization of ERK2-Delta19-25 mutants in serum-starved cells. Assaying these mutants for their ability to affect ERK signaling revealed that ERK2-Delta19-25 mutants acted in a dominant-negative manner to inhibit transcriptional signaling through endogenous ERKs to an Elk1-responsive promoter in transfected COS-1 cells. However, ERK2-Delta19-25 had no effect on the phosphorylation of RSK2, an ERK2 cytoplasmic substrate, whereas a nonactivatable ERK (T183A) that retained these sequences could inhibit RSK2 phosphorylation. These results suggest that the N-terminal domain of ERK2 profoundly affects ERK2 localization, MEK binding, kinase activity, and signaling and identify a novel dominant-negative mutant of ERK2 that can dissociate at least some transcriptional responses from cytoplasmic responses.

FIG. 1

Diagram of ERK2 mutants and crystal structure of ERK2, with mutated regions highlighted. (A) The amino acid sequence of the N terminus of murine ERK2 up to the beginning of the kinase domain is shown, with the open boxes indicating sequences that were deleted from the various mutants and the underlining indicating sequences that were mutated to Ala. The shaded box indicates the kinase domain, and the open box indicates the C terminus. All constructs encode an N-terminal FLAG epitope tag, while mutants ERK2-Δ3-7 and ERK2-Δ10-19 contain an additional nonfunctional KT3 tag at the C terminus, which accounts for their decreased mobility in SDS-PAGE compared to wild-type ERK2. (B) The crystal structure of unphosphorylated ERK2, solved by Zhang and colleagues (47), is shown using the RasMol program. Mutated sequences are colored as follows: ERK2-Δ3-7 is yellow, ERK2-Δ10-19 is green, ERK2-Δ10-25 is green and red, and ERK2-Δ19-25 is red. Lys 52 is shown in blue; Thr 183 and Tyr 185 are shown in purple.

FIG. 2

ERK2-Δ19-25 mutants are phosphorylated in response to mitogens but have little kinase activity. COS-1 cells were transfected with either empty vector, WT-ERK2, or an ERK2 mutant. The following day the cells were serum starved for 4 h before stimulation for 5 min with either 10 ng of EGF per ml or 10% serum. FLAG-ERK immunoprecipitates were used for an in vitro kinase assay using MBP as the substrate. The reaction mixtures were immunoblotted with antibodies for FLAG and phospho-ERK to determine protein expression and phosphorylation. SF, serum-free; P-ERK, phospho-ERK.

FIG. 3

Phosphorylation of ERK2 mutants in response to EGF is MEK dependent. COS-1 cells were transfected and serum starved as described for Fig. 2. For the last hour of serum starvation the cells were treated with either DMSO or 50 μM PD098059 to inhibit MEK activation. The cells were then stimulated for 10 min with either EGF or serum in the presence of either DMSO or PD098059. The cells were harvested, and FLAG-ERK2 immunoprecipitates were immunoblotted with anti-FLAG and anti-phospho-ERK antibodies. SF, serum-free. P-ERK, phospho-ERK.

FIG. 4

Mutation of residues 19 to 25 of ERK2 inhibits binding to MEK. COS-1 cells were transfected with plasmids for HA-MEK2 and FLAG-ERKs as indicated. (A and B) The following day the cells were serum starved for 4 h before harvest at 24 h posttransfection. The cells were lysed in hypotonic buffer, and the proteins were immunoprecipitated with antibodies to the HA epitope. The immunoprecipitations were immunoblotted with antibodies to FLAG and HA. For the lysate blots, an equal amount of soluble cell lysate was run on SDS-PAGE gels and immunoblotted for FLAG-ERK. IP, immunoprecipitate.

FIG. 5

ERK2-Δ19-25 mutants localize to nucleus and cytoplasm of serum starved cells cotransfected with MEK2. COS-1 cells were cotransfected with plasmids for FLAG-ERKs and empty vector (A to D) or FLAG-ERKs (E to H) and HA-MEK2 (I to L). The cells were serum starved before fixing in 4% paraformaldehyde at 48 h posttransfection. Fixed cells were probed with monoclonal M2 anti-FLAG and polyclonal anti-HA antibodies. Empty vector was cotransfected with plasmids for WT-ERK2 (A), ERK2-Δ10-19 (B), ERK2-Δ19-25 (C), or ERK2-Δ19-25-GP (D). Parallel cultures were cotransfected with WT-ERK2 (E) and MEK2 (I), ERK2-Δ10-19 (F) and MEK2 (J), ERK2-Δ19-25 (G) and MEK2 (K), or ERK2-Δ19-25-GP (H), and MEK2 (L).

FIG. 6

Cellular distribution of FLAG-ERK proteins by fractionation. COS-1 cells were cotransfected with plasmids for HA-MEK2 and either a FLAG-ERK2 or empty vector. The cells were washed and serum starved for 4 h before harvest at 48 h posttransfection. Nuclear and cytoplasmic fractions were prepared, and equal cell equivalents of each lysate were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and blotted with antibodies to FLAG and HA. N, nucleus; C, cytoplasm.

FIG. 7

ERK2-Δ19-25-GP has reduced association with ERK2 substrates. (A) COS-1 cells were cotransfected with plasmids for GAL4-Elk1 and either WT-ERK2 or ERK2-Δ19-25-GP. The cells were serum starved for 5 h and stimulated with EGF for 15 min as indicated. FLAG-ERK immunoprecipitates were run on a gel and immunoblotted with antibodies for Elk1 and FLAG. (B) Cells were cotransfected with plasmids for HA-RSK2 and either WT-ERK2 or ERK2-Δ19-25-GP. The cells were starved and stimulated with EGF as described for panel A. Anti-HA immunoprecipitates were run on a gel and immunoblotted with antibodies for FLAG and HA. IP, immunoprecipitate; exp., exposure.

FIG. 8

ERK2-Δ19-25 mutants act as dominant negatives in signaling to an Elk1 responsive reporter. (A) COS-1 cells were cotransfected in triplicate with plasmids for 5X GAL4-luciferase, GAL4-ELK1, HA-RasV12, and increasing amounts of the indicated FLAG-ERK2 construct. The cells were incubated in serum-free DMEM overnight before harvesting at 24 h and determination of luciferase activity. An equal amount of protein from one lysate of each triplicate closest to the mean was immunoblotted with anti-FLAG and anti-HA antibodies to determine the expression of HA-RasV12 and FLAG-ERK2 in each sample. (B) The same experiment as described for panel A was performed, except MEK1 S218/222D was transfected in place of HA-RasV12. (C) 5X GAL4-luciferase, GAL4-ELK1, ERK2 plasmids, and either mutationally activated MEK1 or MEK2 were transfected into COS-1 cells in triplicate and luciferase assays were performed on cell lysates as described for panel B. (D) COS-1 cells were cotransfected in triplicate with 5X GAL4-luciferase, GAL4-ELK1, HA-MEK1 S218/222D, ERK2-Δ19-25 and increasing amounts of WT-ERK2, as indicated. Luciferase activity was determined 24 h after transfection. S/D, S218/222D.

FIG. 9

ERK2-Δ19-25-7A does not inhibit phosphorylation of cytoplasmic ERK substrate RSK2. (A) COS-1 cells were transfected with plasmids for HA-RSK2 and HA-RasV12 and serum starved for 5 h before harvest at 24 h. The MEK inhibitor U0126 was added for the last 2 h before harvest. HA immunoprecipitates were immunoblotted for phospho-RSK and HA-RSK2. Lysates were immunoblotted for HA-RasV12. (B) COS-1 cells were transfected as described for panel A with the addition of cotransfection with the indicated ERK plasmid. HA-RSK2 was immunoprecipitated and immunoblotted with antibodies for HA-phosphorylated RSK. Lysates were immunoblotted for FLAG-ERK and HA-RasV12. P-RSK, phospho-RSK.