ERK2-Δ19-25 mutants are not phosphorylated by active MEK1 in vitro. (A) COS-1 cells were transfected with FLAG-ERK constructs, and FLAG immunoprecipitates from serum-starved cells were each mixed with 5 μg of recombinant MEK1 S218/222D and ATP for 30 min. Purified, recombinant ERK2 protein was included as a positive control for MEK phosphorylation of ERK. Reactions were immunoblotted as indicated. (B) The reactions were identical to those for panel A, except that recombinant WT-MEK1 activated by recombinant B-Raf in vitro was used to phosphorylate the immunoprecipitated ERKs.

Phosphorylation of ERK2-Δ19-25 mutants is induced by EGF but not by mutationally activated MEKs in vivo. (A) COS-1 cells were transfected with FLAG-ERK constructs and incubated in serum-free media overnight. Cells were stimulated with 10 ng of EGF/ml for the indicated times (min). FLAG-ERKs were immunoprecipitated and immunoblotted with antibodies to FLAG and phospho-ERK. Vec, vector. (B) COS-1 cells were cotransfected with FLAG-ERKs and mutationally active HA-MEK1 S218/222D. The cells were serum starved after the transfection, and FLAG-ERKs were immunoprecipitated 24 h after transfection. Immunoprecipitates were immunoblotted with antibodies to FLAG and phospho-ERK. (C) WT-ERK2 or ERK2-Δ19-25-GP was cotransfected with increasing amounts of MEK1 Δ32-51 S218/222D. Cultures were serum starved after transfection and harvested at 24 h. Stimulated cultures received 10 ng of EGF/ml for 10 min. FLAG-ERKs were immunoprecipitated with anti-FLAG antibodies and then immunoblotted with antibodies to FLAG and phospho-ERK.

Ras activity is required for ERK2-Δ19-25 phosphorylation in response to EGF even in the presence of active MEK. (A) COS-1 cells were transfected with WT-ERK2 or ERK2-Δ19-25 and Ras N17, MEK1 S218/222D, or empty vector (Vec). The following day cells were serum starved for 5 h before stimulation with EGF for 10 min. FLAG-ERK immunoprecipitates were immunoblotted with anti-FLAG and anti-phospho-ERK antibodies. SF, serum free (B) Ras V12 requires Rac activity to stimulate ERK2-Δ19-25 phosphorylation. COS-1 cells were cotransfected with the indicated constructs and serum starved after the transfection. Cells were harvested at 24 h after transfection, and anti-FLAG and anti-phospho-ERK immunoblots were performed on anti-FLAG immunoprecipitates.

ERK2-Δ19-25 mutants do not bind to MEK1. (A) Murine ERK2 was subcloned into the pCDNA3 expression vector in frame with a sequence coding for an N-terminal FLAG tag (16). The sequence of the N terminus from residues 16 to 30 is shown up to the first glycine in the kinase domain using the single-letter code for amino acids. Residues that were deleted are underlined, while substitution mutations to alanine are boxed. (B) COS-1 cells were cotransfected with HA-MEK1 and a FLAG-ERK2 mutant. The following day HA-MEK1 immunoprecipitates (IP) were prepared as described in Materials and Methods and were immunoblotted with anti-HA and anti-FLAG antibodies.

Rac signaling increases the association between ERK2 and MEK1. (A and B) COS-1 cells were transfected with FLAG-ERK2, HA-MEK1, and Rac1 L61 as indicated. The cells were serum starved the following day for 4 h before harvest in hypotonic buffer and lysedby centrifugation at 13,000 × g for 20 min. Anti-FLAG (A) or anti-HA (B) immunoprecipitations (IP) were performed from hypotonic extracts. Immunoprecipitated proteins were eluted with FLAG (A) or HA (B) peptides, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotted with anti-FLAG and anti-HA antibodies. (C) Cells were transfected and processed as for panel A, with the substitution of either Cdc42 L61 or Rho L63 for Rac1 L61 as indicated. (D) COS-1 cells were transfected as shown in panel A with plasmids for FLAG-ERK2 and either HA-MEK1, HA-MEK1 S218A, which cannot be activated by Raf, or the activated mutant MEK1 S218/222D. The cells were serum starved and immunoprecipitations were performed as for panel A.

The increase in the MEK-ERK interaction is MEK1 specific and requires T292 and S298 of MEK1. (A) 10T1/2 cells were transfected with HA-MEK1 and either Rac1 L61 (right panel) or empty vector (left panel). The cells were serum starved for 5 h in phosphate-free DMEM with 0.2% fetal calf serum and were labeled with 2 mCi of [³²P]orthophosphate/ml for the last 4 h. The cells were then lysed and HA-MEK1 immunoprecipitated (IP). Tryptic phosphopeptide maps were then performed on the immunoprecipitate and compared to previous MEK1 maps (9). Peptide 1, phospho-S298 peptide; peptide 2, doubly phosphorylated T292 and S298 peptide. (B) COS-1 cells were transfected with WT-ERK2, Rac1 L61, and either MEK1, MEK1 T292A/S298A, or MEK2 as indicated. The following day the cells were harvested in hypotonic buffer and lysed by centrifugation at 13,000 × g for 20 min. Anti-FLAG immunoprecipitates were prepared and eluted with FLAG peptides. Eluted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-HA and anti-FLAG antibodies. (C) Cells were transfected with plasmids encoding WT-ERK2, MEK1, Rac1 L61, and PAK1 K299R as indicated. The following day the cells were serum starved 4 h before harvest and anti-FLAG immunoprecipitates were immunoblotted as for panel A.

(A) Adhesion to fibronectin stimulates the stable association of MEK1 and ERK2 and is PAK dependent. COS-1 cells were transfected with HA-MEK1 and FLAG-ERK2 plasmids. The following day the cells were trypsinized and suspended in serum-free media for 90 min at 37°C and 5% CO₂. Cells were either left suspended (S) or allowed to adhere to a fibronectin-coated dish for the indicated time. The cells were harvested in hypotonic buffer and lysed by centrifugation at 13,000 × g for 20 min. Anti-FLAG immunoprecipitates (IP) were eluted off the antibody overnight at 4°C with FLAG peptide, run on a gel, and immunoblotted with anti-HA and anti-FLAG antibodies. An equal amount of cellular lysate from each sample was immunoblotted for HA-MEK and Myc-PAK. (B) COS-1 cells were transfected with plasmids for FLAG-ERK2 and either HA-MEK1 or HA-MEK2. The cells were placed in suspension (S) as for panel A or suspended and allowed to attach to fibronectin (F)-coated dishes for 20 min. ERK-MEK complexes were immunoprecipitated and immunoblottedas for panel A. (C) Same as for panel B, except the mutant MEK1 T292/S298A was transfected instead of MEK2. (D) PAK activity is required for fibronectin-stimulated ERK activation. REF cells were transfected with FLAG-ERK2 with or without Myc PAK1-K299R. After 48 h cells were suspended as described for panel A and were either left untreated in suspension (S) or allowed to adhere to fibronectin-coated dishes for the indicated times (given in minutes). Extracts were normalized with respect to protein, and anti-FLAG immunoprecipitates were prepared. Resolved immunoprecipitates were blotted sequentially with anti-FLAG and anti-phospho-ERK antisera. Equal amounts of lysate protein were blotted for the dominant-negative PAK1 protein with anti-Myc antisera.