Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the Ste20p homologue GCK and the adapter protein TRAF2

Abstract

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.

FIG. 1.

In vivo binding of endogenous GCK and MEKK1: activation of MEKK1 by coexpressed GCK in vivo. (A) Binding of endogenous GCK and MEKK1. RAMOS cells were lysed and subjected to immunoprecipitation (IP) with anti-GCK and immunoblotting (IB) with anti-MEKK1 or immunoprecipitation with anti-MEKK1 and immunoblotting with anti-GCK. Crude lysates were immunoblotted with the GCK and MEKK1 antibodies as well. (B) GCK activates MEKK1 in vivo. 293 cells were transfected with a low dose of HA-MEKK1 plasmid and with either vector or FLAG-GCK as described in Materials and Methods. MEKK1 was immunoprecipitated with anti-HA and assayed for SEK1(K129R) phosphorylation and autophosphorylation (left panel). The right panel documents expression of the transfected plasmids and the level of GST-SEK1(K129R) used in the MEKK1 assays.

FIG. 2.

GCK stimulates MEKK1 phosphorylation in vivo: similarity to MEKK1 autophosphorylation. 293 cells were transfected with HA-MEKK1 and either vector or FLAG-GCK. A portion of the cells expressing MEKK1 alone and all of the cells expressing MEKK1 and GCK were metabolically labeled with ³²PO₄. The remaining cells expressing MEKK1 alone were left unlabeled. MEKK1 was immunoprecipitated with anti-HA. (A) MEKK1 phosphorylation. (Left) In vitro autophosphorylation of MEKK1 immunoprecipitated from unlabeled cells that did not coexpress GCK; (middle) in vivo phosphorylation of MEKK1 in cells expressing vector or GCK; (right) expression of the MEKK1 polypeptides. (B) Two-dimensional tryptic phosphopeptide mapping of MEKK1 in vivo phosphorylation and in vitro autophosphorylation (auto-P). Expression with GCK or in vitro autophosphorylation is indicated underneath each map. The large arrowheads indicate a prominent labeled spot, probably free phosphate, detected in each map. Smaller arrowheads indicate some of labeled phosphopeptides that are common to MEKK1 in vivo phosphorylation and in vitro autophosphorylation. o, origins.

FIG. 3.

In vitro activation of MEKK1 by purified GCK, GCK CTD, and TRAF2. (A) Purification of GST (lane 1), GST-GCK (lane 2), GST-GCK CTD (lane 3), GST-TRAF2ΔRING (lane 4), and GST-TRAF2 (lane 5). These preparations were used for the in vitro studies described herein (Fig. 3 to 5 and 7). Proteins were purified from transfected 293 cells as described previously (48), and 10% of each preparation was subjected to SDS-PAGE and Coomassie blue staining. (B) GCK stimulates MEKK1 autophosphorylation in vitro but does not itself significantly phosphorylate full-length MEKK1. HA-tagged wild type (wt) or kinase-dead (D1350N) MEKK1 was immunoprecipitated from transfected 293 cells and incubated with soluble GST-GCK and purified as described above, with [γ-³²P]ATP. Incubations in the absence of GCK were performed with GST. Phosphorylation of the MEKK1 polypeptides was analyzed after SDS-PAGE and autoradiography (top). Expression of the MEKK1 polypeptides was determined by immunoblotting with anti-HA (bottom). (C) GCK activates MEKK1 in vitro. The kinase domain of GCK is dispensable for MEKK1 activation. Wild-type or D1350N HA-MEKK1 was immunoprecipitated as for panel B and incubated with an equimolar amount of purified GST-GCK or the GCK CTD (see above) plus [γ-³²P]ATP. Incubations in the absence of GST-GCK or GST-GCK CTD were performed with GST. After a brief incubation, GST-SEK1(K129R) was added. The reaction was stopped with SDS-EDTA, and the samples were subjected to SDS-PAGE and autoradiography (top two panels, left) or immunoblotting with anti-HA. Kinase activity was assessed as MEKK1 autophosphorylation and phosphorylation of GST-SEK1(K129R). The bar graph on the right represent phosphorimager quantitation of the kinase assay results. Quantitation of MEKK1 autophosphorylation for samples not containing MEKK1 represents phosphorimager counts of mock anti-FLAG immunoprecipitations in the region of the gel corresponding to the migration position of MEKK1.

FIG. 4.

Activation of MEKK1 by GCK requires the autophosphorylation phosphoacceptor sites T1381 and T1393. Wild-type HA-MEKK1, MEKK1(T1381A), and MEKK1(T1393A) were immunoprecipitated from transfected 293 cells as for Fig. 3B and incubated with purified GST-GCK (Fig. 3A). Control incubations (minus GCK) were performed with GST. GST-SEK1(K129R) was added, and the results were analyzed as for Fig. 3B. The graph shows a phosphorimager quantitation of the results in the autoradiograms.

FIG. 5.

Activation of MEKK1 by TRAF2 and GCK is not additive; GCK also activates MLK3. (A) TRAF2 activation of MEKK1 in vitro: requirement for the TRAF2 RING domain. HA-MEKK1 (wild-type) was immunoprecipitated from 293 cells as for Fig. 3 and incubated with equimolar concentrations of purified (Fig. 3A) GST-TRAF2, GST-GCK, or GST-TRAF2ΔRING as described for Fig. 3B. Controls were incubated with GST alone. GST-SEK1 was added as for Fig. 3B, and the incubation was continued. Samples were subjected to SDS-PAGE and either autoradiography or immunoblotting as for Fig. 3 and 4. The bar graph shows a phosphorimager quantitation of the kinase assays.

FIG. 6.

Soluble, purified MEKK1 is activated by soluble, purified GCK or TRAF2 in vitro. HA-MEKK1 and FLAG-GCK, GCK(K44M), GCK CTD, and TRAF2 were immunoprecipitated from transfected 293 cells and eluted with the cognate antigen peptide. (A) Silver-stained gels of the purified HA-MEKK1 and FLAG-GCK and TRAF2 constructs. Note that the immunoglobulin G heavy chain [IgG(H)], from the immunoprecipitations, is still present in the eluted protein preparations; however, the free-peptide concentration used in the elutions (100 μM) is far in excess of the IgG concentrations employed in the immunoprecipitation. FLAG-TRAF2 comigrates with the IgG and is not visible in the preparations. (B) Assays for activation of purified MEKK1 by the purified GCK and TRAF2 constructs.

FIG. 7.

Increasing concentrations of TRAF2ΔRING suppresses GCK activation of MEKK1 in vitro. GCK can also activate MLK3 but not MEKK3. (Top, left) Titration of increasing concentrations of purified (Fig. 3A) TRAF2ΔRING suppresses in vitro activation of MEKK1 by GCK. Immobilized MEKK1 was incubated with GCK in the presence or absence of the indicated amounts of TRAF2ΔRING. (Bottom, left) GCK also activates MLK3 but fails to activate MEKK3 in vitro. Immobilized FLAG-MLK3 or Myc-MEKK3 was incubated with the purified GCK or TRAF2 preparation (Fig. 3A) used in the titration experiment; however, samples were run on separate SDS-PAGE gels. The method was as described for Fig. 3 to 5. Activity was measured as phosphorylation of substrate protein [GST-SEK1(K129R)] and autophosphorylation (auto-P). At right is a quantitation of the results.

FIG. 8.

In vivo association of GCK and MLK3. 293 cells were transfected with GST-GCK or GCK CTD and FLAG-MLK3. GCK constructs were purified on glutathione agarose and subjected to SDS-PAGE and immunoblotting with anti-FLAG to detect associated MLK3.

FIG. 9.

Oligomerization is sufficient to trigger MEKK1 activation and phosphorylation in vivo. GCK promotes MEKK1 oligomerization. (A) 293 cells were transfected with GST-SAPK and either empty FKBP12-HA vector or FKBP12-HA-MEKK1. Cells were then treated with vehicle or the indicated concentrations of AP1510 for 1 h. Control cells were treated with vehicle or TNF (100 ng/ml). GST-SAPK was purified on glutathione agarose and assayed. (B) Oligomerization of immunoprecipitated MEKK1 results in activation. FKBP-HA-MEKK1 was expressed in 293 cells and immunoprecipitated with anti-HA. Immunoprecipitates were treated with vehicle or the indicated concentration of AP1510 plus 100 μM [³²P]ATP. Alternatively, a blank immunoprecipitate was identically treated with 100 nM AP1510. SEK1(K129R) was added as a substrate for MEKK1, and the activity of MEKK1 towards the SEK1 polypeptide is shown. (C) Forced oligomerization of MEKK1 enhances its in vivo phosphorylation. 293 cells expressing FKBP-HA-MEKK1 were labeled with ³²PO₄ and treated with vehicle or AP1510 (200 nM). FKBP-HA-MEKK1 was recovered by immunoprecipitation with anti-HA and subjected to SDS-PAGE and autoradiography or to anti-HA immunoblotting. (D) Expression with GCK enhances spontaneous MEKK1 oligomerization in vivo. 293 cells were transfected with FLAG- and HA-MEKK1 plus either vector or untagged GCK. FLAG-MEKK1 was immunoprecipitated (IP) and subjected to SDS-PAGE and immunoblotting (IB) with anti-HA to detect the presence of HA-MEKK1 in FLAG-MEKK1 immunoprecipitates, an indication of MEKK1 oligomerization. FLAG- and HA-MEKK1 immunoprecipitates were also immunoblotted with cognate antibodies to document even expression of the MEKK1 constructs. (E) Sucrose density gradient centrifugation of MEKK1 expressed alone or coexpressed with GCK. Active MEKK1 exists as a high-molecular-weight oligomer, and GCK shifts MEKK1 into this oligomer. Cells were transfected with HA-MEKK1 alone or with FLAG-GCK. Cell extracts were subjected to density gradient centrifugation through sucrose (5 to 60%). Fractions (50 μl) were collected and assayed for HA-MEKK1 immunoreactivity and SEK1 phosphorylating activity. The positions of molecular weight standards are shown.

FIG. 10

Model for TNF and agonist activation of MEKK1 by GCK and TRAF2. Details are discussed in the text. P, phosphorylation of the MEKK1 polypeptide.