Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

Abstract

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk's activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation.

FIGURE 1:

Some Erk1 and Erk2 variants are spontaneously phosphorylated when expressed in HEK293T and NIH3T3 cells. (A) pCEFL vectors carrying cDNAs encoding the indicated Erk1/2 molecules or a control empty vector were introduced into HEK293 cells. At 48 h posttransfection, cells were serum starved for 24 h and harvested. Protein lysates prepared from these cells were analyzed by Western blotting, using antibodies that specifically recognize phospho-Erk or the relevant tags (6xHis-Erk1, HA-Erk2, or GAPDH). (B) The same pCEFL vectors were introduced to NIH3T3 cells. At 48 h posttransfection, the cells were harvested and subjected to Western blot analysis with the indicated antibodies.

FIGURE 2:

Erk1(R84S) induces AP-1– and (AP-1+Ets)–mediated transcriptional activity in HEK293T cells. HEK293T cells were cotransfected with the indicated pCEFL vectors together with (A) AP-1-luc or (B) (AP-1+Ets)–luciferase plasmids. A plasmid encoding Renilla luciferase (pRL-TK) was also added to each transfection mixture. At 48 h posttransfection, cells were harvested, and the lysates were subjected to the luciferase assay as described in Materials and Methods.

FIGURE 3:

Erk1(R84S) is highly expressed and strongly phosphorylated and induces oncogenic transformation of NIH3T3 cells. (A) NIH3T3 cells were transfected with the indicated expression vectors and selected for stable presence of the plasmid using the antibiotic G-418. Four weeks after transfection, cells were harvested. Protein lysates prepared from these cells were subjected to Western blotting, using antibodies that specifically recognize the indicated proteins. (B) NIH3T3 cells were transfected with the indicated vectors and grown in the presence of G-418. Cells were harvested at the indicated time points, and protein lysates prepared were analyzed by Western blot. Asterisk indicates the transfected HA-Erk2. (C) NIH3T3 cells transfected with the indicated vectors were allowed to grow to high density and reach confluence. Continuation of proliferation and appearance of foci was monitored. Cells were fixed and stained with crystal violet 4 wk after transfection. (D) Cells expressing Erk1(R84S) form colonies in soft agar. Cultures of NIH3T3 cells stably expressing the indicated proteins were seeded in triplicate in 96-well plates containing soft agar. Two weeks after plating, cells were stained with MTT and photographed.

FIGURE 4:

The intrinsic activity of Mpk1(R68S) is dependent on the autophosphorylation of the TEY motif. mkk1∆mkk2∆ cells (A), mkk1∆mkk2∆pbs2∆ste7∆ cells (C), or Mpk1∆ cells (E) expressing the indicated Mpk1 molecules were plated in five dilutions on plates containing YPD supplemented with 15 mM caffeine (left) or on plates not containing caffeine (YNB –URA; right). Western blot analysis with the indicated antibodies was performed on protein lysates prepared from mkk1∆mkk2∆ cells (B), mkk1∆mkk2∆pbs2∆ste7∆ cells (D), or Mpk1 cells (F) expressing the indicated Mpk1 molecules.

FIGURE 5:

Erk1(R84S) and Erk2(R65S) are capable of efficient autophosphorylation at the TEY motif. (A) Autophosphorylation capabilities of the purified Erks were assessed by incubating the proteins in a kinase assay mixture with [γ-³²P]ATP and no other substrate. Reactions were terminated at the indicated time points, separated via SDS–PAGE, stained with Coomassie brilliant blue, and exposed to x-ray film. (B) Autophosphorylation of the active variants occurs at the TEY motif. The indicated proteins were subjected to Western blot analysis using antibodies that react with Erk1/2 proteins phosphorylated at their TEY motif (αpErk) or with antibodies that react with the polyhistidine tag (αHis). (C) Intact TEY motif is essential for catalytic activity of Erk2(R65S) toward the substrate MBP. Catalytic activity of purified recombinant Erks carrying mutations at the TEY motif was analyzed with or without preincubation with active MEK1, using [γ-³²p]ATP and MBP as substrates. Reaction mixtures were spotted on filter papers and quantified. Activity of MEK1-activated Erk2(WT) was defined as 100%. In parallel, a sample from each reaction was subjected to SDS–PAGE, stained with Coomassie brilliant blue, and exposed to x-ray film.

FIGURE 6:

The active variants Erk1(R84S) and Erk2(R65S) are autophosphorylated at a novel phosphoacceptor, T207 (in Erk1) and T188 (in Erk2), which is critical for catalytic activity. (A) The indicated Erk proteins (recombinant purified) were subjected to Western blot analysis, using antibodies specifically raised against phospho-Thr-207/Thr-188 (αpT207/T188) and anti-polyhistidine antibody (αHis). (B) The indicated ERKs were introduced into HEK293T cells. At 48 h posttransfection, cells were serum starved for 24 h. Then cells were exposed or not exposed to 50 ng/ml EGF for 10 min, harvested, and subjected to Western blot analysis using the indicated antibodies. (C) Phosphorylation of T207 in Erk1(R84S) is not affected by EGF. NIH3T3 cells stably expressing Erk1(WT) or Erk1(R84S) were collected, and protein lysates were prepared at the indicated time point after EGF addition. Cell lysates were subjected to Western blot analysis using the indicated antibodies. (D) Erk2(T188A), but not Erk2(T188D), manifested dramatic increase in autophosphorylation activity. Autophosphorylation of purified Erk2(R65S), Erk2(T188A), and Erk2(T188D) was tested by incubating the proteins in a kinase assay mixture with [γ-³²P]ATP and no other substrate. Samples were removed from the assay at the indicated time points and subjected to SDS–PAGE. Gels were stained with Coomassie brilliant blue, dried, and exposed to x-ray film. (E) Mutating T188 of Erk2 to either Asp or Ala diminished its catalytic activity. Catalytic activity of the indicated protein was assayed as described in the legend to Figure 5.

FIGURE 7:

T188 is phosphorylated in Erk2(R65S) independently of TEY phosphorylation. (A) Autophosphorylation capabilities of purified recombinant Erks carrying mutations at the TEY motif were tested in the presence of [γ-³²P]ATP as described in the legend to Figure 5A. (B) Purified recombinant Erks carrying mutations at the TEY motif were subjected to Western blot analysis using antibodies specifically raised against phospho–Thr-207/Thr-188 (αpT207/T188), as well as αpErk and αErk antibodies.

FIGURE 8:

Minor conformational changes, apparent only at the site of mutation, are observed when the crystal structure of Erk2(R65S) is compared with that of Erk2(WT). Superimposition of the structures of Erk2(R65S) (yellow) and Erk2(WT) (cyan) at the region of the mutation. The side-chain hydroxyl of S65 in Erk2(R65S) is directed toward Y34 from the P-loop, forming a polar interaction, whereas R65 in Erk2(WT) does not form any interaction with this region. Another hydrophobic interaction, between Y34 and Y62 from the C-helix, is similar in both proteins.