ERK activity is required for NIH 3T3 cell proliferation.
It was first established for fibroblasts that ERK activation is a requisite for growth factor-induced cell growth (33
). However, this is not the case for all cell types; for example, embryonic stem cells grow independently of ERK activation (11
). Similarly, several tumor cell lines proliferate with low levels of ERK activation (20
). Our shRNAs were designed against mouse sequences, and thus we searched a mouse cell line whose proliferation was ERK dependent.
Hence, we tested whether exponential growth of NIH 3T3 cells was ERK dependent. The results of a typical experiment with exponentially growing NIH 3T3 cells treated with MEK inhibitors U0126 and PD184352 are depicted in Fig. . Great care was taken in all experiments, including this one, to measure phosphorylated ERK in cells grown in the same conditions as the ones plated to measure proliferation (12-well plates for counting and 10-cm plates for protein extracts). Addition of MEK inhibitors was sufficient to lower the levels of phospho-ERK1 and phospho-ERK2 after two days of treatment (Fig. , upper blot). For a given concentration, PD184352 was more potent than U0126 to reduce ERK activation. For both inhibitors, a gradual increase of the concentration led to a gradual reduction of ERK activation. Interestingly, in NIH 3T3 cells the level of phospho-ERK2 was more elevated than the level of phospho-ERK1; hence, at high concentrations of inhibitors only the remaining level of phospho-ERK2 was detectable, being higher than threshold of detection. We tried unsuccessfully to lower significantly further the phospho-ERK level by changing the medium with fresh U0126 daily (data not shown).
FIG. 1. Proliferation of NIH 3T3 cells requires MEK/ERK activation. Exponentially growing NIH 3T3 cells were treated with dimethyl sulfoxide (0.2%), with increasing concentrations of U0126 (5 μM, 10 μM, and 20 μM), or with increasing (more ...)
As indicated in Fig. , at 3 days after addition of MEK inhibitors, cell proliferation was markedly reduced, by about 50% with 20 μM U0126 and 66% with 20 μM PD184352. Lower doses of inhibitors were still effective to slow cell growth (about 33% reduction with 5 μM U0126 and 46% reduction with 5 μM PD184352). Globally the rate of cell proliferation correlated with the level of ERK activation. However, even when high levels of inhibitors were used, a significant proliferation of the cells was observed. For example, 20 μM PD184352 diminished ERK activation by about 94% and slowed cell proliferation by about 66%. Changing the growth medium every day in the presence of high concentrations of inhibitor led to the same slow growth (data not shown). Hence, a weak level of ERK activity was sufficient to allow NIH 3T3 cells to proliferate slowly; however, the rate of growth was gradually diminished when ERK activation was gradually decreased, and thus NIH 3T3 cells are a good model to test our hypothesis.
Ablation of ERK1 has no impact on cell proliferation.
NIH 3T3 cells were transfected with the plasmid pSUPER-shERK1 as described in Materials and Methods. Only highly transfected cells were selected owing to a 10-fold excess of shRNA-expressing plasmid above the pBabePuro selection plasmid. After selection of transfected cells with puromycin, puromycin was omitted for the rest of the experiment.
As shown in Fig. , cells expressing the shERK1 plasmid did not display any ERK1 protein. Quantification performed by comparing serial dilutions of control transfected extracts to reach the low level of shERK1-transfected cells indicated about a 95% diminution in ERK1 protein expression in cells expressing shERK1 (data not shown). As expected, pSUPER-shERK1 targeted ERK1 very specifically, since the level of ERK2 was absolutely normal (Fig. ). The cell extract was obtained after 2.5 days of exponential growth; however, the silencing of ERK1 was still maximal at 4 to 5 days postseeding, ERK1 expression started to rebound at between 5 and 6 days postselection (data not shown).
FIG. 2. ERK1 ablation has no impact on cell proliferation. NIH 3T3 cells were transfected with 3 μg of pBabePuro plasmid and 27 μg of control plasmid or 27 μg pSUPER-ERK1 (sh-ERK1). After selection, cells were plated under conditions of (more ...)
The remaining level of ERK1 was sufficiently low that the level of phospho-ERK1 was nearly abrogated (Fig. , lower blot). This was not the case with another plasmid that did not reduce sufficiently the level of the ERK1 protein (data not shown). We took great care in measuring the level of phospho-ERKs in exponentially growth conditions; thus, 10-cm diameter plates were seeded at the density of the 12-well plates used for measuring cell proliferation, and lysis was performed at 2.5 days postseeding. This is a critical step, since confluence markedly affects ERK activation measured by the phospho-ERK level (data not shown).
Cells lacking ERK1 grew at the rate of control transfected cells for at least 4 days postseeding (Fig. ). The results are representative of seven independent experiments which all indicated that removal of ERK1 had no impact on cell proliferation. For each experiment, puromycin-resistant cells were seeded at three cell densities, and great care was taken to compare only plates that displayed exactly the same number of cells at 6 h postseeding. No difference in cell attachment was noted (data not shown). Hence, silencing of ERK1 alone has no impact on NIH 3T3 cell proliferation.
Reducing ERK2 markedly slows cell proliferation.
Plasmids expressing shRNA targeting ERK2 were transfected and selected as described above and in Materials and Methods. Two plasmids expressing shRNA sequences unrelated to ERK2 very efficiently reduced the level of the ERK2 protein (Fig. ). Quantification performed by comparing serial dilutions of control transfected cell extracts with shRNA-expressing extracts indicated that the ERK2 level was reduced by about 90% to 95% in transfected cells (data not shown). Selectivity towards ERK2 is very high, since the level of ERK1 was not affected upon transfection with either plasmid targeting ERK2 (Fig. ).
FIG. 3. ERK2 ablation slows cell proliferation. NIH 3T3 cells were transfected with 3 μg of pBabePuro plasmid and 27 μg of control plasmid, 27 μg pSUPER-ERK2 (sh-ERK2), or 27 μg pSUPER-ERK2bis plasmids (sh-ERK2bis). After selection, (more ...)
The level of phospho-ERK2 was markedly reduced in cells that were transfected with one or the other plasmid targeting ERK2 (50% reduction [data not shown]). Reducing the level of phospho-ERK2 in exponentially growing cells was sufficient to reduce markedly the rate of cell proliferation (Fig. ). The two sequences were equally effective in reducing cell proliferation; however, a third sequence that did not reduce the level of phospho-ERK2 as much had limited impact on cell proliferation (data not shown).
The results in Fig. are representative of four independent experiments with one or the other plasmid that targets ERK2, and they indicate that ERK2 is required for NIH 3T3 cell proliferation to proceed normally.
Removal of both ERK1 and ERK2 seems equally effective as removal of only ERK2 in slowing cell proliferation.
A lack of an effect of ERK1 silencing could be explained by the weak contribution of ERK1 to the total ERK activity, as indicated by the low ratio of phospho-ERK1 to phospho-ERK2 in the control transfected cells in all the experiments presented above. Hence, we decided to lower the ERK1 expression in limiting conditions of ERK2 activation. Initially we tried to silence ERK1 completely in a background of mild ERK2 silencing, and we observed no increase of the effect on cell proliferation for ERK1 and ERK2 silencing over the sole silencing of ERK2 (data not shown). Figure depicts one experiment where ERK1 was expressed or not in conditions where ERK2 was maximally silenced.
FIG. 4. Double silencing of ERK1 and ERK2 is equally effective as silencing ERK2 alone in slowing cell proliferation. Cells were transfected with 3 μg of pBabePuro plasmid and 27 μg of control plasmid, 13 μg pSUPER-ERK2 plus 13 μg (more ...)
Cells transfected with the plasmid expressing shERK2 displayed a very low level of ERK2 expression, while ERK1 expression was equal to that of control transfected cells (Fig. ). In cells expressing both shERK1 and shERK2, expression of both ERK isoforms was barely detectable compared to that in control transfected cells. Importantly, the very low level of ERK2 expression was barely detectable for single and double silencing.
Interestingly, silencing of ERK2 alone strongly reduced ERK2 phosphorylation while concomitantly increasing ERK1 phosphorylation compared to that in control transfected cells (Fig. , middle blot). Furthermore, when both ERK1 and ERK2 were silenced simultaneously, although the levels of ERK1 and ERK2 were barely detectable, the phosphorylation level of ERK1 and ERK2 was not as diminished as expected: ERK1 phosphorylation was similar to that in control transfected cells, and ERK2 phosphorylation was reduced compared to that in control transfected cells but was markedly higher than that in cells where ERK2 alone was silenced. Interestingly, the ratio of phospho-ERK1 to phospho-ERK2 is inverse for single ERK2 silencing and double ERK1 and ERK2 silencing.
Removal of ERK2 alone or simultaneous removal of the two ERKs lowered the proliferation of NIH 3T3 cells compared to control transfected cells (Fig. ). However, cells grew at the same rate whether ERK2 expression was abrogated alone or expression of ERK1 and ERK2 was abrogated simultaneously. At first glance, this experiment confirmed that removal of ERK1 had no impact on cell proliferation. However, clearly there was no correlation between the level of ERK2 activation and the rate of cell proliferation: double-silenced and single-ERK2-silenced cells grew at the same rate despite different levels of phospho-ERK2.
Both isoforms compete for upstream activating signals.
The dramatic inversion of the ratio between the phospho-ERK isoforms in ERK2-silenced cells versus ERK1- and ERK2-silenced cells (Fig. ) prompted us to evaluate more closely this shift of activation among isoforms.
A typical experiment with exponentially growing cells in which either ERK1 or ERK2 expression has been reduced is presented in Fig. . The immunoblot that displays ERK levels confirms that the vectors ablate expression of their target very effectively and highly specifically, since expression of the targeted isoform is nearly abolished while expression of the other ERK isoform is unaltered. When assessing the level of phospho-ERK in these cells, one must note first that removal of ERK1 increased activation of ERK2, as was reported for mouse embryo fibroblasts derived from knockout animals (32
). Furthermore, this experiment indicated unambiguously that removal of ERK2 led to increased activation of ERK1 compared to that in control transfected cells. Can this phenomenon be observed only in exponentially growing cells? To answer this question, cells transfected in this experiment were also plated at high density to reach confluence at 1.5 days postseeding, serum deprived overnight, and stimulated with 10% FCS. Other experiments indicate that confluence did not affect the level of silencing (data not shown); hence, at the time of stimulation, silencing of ERK1 or ERK2 was identical as shown by immunoblotting for total ERK (Fig. ). Removal of ERK1 also increased phospho-ERK2 levels during acute stimulation (Fig. ). The overactivation of ERK2 was more obvious after 3 and 5 h of FCS stimulation.
FIG. 5. The remaining ERK isoform is overactivated. Cells were transfected with 3 μg of pBabePuro plasmid and 27 μg of control plasmid, 27 μg pSUPER-ERK1 (sh-ERK1), or 27 μg pSUPER-ERK2 (sh-ERK2). After selection, cells were plated (more ...)
The phospho-ERK1 level was much higher in cells lacking ERK2 than in control cells at all time points of stimulation (Fig. ). The phospho-ERK2 level was still detectable owing to a residual pool of ERK2 (Fig. ). After 5 h of serum stimulation, removal of ERK2 led to inversion of the activation ratio between the isoforms compared to that in control transfected cells. The results indicate that ERK activation was transferred to the residual pool of ERK molecules, in exponentially growing cells as well as during acute stimulation (Fig. ).
ERK1 becomes a positive activator of cell proliferation when ERK2 activation is severely limiting.
It may be concluded from the results shown in Fig. that MEK activates the remnant pool of ERKs present in the cell, independently of isoforms. Hence, evaluating the role of ERK1 at limiting levels of ERK2 activation is difficult, since the silencing of ERK1 can “reactivate” the remnant pool of ERK2. We decided to lower further the level of the ERK2 protein in the doubly transfected cells in order to compare the same level of phospho-ERK2 in cells lacking ERK2 alone or lacking both ERK1 and ERK2. Figure displays the results of one such experiment. ERK2 was nearly undetectable in cells transfected with 27 μg or 57 μg of shERK2 plasmid and in cells transfected with 40 μg of shERK1 plasmid in the presence of 57 μg of shERK2 plasmid (Fig. , upper blot). Only cells expressing the shERK1 plasmid showed a drop in the ERK1 level compared to control transfected cells.
FIG. 6. At a constant level of ERK2 activity, ERK1 silencing further slows cell proliferation. NIH 3T3 cells in a 10-cm dish were transfected with a total of 100 μg of plasmid containing 3 μg of pBabePuro plasmid for selection and 97 μg (more ...)
When ERK2 alone was lowered, the phospho-ERK1 level was higher than that in control transfected cells; in contrast, in cells lacking ERK1 and ERK2, the phospho-ERK1 level was much lower than that in control transfected cells (Fig. ). The phospho-ERK2 level was lowered in cells transfected with 27 μg of shERK2 plasmid compared to control transfected cells; it became even lower when cells were transfected with 57 μg of shERK2 plasmid alone. In cells lacking ERK1 and ERK2, the phospho-ERK2 level was not lowered as much as in cells transfected with 57 μg of shERK2 alone but was nearly as low as in cells transfected with 27 μg of shERK2. Hence, we decided to compare the proliferation of cells transfected with 27 μg of shERK2 alone with that of cells doubly transfected. In these two populations of cells the level of phospho-ERK2 was nearly identical; however, in the doubly transfected cells phospho-ERK1 was markedly reduced. Thus, we were then able to assess the role of ERK1 when ERK2 activation was low and comparable in two populations of cells (Fig. , lanes 2 and 4).
Removal of ERK2 diminished the rate of cell proliferation compared to control transfected cells (Fig. ). Interestingly, the removal of ERK1 and ERK2 reduced cell proliferation more than the sole removal of ERK2, despite an equivalent level of active ERK2 (Fig. ). Hence, for the first time we demonstrate that ERK1 plays a positive role in the control of cell proliferation. This role of ERK1 was unmasked in this experiment designed to avoid “reactivation” of ERK2 in doubly transfected cells.
Cells grew at the same rate with different levels of phospho-ERK2 (Fig. ) but also grew at different rates with nearly equivalent levels of phospho-ERK2 (Fig. ). These two observations indicate that the rate of cell proliferation was not correlated with the level of ERK2 activation. To determine whether the rate of proliferation was correlated with the level of total ERK activation, we decided to evaluate the total level of phospho-ERK in Fig. . To avoid problems of threshold detection of immunoblots, we intended to combine the phospho-ERK1 and phospho-ERK2 signals as one band for quantification of the chemiluminescence by camera capture. In Fig. , one gel is presented and three independent gels were used for quantification. Silencing of ERK2 with 27 μg of shERK2 reduced the total ERK phosphorylation by 30% over that in control transfected cells, whereas silencing of ERK2 and ERK1 reduced total ERK phosphorylation by about 45%. Hence, we observe a correlation between the rate of cell proliferation and the level of total ERK phosphorylation.
Removal of ERK1 alone or ERK2 alone is sufficient to abrogate IEG transcription.
To confirm that ERK1 and ERK2 are both positive contributors of ERK signaling, we decided to study the effect of removal of the ERK isoforms on another ERK-dependent biological response. We chose to study IEG transcription since it is well documented that transcription of many IEGs is ERK dependent and quantitative measurement is easily performed. shRNA-expressing plasmids were transfected, and cells were selected and plated at high density for 1 day prior to serum depletion overnight. Following 45 min of serum stimulation, the mRNA levels of several genes were measured by quantitative RT-PCR.
Transfection with shRNA efficiently silenced specifically ERK1 or ERK2, since nearly all of the targeted isoform was removed while the other was expressed at normal levels (Fig. ). In this experiment, we sought to silence both isoforms at the same time to compare the biological impact of this double silencing with the impact of single silencings.
FIG. 7. Both ERK1 and ERK2 contribute to stimulate IEG transcription. NIH 3T3 cells in a 10-cm dish were transfected with a total of 100 μg of plasmid containing 3 μg of pBabePuro plasmid for selection. Cells were transfected with 97 μg (more ...)
Hence, we transfected higher levels of shRNAs-expressing plasmids against ERK1 and ERK2 in the double silencing than in the single silencing, to counteract compensation on the remaining ERKs as shown in Fig. . The middle immunoblot of Fig. represents the level of phospho-ERKs in transfected cells stimulated or not for 45 min with FCS. ERK1 silencing decreased markedly the level of phospho-ERK1. As expected ERK2 silencing decreased markedly the level of phospho-ERK2 and concomitantly increased the level of phospho-ERK1. In doubly silenced cells, both phospho-ERK1 and phospho-ERK2 were lower than in control transfected cells. Furthermore, the levels of phospho-ERK2 were comparable in cells where only ERK2 was silenced and in cells where both ERK1 and ERK2 were silenced, due to attempts to avoid compensation by transfecting larger amounts of plasmids in the double silencing. Unfortunately, in this experiment we were unable to reduce ERK1 phosphorylation in the double silencing as effectively as in the single ERK1 silencing despite a larger amount of sh-ERK1 plasmid transfected in doubly transfected cells. This observation stresses once more the difficulty of avoiding compensatory activation of the remaining pool of ERKs.
Figure represents the quantification of the respective phospho-ERK levels in Fig. . Interestingly, when observing the phospho-ERK levels in stimulated cells, a gradation in the reduction of phospho-ERKs was observed. Removal of ERK1 decreased phospho-ERKs by about 25%, removal of ERK2 decreased phospho-ERKs by about 40%, and removal of both ERK1 and ERK2 decreased both phospho-ERKs by about 70% (Fig. ). The total active ERK level is obtained by adding the residual activated pool of the two ERKs. Interestingly, the contribution of ERK1 or ERK2 to the total phospho-ERK level appears to be reversed during ERK1 silencing versus ERK2 silencing. In addition, when both ERK1 and ERK2 are silenced, the ratio between active ERK1 and active ERK2 returns to the ratio in control transfected cells, although the total level of active ERKs is much reduced.
Stimulation of serum-deprived cells for 45 min led to a massive induction of the levels of junB (Fig. ) and egr1 (Fig. ) mRNAs (11.4- and 13.8-fold, respectively). In preliminary experiments we demonstrated that this induction was ERK dependent, since it was markedly reduced on MEK inhibition with 50 μM U0126 or 2 μM PD184352 (data not shown).
Silencing of either ERK1 or ERK2 alone was sufficient to lower the mRNA level of junB compared to that in control transfected cells, while silencing of both ERK1 and ERK2 lowered further the junB mRNA level, to below that of ERK2 alone (Fig. ).
Since ERK2 phosphorylation was nearly the same in the single silencing with sh-ERK2 as in the double silencing, there is no correlation between the level of junB mRNA expression and ERK2 activation. In contrast, in the double silencing there was a decrease in total ERK phosphorylation compared to single silencing, and hence this experiment confirms that total ERK activation correlates with junB mRNA induction.
Regulation of the egr1 mRNA level was somewhat different when the same extracts were examined. First, silencing of ERK1 or silencing of ERK2 was nearly equally effective in reducing the egr1 mRNA level (Fig. , bars 3 and 4). Second, induction of the egr1 mRNA level was reduced equally when ERK2 was silenced alone or in conjunction with ERK1 (Fig. , last bar). This experiment confirms that ERK1 plays a positive role in increasing the mRNA levels of ERK-responsive genes. However, there is not always a straightforward correlation between the level of total ERK activity and induction of the mRNA level (as shown for egr1 mRNA induction). Experimental variation certainly plays a role, and it is also obvious that ERK-independent signaling pathways contribute to induce the mRNA levels of many IEGs; hence, full mRNA induction may require full ERK activation, but a threshold ERK activity may be sufficient for robust induction to occur.
Stoichiometric ratio of isoforms in NIH 3T3 cells.
The results presented above (Fig. and ) indicate that both ERK isoforms are positive activators of cell proliferation and IEG transcription; furthermore, the level of phosphorylated ERK1 is consistently lower than the level of phospho-ERK2 in NIH 3T3 cells (Fig. ). Hence, when ERK activation is not limiting, removal of ERK1 may have no more effect than removal of 20 to 30% of ERK2. Is the low level of phospho-ERK1 relative to the level of phospho-ERK2 due to inefficient activation? To understand better the consequences of the lack of ERK1 or of ERK2, we sought to determine precisely the stoichiometric ratio between ERK1 and ERK2 in our model system and to compare it to the precise ratio between phospho-ERK1 and phospho-ERK2.
We normalized the immunoblot signals for ERK1 and ERK2 by comparing NIH 3T3 cell extracts with standard quantities of exogenously expressed ERK1 and ERK2. We used epitope-tagged ERK1 and ERK2 expressed in mammalian cells as standards to ensure proper posttranslational modification. Since most ERK antibodies are directed towards the N- or C-terminal end of the molecule, two sets of standards were used to avoid bias. Hence, the full-length mouse ERK1 and ERK2 were epitope tagged either at the N terminus with the HA epitope (9 amino acids) or at the C terminus with the VSVG epitope (11 amino acids).
Figure shows representative immunoblots that allowed us to quantify the ratio between ERK1 and ERK2 in NIH 3T3 cells. The chemiluminescence was acquired with radiography film (higher resolution) and with light acquisition equipment for precise quantification and calculation. For Fig. , the different gels were loaded with identical extracts, either from HEK293 cells transfected with epitope-tagged ERKs or from NIH 3T3 cells.
FIG. 8. Quantification of the relative levels of ERK1 and ERK2 and the relative levels of activated ERK1 and activated ERK2. HEK293 cells were transfected with plasmids expressing mouse ERK1 or mouse ERK2 that was tagged at the N terminus with the HA epitope (more ...)
For Fig. , normalization was performed with exogenously expressed ERK1 and ERK2 that were epitope tagged at the N terminus. Gels were loaded with extracts from HEK293 cells transfected with mouse HA-ERK1 or mouse HA-ERK2 or with nontransfected NIH 3T3 cell extracts. In the linear range of the HA signal, HA-ERK2 was 10% more abundant than HA-ERK1 in the mix (Fig. , upper blot). The monoclonal antibody mix 1 recognized preferentially HA-ERK2 (HA-ERK1 = 0.486 × ERK2) (Fig. , lower blot). For the NIH 3T3 extracts, only the loading of 15 μg and 30 μg gave linear results (data not shown), with a much stronger signal for ERK2 than for ERK1 (ERK1apparent = 0.132 × ERK2apparent). After correction for the loading of the HA isoforms (10% difference) and for the affinity of the antibodies, the ratio was determined to be ERK1normalized = × 0.3 ERK2normalized.
The signal detected with the HA antibody in Fig. indicated that less HA-ERK1 than HA-ERK2 was loaded on the gel (HA-ERK1 = 0.81 × HA-ERK2). However, the signal with a polyclonal mix of anti-ERKs antibodies (E1B and ERK1 no. 61), was stronger for HA-ERK1 than for HA-ERK2 (HA-ERK1 = 1.36 × HA-ERK2). This ratio indicated that on an immunoblot where there was more ERK2 than ERK1, this mix of antibodies recognized more ERK1. Despite this bias, in extracts of NIH 3T3 cells ERK2 was recognized to a greater extent than ERK1 (ERK1apparent = 0.44 × ERK2apparent). After correction for the loading and the isoform bias of the mix of antibodies, this normalization in NIH 3T3 cells corresponded to ERK1normalized = 0.26 × ERK2normalized.
In Fig. , normalization was performed with exogenously expressed ERK1 and ERK2 that were epitope tagged at the C terminus. Gels were loaded with extracts from HEK293 cells transfected with mouse ERK1-VSVG or mouse ERK2-VSVG or with nontransfected NIH 3T3 cell extracts. Equal loading of the two tagged isoforms was obtained, as shown by equal signals detected with the VSVG antibody (Fig. , upper blot). Parallel revelation with the second mix of monoclonal ERK antibodies on a gel loaded equally to the first showed a ratio between ERK1 and ERK2 that differed slightly from that obtained with the mix 1 used for Fig. , middle panel. Indeed, the signal of ERK1-VSVG was stronger than that of ERK2-VSVG when revealed with monoclonal mix 2, indicating that the mix had more affinity for ERK1. In contrast, the signal obtained for NIH 3T3 cells extracts was much stronger for ERK2 than ERK1 (Fig. , middle panel). This indicates that there was more ERK2 than ERK1 in NIH 3T3 cells (ERK1normalized = 0.22 × ERK2normalized). The lower blot in Fig. shows the ERK-specific signals obtained on a gel loaded equally incubated with a mix of polyclonal antibodies. This polyclonal mix recognized even more ERK1, since the signal was stronger for ERK1-VSVG than for ERK2-VSVG although equal quantities were loaded on the gel. As shown with 30 μg of NIH 3T3 cell extracts and with a longer exposure captured on a charge-coupled device camera rather than on film, this mix of antibodies recognized slightly more ERK2 than ERK1. Again, this result indicated that there was more ERK2 than ERK1 in these cells (ERK1normalized = 0.18 × ERK2normalized).
Overall, the average calculation with the four different approaches in NIH 3T3 cells gave ERK1normalized = 0.245 × ERK2normalized. The results of quantification were nearly identical when exogenous ERK standards were tagged at the N terminus or C terminus. If total ERK is adjusted to 100%, ERK1 = 20% and ERK2 = 80% in NIH 3T3 cells.
We then sought to measure as precisely as possible the ratio of activated ERK isoforms in NIH 3T3. Note that the phospho epitope is identical in ERK1 and ERK2, and hence no normalization is necessary for SDS-polyacrylamide gel electrophoresis with proteins of nearly the same molecular weight. We performed immunoblotting experiments with anti-phospho-ERK antibodies in extracts from exponentially growing and 24-hour-arrested cells stimulated for either 7 min or 45 min with 10% FCS. Figure shows the chemiluminescent signal measured with the anti-phospho-ERK antibody in diluted extracts of NIH 3T3 stimulated for 45 min with 10% FCS. The average ratio is phospho-ERK1 = 0.25 × phospho-ERK2. For cells stimulated for 7 min the ratio was 0.27, and for exponentially growing cells the ratio was 0.27 (data not shown). If total phospho-ERK is adjusted to 100%, then phospho-ERK1 = 20% and phospho-ERK2 = 80% in NIH 3T3 cells.
In conclusion, we demonstrate for the first time that the ratio between the activated ERK isoforms correlated exactly with the ratio of their relative expression. In addition, we never observed a preferential activation of one isoform when stimulating arrested NIH 3T3 with various agonists (fibroblast growth factor, platelet-derived growth factor, thrombin, and insulin, alone or in combination) (data not shown). Therefore, we propose that ERK1 and ERK2 compete equally for phosphorylation by MEK1/2 and that the ratio of phospho-ERK1 to ERK2 reflects the mass ratio of ERK1 to ERK2.
Comparison of the in vitro specific activities of HA-ERK1 and HA-ERK2.
After determining that ERK1 and ERK2 are activated relative to each other with respect to their expression ratio, we sought to compare their specific activities in vitro. For this, CCL39 hamster fibroblasts were transfected with mouse HA-ERK1 or mouse HA-ERK2 and stable clones obtained. Cells were serum starved for 24 h and then stimulated for 5 min with 10% FCS to maximally activate ERKs (endogenous ERKs and HA-ERKs). First we determined that GST-Elk307-428
phosphorylation increased linearly for at least 30 min when the immunoprecipitated HA-ERKs were incubated in kinase buffer with an excess of GST-Elk307-428
(evaluation of excess by amido black staining [data not shown]). Then, increasing amounts of immunoprecipitated HA-ERKs were incubated with GST-Elk307-428
. The expression levels of HA-ERK1 and HA-ERK2 were distinct in the two chosen stable clones, and hence we decided to measure simultaneously in each kinase assay lysate the amount of active ERK immunoprecipitated and the amount of phosphorylated GST-Elk307-428
. The phospho-ERK epitope is identical for ERK1 and ERK2, and therefore the signal is directly indicative of the amount of active ERK. Phosphorylation of the substrate GST-Elk307-428
was determined by immunoblotting with the anti-phospho-Elk-Ser383 antibody. To minimize bias induced by variation in protein transfer for different gels, the levels of HA-ERKs was determined for a single SDS-polyacrylamide gel, and similar levels of phospho-Elk307-428
were detected in a single SDS-polyacrylamide gel. An augmentation of phospho-HA-ERKs was observed in kinase assays performed with increasing quantities of cell extracts (Fig. , upper blot). Phospho-Elk1307-428
levels were determined in the same kinase assay (Fig. , lower blot). We observed that comparable levels of phospho-HA-ERK1 and phospho-HA-ERK2 induced comparable phosphorylation of the substrate GST-Elk307-428
(compare lane 5 with lane 13). Figure shows the linear relationship between the level of phospho-ERKs and the phosphorylation of GST-Elk1307-428
, each measured in the same kinase assay lysate. The slope of the linear regression is virtually identical for phospho-HA-ERK1 and phospho-HA-ERK2 to phosphorylate GST-Elk1307-428
. This result demonstrates for the first time that the in vitro kinase activities of HA-ERK1 and HA-ERK2 are almost identical. Considering that it is impossible to obtain cells lacking ERK2 from erk2−/−
mouse embryos (36
) or to purify to homogeneity ERK1 separate from ERK2, our comparison of the specific activities of transfected HA-ERK1 and HA-ERK2 seems to be at present the most precise way to compare the intrinsic kinase activities of ERK1 and ERK2.
FIG. 9. Determination of HA-ERK1- and HA-ERK2-specific kinase activities. HA-ERK1 or HA-ERK2 was immunoprecipitated from increasing volumes of cell lysate obtained from CCL39 cells that stably express HA-ERK1 or HA-ERK2. An in vitro kinase assay using the GST-Elk-1 (more ...) Close correlation between expression of the ERK isoforms and their relative activation in brain structures.
A consequence of demonstrating a correlation between the relative expression of the ERK isoforms and their relative activation is the ability to perform phospho-ERK immunoblotting to study the relative levels of ERK isoforms. We tested whether this correlation holds true in mouse tissues. Since several tissue arrays indicate that the greatest disparities in ERK mRNA expression occur in brain structures, we measured the relative levels of ERK1 and ERK2 and their phosphorylation state in brain extracts.
ERK1 and -2 levels in eight brain structures were detected with either the monoclonal ERK antibody mix 2 (Fig. , panel 1) or the polyclonal ERK antibody mix (Fig. , panel 2). At first sight, the relative abundances of ERK1 and ERK2 seemed to differ; however, as expected from the normalization determined for Fig. , the polyclonal mix recognized ERK1 with greater affinity (Fig. , lower blot). Direct comparison was thus impossible without normalization to adjust for the specific affinity for each isoform. After taking into account the higher affinity of the polyclonal mix of antibodies for ERK1, the calculated ratio observed between ERK1 and ERK2 (Fig. , panel 2) resembled very closely that obtained with the monoclonal mix 2 (Fig. , panel 1) (quantification not shown). In any case, both antibody mixes indicated that some brain structures, such as the superficial cortex, seemed devoid of ERK1 (13 times more ERK2 than ERK1 [calculation not shown]), whereas other areas, such as the medulla, contained more similar levels of the ERK isoforms (only twofold more ERK2 than ERK1). This result is in accordance with previous studies indicating that the brain stem, medulla, and sciatic nerve express relatively more ERK1 than ERK2 than most other tissues (31
). Panels 3a and 3b in Fig. show the relative phosphorylation states of ERK1 and ERK2 in the same brain extracts with long and short exposures of the blot, respectively. Although there were great disparities in the total ERK phosphorylation between samples, for all extracts the ratio between phospho-ERK1 and phospho-ERK2 very tightly correlated with the ratio between ERK1 and ERK2. For example, the medulla showed the highest level of ERK1 relative to ERK2 (blot 1) and also the highest level of phospho-ERK1 relative to phospho-ERK2 (blot 3a). In contrast, in the superficial cortex there was little ERK1 relative to ERK2 (blot 1) and there was little phospho-ERK1, as best seen with dilutions of the superficial cortex sample to avoid saturation of the signal (blot 3a) or with a shorter exposure of the same immunoblot (blot 3b).
FIG. 10. Correlation between the levels of ERK isoforms in brain and their relative phosphorylation. Proteins from mouse brain were extracted in Laemmli sample buffer as described in Materials and Methods. Extracted brain structures: Ch, cerebellar hemisphere; (more ...)
The results presented in Fig. indicated that disparities between ERK1 and ERK2 expression in mouse tissues closely mimic the relative activation states of the two isoforms. In addition, these observations confirm that measuring phospho-ERKs allows prediction of the ratio between ERK1 and ERK2. This conclusion was drawn from experiments with mouse tissues, since the normalization of the antibodies was performed with mouse isoforms; however, the total conservation of the phospho epitope across species leads us to propose that measuring phospho-ERKs will predict the ERK1/ERK2 ratio at least in other mammals.
Normalization of protein loading was performed by measuring the abundance of tubulin and RabV proteins. Although there were minor loading differences, there were marked differences in total ERK expression. For example, in the pineal gland there was little ERK, whereas in the olfactory bulb ERK was very abundant. Interestingly, MEK1 seemed to be expressed at higher levels in tissues where ERK was abundant, and MEK2 was virtually not detected in brain extracts with our antibody (data not shown).
Genetic evidences for dosage requirement of total ERK activity.
Mice with erk1 gene invalidation are viable and reproduce normally, while invalidation of the erk2 gene leads to early embryonic lethality. ERK isoforms may have unique functions, explaining the contrast in phenotypes. Alternatively, low levels of ERK1 expression in some cells may explain the inability of ERK1 to compensate for a loss in ERK2, whereas ERK2 can compensate for ERK1 in nearly all cells. To test this hypothesis, we assessed whether it is detrimental or not to loose erk1 when only one allele of erk2 is expressed. Hence, we bred animals lacking one erk2 allele with animals lacking both erk1 alleles.
The erk1−/−; erk2+/+ animals and the erk1+/+; erk2+/− animals were backcrossed for nine and five generations, respectively, with C57BL/6 mice. When erk1−/−; erk2+/+ animals are crossed with erk1+/+; erk2+/− animals, 50% of the offspring ought to be double heterozygous animals (erk1+/−; erk2+/−). However, this breeding program led to the birth of 128 animals of which only 4 were double heterozygous; the remaining 124 animals were erk1+/−; erk2+/+. The frequency of birth of double heterozygous animals was thus only 3.4% instead of the 50% expected. This result indicated that it was very unfavorable to diminish the quantity of erk1 alleles when only one erk2 allele is present. This experiment was performed in the genetic background of C57BL/6 mice; however, the birth of double heterozygous animals was also observed in three other mouse genetic backgrounds (S. Meloche, personal communication).
Very interestingly, 59 pups were obtained by crossing the double heterozygous animals with each other (we were fortunate to obtain two females and two males in the F1 generation). Among the 59 animals that were born, 19 were double heterozygous (35%). As expected, no erk2−/− animals were born; furthermore, no animals were born with only one allele of erk2 and no allele of erk1. Hence, only 60% of the expected offspring could survive. Taking into account the survival rate, the frequency of double heterozygous pups should be 25% of the total expected offspring, i.e., 25 out of the 60 animals that could survive, which is 41%. Hence, the observed frequency of double erk heterozygotes born from double heterozygous parents is very close to the calculated frequency (35% versus 41%). Consequently, although obtaining double heterozygous animals was very detrimental in the F1 generation, transmission of double erk heterozygocity to the next generation became normal. In accordance with our observation in C57BL/6 mice, in three other independent genetic backgrounds no animals were ever born with only one allele of erk (S. Meloche, personal communication). In conclusion, erk gene dosage seems to be crucial for mouse survival; no animal can survive with one erk allele only, while animals survive with a minimum of only two erk2 alleles or with a minimum of one allele of erk1 and one allele of erk2.