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Originally published In Press as doi:10.1074/jbc.M410031200 on November 16, 2004

J. Biol. Chem., Vol. 280, Issue 5, 3832-3837, February 4, 2005
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Live Cell Imaging of ERK and MEK

SIMPLE BINDING EQUILIBRIUM EXPLAINS THE REGULATED NUCLEOCYTOPLASMIC DISTRIBUTION OF ERK*

W. Richard Burack{ddagger} and Andrey S. Shaw

From the Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, August 31, 2004 , and in revised form, October 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In response to epidermal growth factor (EGF), the mitogen-activated protein kinase ERK2 translocates into the nucleus. To probe the mechanisms regulating the subcellular localization of ERK2, we used live cell imaging to examine the interaction between MEK1 and ERK2. Fluorescence resonance energy transfer (FRET) studies show that MEK1 and ERK2 directly interact and demonstrate that this interaction in the cytoplasm is largely responsible for cytoplasmic retention of ERK2. Stimulation with EGF caused loss of FRET as ERK separated from MEK and moved into the nucleus. FRET was recovered as ERK returned to the cytosol, indicating ERK reassociation with MEK in the cytoplasm. The EGF-induced transit of ERK through the nucleus was complete within 20 min, and there was no significant movement of MEK into the nucleus. Fluorescence recovery after photobleaching experiments was used to assess the rate of movement of MEK and ERK. The steady-state rate of ERK entry into the nucleus in resting cells was energy-independent and greater than the rate of ERK entry upon EGF stimulation. This suggests that the rate constant for ERK transport across the nuclear membrane is not limiting nuclear entry. Thus, we suggest that the movement of ERK into and out of the nucleus in response to agonist occurs primarily by diffusion and is controlled by interactions with binding partners in the cytosol and nucleus. No evidence of ERK dimerization was detected by FRET methods; the kinetics for nucleocytoplasmic transport were unaffected by mutations in the ERK putative dimerization domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The canonical mitogen-activated protein kinase, extracellular signal-regulated kinase 2 (ERK2),1 has physiologically critical targets in both the cytoplasm and nucleus (1, 2). Furthermore, the distribution of ERK2 between cytoplasm and nucleus and the duration of ERK2 residence in the nucleus are critical determinants of the signal mediated by mitogen-activated protein kinases (3–6). Whereas ERK2 is activated in the cytoplasm, it moves to the nucleus after it is phosphorylated by a mechanism, which has been reported to include at least two pathways (7–9). Both passive movement of a monomeric form and active transport of a dimeric form have been invoked to explain this nuclear influx (10). Disruption of the MEK/ERK interaction causes ERK to accumulate in the nucleus, suggesting that the MEK/ERK interaction maintains ERK in the cytoplasm (11).

Although some fraction of endogenous MEK and ERK must interact directly in vivo to allow for ERK activation, the affinity appears too low to be accurately determined by methods using recombinant proteins in vitro.2 A direct interaction is only inferred from the requirement to co-express MEK with ERK to localize ERK to the cytoplasm; overexpression of ERK alone results in nuclear localization of ERK (4).

Several models for how ERK2 nucleocytoplasmic distribution is regulated have been proposed (3, 4, 12). Differences among these models concern the energy dependence of the translocation and the potential role of MEK as an export shuttle factor or as a cytoplasmic "anchor." ERK2 interacts directly with the nuclear pore complex in the absence of importins and can be imported into the nucleus in the absence of ATP in permeabilized cell systems (8, 9). Other data have suggested that there is a specific role for active transport of a dimeric active form of ERK (7). However, it is unclear if dimerization is required for the rapid, agonist-induced entry of ERK into the nucleus. The exit of ERK from the nucleus has been less studied, but some experiments suggest that MEK may function as a nuclear export shuttle factor for ERK (11, 14, 15). Additionally, MEK and PEA-15 have been proposed to alter ERK localization simply by blocking regions of ERK required for interaction with the nuclear pore (5, 16). It has also been proposed that the steady state distribution of ERK between cytoplasm and nucleus is determined by the regulated expression of compartment-specific binding partners (17). Here we test these possibilities by using in vivo FRAP and FRET techniques to monitor the interactions and movements of ERK and MEK.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors—Nondimerizing A206K variants of GFP, CFP, and YFP were made by oligonucleotide-directed mutagenesis in the Clontech C1 vector (18). All fluorescent fusion proteins were made as fusions to the C terminus of GFP (or variants). Mouse ERK2 was cloned into the XhoI/BamHI sites of the MCS; coding sequence of ERK2 starts at alanine 2. The dimer-deficient form of ERK2 (dmERK) was the H176E L4A mutant (10). Mouse MEK1 was cloned into the HindIII/BamHI sites of the MCS; coding sequence of MEK1 starts at proline 2. Full-length MEK1 was expressed using pCDNA3.1. CFP and YFP fusions with MEK1 lacking the nuclear export sequence (deletion 32–44) were gifts from R. Lewis (University of Nebraska). Membrane-targeted CFP-MEK1 consists of the first 11 residues of murine Lck (MGCVCSSNPED) followed by a 5-amino acid linker (EPVAT) and then methionine 1 of CFP.

Cell Culture—All experiments were performed in transiently transfected HEK 293 cells. For microscopy, 105 cells were plated on acid-washed 2.5-cm coverslips on day 1, transfected with 2–4 µg of DNA on day 2 (Superfect; Qiagen), and used at 16–24 h. For imaging, cells were transferred to HEPES-buffered saline with 1% human serum albumin. For ATP depletion, the medium was supplemented with 10 mM sodium azide and 2 mM 2-deoxyglucose. EGF was applied by replacing the buffer with prewarmed buffer including 20 ng/ml EGF.

All live cell imaging was performed at 37 °C using a directly heated coverslip system (Bioptechs, Scranton, PA). Image analysis was performed using IPlab (Scanalytics). Scanning confocal microscopy was performed on a Zeiss LSM 510, and epifluorescence was performed on a Zeiss Axiovert 200 both with a 63 x 1.4 NA objective. For imaging FRET by epifluorescence, we used a photometrics PXL camera, a 150-watt xenon arc lamp, automated filter wheels (providing specific filters for CFP and YFP excitation and emission), and a single multiband dichroic similar to Chroma set 86002v2 (Rockingham, VT). There was minimal cross-talk of CFP to the YFP channel. Three images were acquired sequentially (EX CFP/EM CFP; EX CFP/EM YFP; EX YFP/EM YFP). The normalized FRET was calculated from these images; FRET images are shown as an "intensity-modulated display," where the grayscale corresponds to the concentration of acceptor and the color scale corresponds to the normalized FRET (19).

For quantification of FRET by "donor enhancement with acceptor bleaching," YFP was bleached with a 514-nm line of a krypton/argon laser for both confocal and epifluorescence studies. Similar results were obtained on both instruments. CFP was excitation at 458 for confocal images and with a 435 ± 15-nm filter for epifluorescence.

For FRAP studies of movement within the cytoplasm or nucleus, YFP was bleached with both the 488-nm and 514-nm lines simultaneously at full power applied to a 1 x 1-µm square defined to contain 4 x 4 pixels. This region was bleached for <3 s, and then the same region was imaged at less than 1% of the bleach power. The image acquisition rate approaches 1 kHz. Approximate diffusion constants were calculated as if the system were two dimensional and the bleach region were circular. These calculated diffusion constants were within the range considered typical of cytoplasmic proteins (1–3 x 108 cm2/s).

The FRAP studies of movement across the nuclear membrane were performed on both the scanning confocal and epifluorescence microscopes and gave similar results. On the scanning confocal, the nucleus was outlined and bleached in a manner similar to that described above except that the region bleached was much larger and generally required ~30 s of repeated scanning to get at least a 50% reduction in nuclear intensity. Images of the entire cell taken immediately following the bleach showed that the nucleus had a homogenous intensity; the nuclear membrane was clearly defined as the abrupt boundary between the relatively dim nucleus and the relatively bright cytoplasm. To prove that the recovery of fluorescence corresponds to movement across the nuclear membrane rather than movement between image planes within the nucleus, we performed controls in which less than one-fourth of the nucleus is bleached for 30 s; images of the entire cell taken immediately following the bleach showed that the nucleus had a homogenous intensity; again the nuclear membrane was clearly defined as the abrupt boundary between the relatively dim nucleus and the relatively bright cytoplasm. This control confirms that the fluorescent proteins move within a single compartment much faster than they cross the nuclear membrane; this result considerably simplifies the analysis. In the FRAP studies of movement across the nuclear membrane performed by epifluorescence, the entire nucleus was bleached with a short (<3-s) exposure to the 514 line of a 2-milliwatt krypton/argon laser. The beam shape was roughly Gaussian with a full width at half-height corresponding approximately to the width of the nucleus. Again, control experiments showed that movement within the nucleus and cytoplasm was rapid compared with movement across the membrane. Therefore, FRAP measurements could be obtained regardless of exactly where in the cell the beam was focused, provided that bleaching intensity was greater in the nucleus than the cytoplasm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Targeting of MEK to the Plasma Membrane Directs ERK to the Plasma Membrane—The requirement for MEK overexpression to maintain overexpressed ERK in the cytoplasm is well described and reproduced in Fig. 1, A and B. In the absence of MEK overexpression, ERK localizes primarily to the nucleus (Fig. 1A). Co-expression with MEK results in ERK localization in the cytoplasm. To determine whether the localization of ERK is controlled directly by MEK, a mutated form of MEK was generated that targets MEK to the plasma membrane. The membrane-targeted form of MEK was made by fusing sequences encoding the myristoylation and palmitoylation sequences of the Src family tyrosine kinase, LCK, to the N terminus of YFP or CFP, which was in turn fused to the amino terminus of MEK-1. When expressed in HEK 293 cells, this protein was localized at the plasma membrane (Fig. 1C). Coexpressed YFP-ERK2 was also strongly localized to the plasma membrane (Fig. 1D).



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  		FIG. 1.
Membrane-targeted MEK1 recruits YFP-ERK2 to the plasma membrane. HEK 293 cells were transfected with vectors encoding YFP-ERK (A; YFP image), YFP-ERK and MEK (B; YFP image), or simultaneously with LCK-CFP-MEK (C; CFP-image) and YFP-ERK (D; YFP image). Confocal images were acquired from live cells at ~16 h after transfection.

 
ERK and MEK Directly Interact in the Cytoplasm—A fluorescence resonance energy transfer (FRET) assay was used to determine whether MEK and ERK might directly associate. FRET requires the two fluorophores to be in close proximity, within ~4 nm of one another. YFP-ERK was co-expressed with CFP-MEK, and FRET was quantified by measuring the enhancement of donor (CFP) fluorescence upon photobleaching of the acceptor (YFP). FRET was detectable with wild type MEK (Fig. 2, column 1) and a mutated form of MEK lacking the nuclear export sequence, {Delta}NES-MEK (Fig. 2, column 3). Whereas the fluorescence intensity of ERK in the nucleus and cytoplasm varied with the localization of MEK (>80% cytoplasmic for wild type and >80% nuclear for {Delta}NES-MEK), the FRET was not significantly different in these two compartments (data not shown). A direct interaction at the plasma membrane of YFP-ERK with the Lck-CFP-MEK was also easily detectable (Fig. 2, columns 4 and 5). Importantly, no significant FRET signal was detected when the unfused fluorescent proteins were overexpressed together or when YFP was overexpressed with CFP-ERK (and vice versa) (Fig. 2, columns 2 and 5).



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  		FIG. 2.
MEK and ERK directly interact in unstimulated live cells. CFP-ERK (A) or Lck-CFP-MEK (B) are the FRET donors. FRET was detected by the enhancement of donor fluorescence upon bleaching YFP in the entire cell. In the presence of YFP-MEK, the CFP-ERK was largely (but not exclusively) in the cytoplasm. In the presence of YFP, the CFP-ERK was distributed between the nucleus and cytoplasm. In the presence of YFP-{delta} NES-MEK, the CFP-ERK was almost entirely within the nucleus. The percentage change in donor fluorescence and the percentage of acceptor bleach are shown with S.D. values. p values were calculated using a two-tailed Student's t test.

 
Since ERK moves from cytoplasm to nucleus in response to agonist, FRET measurements were used to assess the temporal relationship between dissociation of MEK and ERK and the movement of ERK into the nucleus. After application of EGF, there was a short lag period followed by an abrupt movement of ERK into the nucleus and a corresponding loss of FRET. The central panel in Fig. 3 shows a field of HEK 293 cells treated with EGF in which the majority of cells show nuclear accumulation of ERK. There was no significant movement of MEK into the nucleus in response to agonist (Fig. 3, top panels). Therefore, the loss of FRET signal suggests that MEK·ERK complexes dissociate before ERK enters the nucleus and that ERK is not associated with MEK in the nucleus. By 600 s after application of EGF, ERK returned to the cytoplasm, and its localization in the cytoplasm was accompanied by increased FRET. The time course of fluorescence and FRET is shown for a representative single cell in Fig. 4. The duration of the nuclear flux was similar to that described in a prior live cell imaging study of GFP-ERK fluxes in PC12 and NIH3T3 cells (20).



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  		FIG. 3.
MEK and ERK dissociate with EGF stimulation and promptly reassociate when ERK returns from the nucleus to the cytoplasm. HEK 293 cells were transfected with CFP-MEK and YFP-ERK at ~16 h while maintained at 37 °C, and 20 ng/ml EGF was applied. At 90 s after the addition of EGF, the YFP-ERK is within the nucleus (the arrowhead marks the nucleus; see central panel) and returns to the cytoplasm by 600 s after EGF. The CFP-MEK remains in the cytoplasm without significant nuclear fluorescence. FRET is markedly decreased at 90 s and returns to the basal state by 600 s. FRET is shown as an intensity-modulated display in which the normalized FRET represented by an arbitrary color scale and the brightness is directly proportional to the total acceptor intensity when excited directly at 488 nm.

 



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  		FIG. 4.
Time course of FRET and CFP quenching in HEK 293 cells expressing CFP-MEK and YFP-ERK in response to EGF. Cells were prepared as in Fig. 3. There is a drop in the normalized FRET calculated for the entire cell starting at about 60 s after the addition of EGF. Simultaneously, the total CFP fluorescence increased, indicating dequenching of the donor.

 
The Association of YFP-ERK with MEK in the Cytoplasm Does Not Decrease the Mobility of YFP-ERK—The mobility of MEK and MEK·ERK complexes within the cytoplasm were studied by FRAP techniques. A small region of the cell (~1 µm2) was bleached, and the recovery was measured in the millisecond time domain. The mobilities of YFP-MEK and YFP-ERK were indistinguishable from the mobility of YFP (Fig. 5A). Therefore, overexpressed YFP-MEK and YFP-ERK appears to diffuse freely in the cytoplasm. When YFP-ERK was overexpressed in the presence of MEK, such that YFP-ERK was retained in the cytoplasm, the mobility of YFP-ERK was again indistinguishable from that of YFP or of YFP-MEK alone. As a control, FRAP studies of the membrane-tethered form of MEK indicated a slow recovery consistent with a membrane-associated molecule that must diffuse within a relatively more viscous medium (single curve shifted to the right in Fig. 5, A and B). YFP-ERK mobility within the nucleus was also indistinguishable from that of YFP alone. These results suggest that MEK and ERK are freely mobile in the cell and are not associated with static cellular structures.



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  		FIG. 5.
CFP-MEK and YFP-ERK diffuse freely in the cytoplasm as assessed by fluorescence recovery after photobleaching. HEK 293 cells were transfected with YFP, Lck-YFP-MEK, YFP-MEK, or untagged MEK and YFP-ERK. At 16 h, the cells showed the expected cytoplasmic expression of the YFP-MEK and the expected cytoplasmic distribution of the YFP-ERK in the presence of overexpressed MEK. A 1-µm2 region was photobleached using the 488-nm line. The recovery of fluorescence in this small region was measured at a maximum rate of 1 kHz. A shows the best fits with 95% confidence limits. The mobilities of YFP-MEK and YFP-ERK (in the presence of MEK) are indistinguishable from the mobility of YFP. The mobility of the membrane-targeted form of MEK is shown as the single curve to the right (with 95% confidence limits). B shows the same curves with a small fraction of the data points (less than 5%).

 
In the Absence of MEK, the Accumulation of YFP-ERK in the Nucleus Is Energy-independent—When GFP alone was overexpressed in HEK 293 cells, quantitative confocal imaging indicated that the concentrations of GFP in the nucleus and cytoplasm were approximately equal (data not shown). (The apparent excess of GFP in the nucleus seen in wide field images is probably due to the greater thickness of cells in the region of the nucleus.) In contrast, overexpressed YFP-ERK preferentially accumulated in the nucleus to about 1.5–2 times the concentration in the cytoplasm. Treatment with azide and 2-deoxyglucose to deplete ATP did not affect the localization of YFP-ERK in the nucleus (Fig. 6, bottom). The ATP independence indicates that energy is not required for nuclear localization of YFP-ERK or that both import and export rates are proportionally affected by the ATP depletion.



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  		FIG. 6.
The steady state distribution of YFP-ERK between cytoplasm and nucleus is energy independent. HEK 293 cells were transfected with YFP-ERK and CFP-MEK. At 16 h, the cells showed the expected cytoplasmic expression of the YFP-MEK and the expected cytoplasmic distribution of the YFP-ERK in the presence of overexpressed MEK. The cells were imaged before and after the addition of a medium containing 10 mM azide and 5 mM 2-deoxyglucose. Only YFP-ERK data for four cells are shown. (Each cell is represented by a distinct symbol.) Greater then 20 additional cells were also scored and were similar. The data for CFP-MEK were essentially identical. The upper panel shows that a 30-min incubation in 10 mM azide plus 5 mM 2-deoxyglucose is sufficient to entirely ablate EGF-induced activation of ERK, indicating that the ATP depletion is significant. The specimens were assessed using an anti-phospho-ERK antibody and total ERK (p42 and p44 are not resolved).

 
The Rates of YFP-ERK Movement into and out of the Nucleus Are Similar to the Rates for YFP and Are Unaffected by ATP Depletion—FRAP measurements were used to measure the rates of transport of YFP-ERK into the nucleus before and after ATP depletion. In these experiments, since the entire nucleus was bleached, recovery represents the movement of cytoplasmic YFP-ERK across the nuclear membrane. The rates of recovery of nuclear fluorescence for YFP and YFP-ERK were indistinguishable (Fig. 7A). This suggests that the nuclear import of ERK is not mediated by a specific import mechanism and may be energy-independent. These data are also consistent with previous work using permeabilized cells suggesting that ERK import into the nucleus occurs by facilitated diffusion.



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  		FIG. 7.
YFP-ERK movement across the plasma membrane is energy-independent and occurs at a rate indistinguishable from YFP. Cells were prepared as in previous figures. FRAP was determined after bleaching the entire nucleus of cells with either overexpressed YFP (triangles) or YFP-ERK (circles). A, YFP recovery was indistinguishable from YFP-ERK recovery. The extent of bleach was similar in both cases. B, the rate of YFP-ERK translocation in individual cells before and after energy depletion was not distinguishable. Data for three cells are shown. Nuclear FRAP was performed on each cell, and recovery was recorded until essentially complete (~5 min). The medium was then changed to contain 10 mM azide plus 2 mM 2-deoxyglucose, and the cells were incubated for 45 min. Nuclear FRAP measurements were then repeated. Filled symbols, before ATP depletion; open symbols, after ATP depletion. Similar results were obtained with 10 additional cells.

 
To test whether ERK movement across the membrane is energy-independent, we performed nuclear FRAP on individual cells before and depletion of ATP. Whereas ATP depletion did not alter the steady state distribution of YFP-ERK between nucleus and cytoplasm (Fig. 6, graph), the rate of transport was also unaffected by ATP depletion (Fig. 7B). Since the steady-state distribution and the nuclear entry rates were unaffected by ATP depletion, we infer that the nuclear export rate of ERK was also unaffected by ATP depletion.

Traversing the Nuclear Membrane Is Not the Rate-limiting Step for Agonist-induced Nuclear Accumulation of YFP-ERK— The bulk rates of YFP-ERK entry into the nucleus for cells either at steady-state (resting) or equilibrium (the rate observed after energy depletion) were much greater than that induced by EGF. For cells with both overexpressed MEK and YFP-ERK, the maximum rate of YFP-ERK entry into the nucleus after the addition of EGF was determined by the maximum change in nuclear fluorescence intensity between any two time-consecutive time points, which occurred about 120 s after the addition of EGF. (To qualitatively compare this maximum rate to the rates seen for steady-state, non-agonist-induced flux of ERK into the nucleus, cells expressing YFP-ERK only were selected, which had a cytoplasmic intensity less than half of that seen in matched cells co-expressing MEK and YFP-ERK. This selection is necessary to assure that a more rapid kinetics of nuclear entry is not simply the trivial result of a larger pool of available YFP-ERK. Cells with this relatively low cytoplasmic concentration of YFP-ERK were easily found, because overexpressed YFP-ERK accumulated in the nucleus, not the cytoplasm, as shown in Fig. 1, A and B). For the steady state or equilibrium state (after ATP depletion), the maximum bulk rate of YFP-ERK movement into the nucleus was determined by photobleaching the nucleus and measuring the recovery at short time intervals. Invariably, this rate of increase of total nuclear fluorescence determined by FRAP of steady-state (or equilibrium) cells overexpressing YFP-ERK was severalfold higher than the maximum rates we ever recorded for YFP-ERK accumulation in the nucleus in response to agonist in cells overexpressing both MEK and YFP-ERK.

ERK Dimerization Is Not Required for the Rapid Influx of ERK into the Nucleus in Response to Agonist—It has been suggested that ERK dimerization is required for energy-dependent entry into the nucleus. We were therefore interested to test whether mutated forms of ERK unable to dimerize were impaired in their ability to transport to the nucleus in response to agonist. A mutated form of YFP-ERK (dmERK) with mutations in the putative dimer interface of ERK, expressed in the absence of MEK, distributed to the nucleus in a manner indistinguishable from that for YFP-ERK. The rate of diffusion within the cytoplasm (as measured by cytoplasmic FRAP of 1 µm2) and the movement across the plasma membrane (as measured by nuclear FRAP) were both unchanged. When overexpressed with MEK in HEK 293 cells, YFP-dmERK was retained in the cytoplasm in a manner indistinguishable from that for YFP-ERK, nor was any defect in the rate of ERK movement into the nucleus in response to EGF seen.

Because these studies required comparisons between cells and therefore might miss subtle effects of ERK dimerization on the rates of ERK translocation into the nucleus, we measured the movement of both wild type ERK and dmERK co-expressed in the same cells. Again, we found no difference in the rate of CFP-wild type ERK and YFP-dmERK translocation into the nucleus in response to EGF (Fig. 8). There was no reproducible or significant difference in the lag time to entry, the fraction of the two forms that entered the nucleus, or the duration of nuclear localization. This suggests that dimerization of ERK is not required for nuclear entry or exit.



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  		FIG. 8.
ERK dimerization is dispensable for the rapid translocation of ERK into the nucleus. HEK 293 cells were transfected with pcDNA expressing MEK and wild type YFP-wild type ERK, or CFP-wild type ERK (filled) and YFP-dimer mutant ERK (open). CFP and YFP were imaged simultaneously before and after the addition of EGF.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrated that ERK and MEK directly interact and that this interaction is largely responsible for cytoplasmic localization of overexpressed ERK in resting cells. Measuring the lateral mobility of ERK bound or unbound to MEK showed high mobility, suggesting that MEK does not restrict ERK to any specific region of the cytoplasm, nor does it "anchor" ERK to a cytoplasmic structure. We also found that the rate of ERK translocation into the nucleus was similar to the rate of translocation of GFP, consistent with data suggesting that ERK is not actively transported into the nucleus. Confirming this, energy depletion did not change the steady state distribution of ERK, nor did it affect the rate of nuclear translocation. These findings suggest that a simple model in which MEK functions as a buffer can explain the localization and nuclear translocation of ERK during signal transduction.

This simple model assumes that association with MEK (and other ERK ligands) reduces the concentration of "free" ERK, and only "free" ERK (but not the MEK-ERK complex) can cross the nuclear membrane. Translocation would be initiated by release of ERK from MEK (increasing free ERK in the cytoplasm), followed by facilitated or simple diffusion of ERK into the nucleus, and then binding of ERK to targets in the nucleus. Nuclear versus cytoplasmic distribution could be controlled by the number and affinity of binding partners in each compartment. The accumulation of overexpressed GFP-ERK in the nucleus compared with the cytoplasm suggests that there is a relative abundance of ERK binding targets in the nucleus compared with the cytoplasm. We hypothesize that the phosphorylation state of MEK and ERK controls the affinity of the complex in the cytoplasm. Whereas the major binding partner of ERK in the nucleus is not known, it has been proposed that dimeric active ERK can bind an abundant class of dimeric transcription factors (10). In our model, movement of ERK back into the cytoplasm can be explained by dephosphorylation of ERK and reacquisition of affinity for MEK. It is also possible that ERK dephosphorylation regulates its affinity for targets in the nucleus.

The presence of a canonical NES in MEK has suggested that MEK might serve as an export shuttle for ERK (11, 14). This suggestion is partially based on experiments using Leptomycin B treatment, which causes nuclear accumulation of both MEK and ERK (14, 21). For several reasons, we do not think that it is likely that MEK serves as an export shuttle for ERK. First, we did not detect significant amounts of GFP-MEK in the nucleus in resting cells, nor did we see an increase in nuclear MEK in agonist-stimulated cells. We also did not detect FRET between MEK and ERK in the nucleus. Second, plasma membrane-targeted MEK, which presumably cannot rapidly traffic through the nucleus, was sufficient to localize ERK in the cytoplasm. Third, since NES-dependent export requires energy, we might have expected MEK to accumulate in the nucleus after energy depletion, but there was no change in GFP-MEK localization after energy depletion. MEK has been previously shown to accumulate in the nucleus with progression from G2 to M phase (22). Therefore, the MEK NES may have functions that are independent of its association with ERK.

The FRAP data suggest that MEK does not function to "anchor" ERK to a specific immobile structure in the cell. It is possible that MEK could link ERK to scaffolds like KSR (kinase supressor of Ras) in the cytoplasm and that overexpressed MEK has saturated binding to scaffolds. Alternatively, it is possible that the scaffolds are freely mobile. Regardless, the interaction of ERK with freely mobile MEK in the cytoplasm is sufficient to account for the cytoplasmic localization of ERK.

Although we found no evidence of FRET between molecules of ERK, the crystal structure of the doubly phosphorylated active form of ERK, but not the dephosphorylated form, suggests that active ERK forms dimers (10, 13). It has been proposed that ERK dimers interact with dimeric transcription factors, causing retention of active ERK in the nucleus (10). Others have suggested that activated ERK forms dimers in the cytoplasm, and it is the dimeric ERK that is then actively transported into the nucleus (7). In our studies, we failed to detect FRET between YFP- and CFP-tagged ERK upon activation with EGF or when coexpressed with a constitutively active form of MEK (data not shown). Similar kinetics of EGF-induced nuclear entry for both wild type and a dimer interface mutant suggest that dimerization is not necessary for nuclear import. We cannot rule out, however, the possibility that dimeric ERK is required for interaction with substrates in the nucleus.

Our model suggests that the bulk rate of ERK entry into the nucleus increases only because the amount of free ERK increases upon agonist-induced dissociation from MEK or other ligands. Whereas enhancing the rate constant for facilitated diffusion (or an increased number of pores) could produce the kinds of data seen in our experiments, it is not necessary to invoke this additional mechanism. Because the rate of ERK entry into the nucleus in resting cells was severalfold higher than the rate of ERK entry in response to agonist, the rate constant for nuclear import was not limiting ERK entry into the nucleus. Therefore, the release of ERK from the ERK/MEK complex is probably rate-limiting for ERK translocation.

The relatively complex movement of ERK into and out of the nucleus in response to an agonist can be substantially explained by this simple model, which assumes that the affinity of ERK for MEK changes with the phosphorylation state of one or both enzymes. The model assumes the existence of an abundant nuclear binding site for ERK. Furthermore, the model leaves open a role for modulation of the affinity of these nuclear binding sites by changes in phosphorylation of ERK or the binding sites themselves. Ongoing experiments address the role of kinase activity and phosphorylation state in the context of this model.


   FOOTNOTES
 
* This work was supported by NCI, National Institutes of Health, Grant RO1-CA102441. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. E-mail: rburack{at}path.wustl.edu.

1 The abbreviations used are: ERK2, extracellular signal-regulated kinase 2; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; FRAP, fluorescence recovery after photo-bleaching; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; EGF, epidermal growth factor; NES, nuclear export sequence; dmERK, dimer mutant ERK. Back

2 S. Bruck and A. Shaw, unpublished results. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Charles Spencer, David van Nostrand, and Sumita Aurora.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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