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First published online January 21, 2009
doi: 10.1242/10.1242/jcs.036319

Journal of Cell Science 122, 425-435 (2009)
Published by The Company of Biologists 2009
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Positive- and negative-feedback regulations coordinate the dynamic behavior of the Ras-Raf-MEK-ERK signal transduction pathway

Sung-Young Shin1,*, Oliver Rath2,*, Sang-Mok Choo3, Frances Fee2, Brian McFerran2,{ddagger}, Walter Kolch2,4,§ and Kwang-Hyun Cho1,§

1 Department of Bio and Brain Engineering and KI for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
2 Beatson Institute for Cancer Research, Cancer Research UK, Glasgow, UK
3 School of Electrical Engineering, University of Ulsan, Ulsan, Korea
4 Institute of Biomedical and Life Science, University of Glasgow, Glasgow, UK


Figure 1
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  		Fig. 1. The Ras-Raf-MEK-ERK signaling pathway and its feedback regulation mechanism. Activated ERK (ppERK) triggers two major feedback loops: the positive-feedback loop resulting from inactivation of the inhibitory protein RKIP and the negative-feedback loop formed by inactivation of the Ras activating exchange factor complex Grb2-SOS.

 

Figure 2
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  		Fig. 2. ERK phosphorylates RKIP generating a positive-feedback loop. (A) RKIP is a substrate for ERK. Recombinantly produced purified RKIP protein was phosphorylated in vitro with purified Raf-1, MEK and ERK proteins exactly as described previously (Yeung et al., 2000Go). R, R*, M and E represent Raf-1, activated Raf-1, MEK and ERK, respectively. RKIP is phosphorylated only when ERK is present in the reaction. When ERK is activated by preincubation with activated Raf-1 and MEK (R*+M+E) RKIP phosphorylation is enhanced. (B) S99 of RKIP is an ERK phosphorylation site. Wild-type RKIP and the indicated mutants were expressed in E. coli, purified and phosphorylated by activated ERK in vitro. Mutation of S99 substantially reduces the phosphorylation of RKIP by ERK. (C) RKIP inhibitory function is inhibited by ERK phosphorylation generating a positive-feedback loop. Recombinant purified proteins were used to reconstitute the Raf-1–MEK–ERK phosphorylation cascade in vitro as described in the Materials and Methods section. MEK phosphorylation in the presence of increasing amounts of recombinant RKIP was assayed by ppMEK antibodies. `-feedback' indicates that MEK phosphorylation by Raf-1 was assayed in a reaction containing Raf-1, MEK and increasing amounts of RKIP. The `+feedback' condition in addition contained ERK which permits RKIP inactivation by ERK. The experiment is representative of three repeats and a quantification is shown on the right. The blue line represents MEK phosphorylation in the absence of ERK, i.e. the absence of feedback inhibition of RKIP. The red line indicates MEK phosphorylation in the presence of ERK where feedback is enabled. (D) Phosphorylated RKIP does not bind to Raf-1. Recombinant RKIP was phosphorylated by ERK in the presence of [{gamma}-32P]ATP as in B. Increasing amounts of RKIP (1.1 µg, 3.3 µg, 10 µg) were incubated with equal amounts of GST or GST–Raf-1 immobilized on glutathione-Sepharose beads. After washing the binding assay was separated by SDS-PAGE and bound RKIP was detected by immunoblotting, and phosphorylated RKIP was detected by autoradiography of the immunoblots. `Input' contains 10% of the RKIP used in the binding assay.

 

Figure 3
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  		Fig. 3. Oscillations of ERK activity. (A) ERK activation profile in COS-1 cells in response to TPA treatment. Serum starved COS-1 cells were treated with 100 ng/ml TPA for the indicated time points. Cell lysates were immunoblotted with ppERK followed by ERK antibodies. Lower panel, western blots of two independent representative experiments. Upper panel, the blots were quantified by laser densitometry, ppERK was corrected for ERK loading, and plotted. (B) Comparison of biochemically measured with predicted ERK activation. The biochemically measured ERK activities from panel A are shown as red rectangles with the error bars corresponding to the s.d. from two independent experiments. The measured ERK activity was scaled down for comparison with the simulation data. The ERK activation profile predicted by the PSRA parameter estimation method is shown as dashed blue line obtained from a nominal parameter values (see Table 2). Blue circles with error bars denote in silico simulation data obtained from repeated simulations (n=50) over a 30% random variation of parameters.

 

Figure 4
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  		Fig. 4. Dynamic analysis of the positive- and negative-feedback mechanisms by computational simulation. `No positive feedback' indicates that the positive-feedback loop has been removed by inhibiting ERK mediated RKIP phosphorylation. `No negative feedback' indicates that the negative feedback loop has been removed by inhibiting ERK mediating SOS phosphorylation. `RKIP removal' indicates that RKIP has been removed completely, eliminating both the positive feedback and the inhibitory action of RKIP. The control had none of the feedback loops or RKIP removed. (A) Simulations for persistent stimulations (300 minutes). (B) Simulations for transient stimulations (30 minutes).

 

Figure 5
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  		Fig. 5. RKIP regulates the oscillatory behavior of cellular responses. The oscillatory behaviors of ppMEK (A) and ppERK (B) are suppressed along with the increase of RKIP concentration. The oscillation amplitude of activated Raf-1 levels (C) is increased whereas the oscillatory pattern vanishes as RKIP concentration increases.

 

Figure 6
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  		Fig. 6. RKIP induces a switch-like behavior of MEK activity. The mathematical model was modified such that Raf-1 activation could be controlled independent of upstream inputs. (A) Under conditions of constant Raf-1 activity achieved by simulating the expression of the constitutively active Raf{Delta}N the levels of ppMEK decreased sigmoidally as RKIP increased. (B) The steady-state response curves of ppMEK. The switch-like behavior in response to a gradual increase of RKIP levels disappeared when the positive-feedback loop was eliminated.

 

Figure 7
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  		Fig. 7. Experimental verification of the switch-like behavior of phosphorylated MEK. (A) COS-1 cells were transfected with a constant amount of Raf{Delta}N in order to activate MEK and different amounts of RKIP plasmid as indicated in order to generate a linear increase in RKIP protein levels. Cells were lysed 2 days after transfection, and steady-state MEK activation was assayed by western blotting with phosphospecific MEK antibodies. Total levels of MEK and RKIP proteins were also assayed by western blotting. Quantification of blots by laser densitrometry is shown below as normalized scan units. (B) LS174T colon cancer cell lines with a doxycycline (Dox)-regulatable Flag-RKIP or Flag-RKIP S99A expression system were induced with the indicated amounts of Dox to achieve a graded overexpression of RKIP. As a result of the Flag-tag, the induced RKIP migrates just above the endogenous RKIP band. Cells were stimulated with 10 ng/ml TPA for 15 minutes, ppMEK, MEK and RKIP levels were assayed by immunoblotting (upper panels) and quantified by laser densitometry (lower panels; expression is given as normalized scan units on a logarithmic scale). The experiment was repeated twice with consistent results.

 

Figure 8
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  		Fig. 8. RKIP determines the dynamics of phosphorylated ERK depending on stimulation strength. (A) At low RKIP concentrations ERK activity exhibits damped oscillations. (B) At a high RKIP concentration, total initial ppERK levels are significantly suppressed and the oscillations become dampened compared with those observed under conditions of low RKIP expression.

 

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© The Company of Biologists Ltd 2009