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. 2000 May 23;97(11):5818-23.
doi: 10.1073/pnas.97.11.5818.

Scaffold proteins may biphasically affect the levels of mitogen-activated protein kinase signaling and reduce its threshold properties

A Levchenko  1 , J Bruck, P W Sternberg
Affiliations

Scaffold proteins may biphasically affect the levels of mitogen-activated protein kinase signaling and reduce its threshold properties

A Levchenko et al. Proc Natl Acad Sci U S A. .

Abstract

In addition to preventing crosstalk among related signaling pathways, scaffold proteins might facilitate signal transduction by preforming multimolecular complexes that can be rapidly activated by incoming signal. In many cases, such as mitogen-activated protein kinase (MAPK) cascades, scaffold proteins are necessary for full activation of a signaling pathway. To date, however, no detailed biochemical model of scaffold action has been suggested. Here we describe a quantitative computer model of MAPK cascade with a generic scaffold protein. Analysis of this model reveals that formation of scaffold-kinase complexes can be used effectively to regulate the specificity, efficiency, and amplitude of signal propagation. In particular, for any generic scaffold there exists a concentration value optimal for signal amplitude. The location of the optimum is determined by the concentrations of the kinases rather than their binding constants and in this way is scaffold independent. This effect and the alteration of threshold properties of the signal propagation at high scaffold concentrations might alter local signaling properties at different subcellular compartments. Different scaffold levels and types might then confer specialized properties to tune evolutionarily conserved signaling modules to specific cellular contexts.

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Figures

Figure 1
Figure 1
Various kinase–scaffold combinations (scaffold species) Ci postulated by the model and transitions among them. Transitions by kinase association or dissociation (magenta arrows; only transitions for C1 are shown for clarity) and intrascaffold reactions (red arrows) are allowed in the model. P designates phosphorylation of a kinase.
Figure 2
Figure 2
The role of scaffold proteins in kinetics and input–output sensitivity of MAPK activation. (A) Kinetics of MAPK activation as a function of a two-member scaffold concentration. The scaffold concentrations considered are indicated in the figure. The dashed line represents the kinetics of unscaffolded reaction. (B) Dependence of MAPK activation on the level of signaling. For each scaffold concentration, the kinetics of MAPK activation was computed as in A, and integral over the first 100 sec was plotted as a function of the signal. The level of the signal corresponds to the concentration in micromolars of a MAPKKK activator. The dashed curve represents unscaffolded reaction. (C) Input–output relationships for various scenarios of reactions in the scaffold. The processive (all values are divided by four), intermediate, and distributive (multiplied by 108) reactions are considered (see details in the text). MAPK activation with no scaffold is presented by the dashed curve for comparison.
Figure 3
Figure 3
Existence of an optimal scaffold concentration. (A) Dependence of MAPK activation (red) and functional scaffold–kinase complexes C6 (blue) and C8 (dashed magenta) on the two-member scaffold concentration. MAPK activation at 100-fold lower MAPKK phosphatase concentration is shown in light blue. C6 is normalized by dividing by 40 and C8 by multiplication by 17 for better comparison and illustration purposes. The results are compared with the plot of 0.15/[Scaffold] (green). (B) The optimum of MAPK activation or C6 formation is not sensitive to the dissociation constant KD of MAPK–scaffold interaction. The lighter areas in the contour plots correspond to higher MAPK or C6 concentrations. (C and D) The optimal scaffold concentration (two-member scaffold) is a function of MAPKK (C) and MAPK (D). Both three-dimensional and contour plots of MAPK activation are shown.
Figure 4
Figure 4
The scaffold membership and cooperativity of MAPKK and MAPK binding to scaffold affect MAPK activation. (A) Comparison of the effects of two- (red) and three- (blue) member scaffolds on the signaling properties. Assumptions of the three-member scaffold model are the same as the two-member model considered above with the additional assumption of MAPKKK binding to the scaffold. The dashed line illustrates that it takes considerably more two-member than three-member scaffolds to inhibit activity to the same level at high scaffold concentrations. (B) Cooperativity does not affect the property of existence of an optimal scaffold concentration but results in a shift of the optimum. No cooperativity (red), partial positive (green), and partial negative (blue) cooperativity are shown. In partial cooperativity, only inactive kinases interact. The dashed lines of the corresponding color represent C6 normalized as in Fig. 3. (C) Full (red) and partial (green) positive cooperation is compared with noncooperative binding (blue). Inset illustrates the concomitant dependence of C3 (see Fig. 1). In all figures, cooperativity is defined as an increase or decrease in the second kinase association constant by the factor of 10 when the first kinase is bound. See more information in the text.
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