Review

Scaffold Proteins: Hubs for Controlling the Flow of Cellular Information

Science  06 May 2011:
Vol. 332, Issue 6030, pp. 680-686
DOI: 10.1126/science.1198701

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  1. Fig. 1

    Scaffold proteins organize cellular information flow. (A) Spatial organization is necessary to achieve high-fidelity intracellular information transfer. Proteins can be assembled into specific complexes by compartmentalization (organelle targeting), by membrane localization, and by scaffold proteins. (B) Intracellular signaling pathways often use scaffold proteins. Canonical examples include Ste5, essential to the yeast mating MAPK pathway, and KSR, which directs signaling in the mammalian Ras-Raf-MEK-MAPK pathway. (C) Scaffold proteins also play an important role in organizing cell-cell communication junctions, such as neuronal synapses. The PDZ scaffold, PSD-95, controls NMDA and AMPA glutamate receptor targeting to the synapse. (D) Assembly-line processes such as protein folding use scaffold proteins. The HOP protein promotes transfer of unfolded proteins between Hsp70 and Hsp90 chaperones.

  2. Box 1.

    Structure and mechanisms of a canonical scaffold: the MAPK scaffold protein Ste5. (A) The Ste5 scaffold protein is composed of modular interaction domains, some of which mediate essential steps in the three-tiered mating MAPK signaling cascade, and some of which function in higher-order regulatory behaviors. (B) Core steps of mating pathway (see also movie S1): Binding of α-factor peptide to its receptor (Ste2) leads to activation of the guanine nucleotide binding protein (G protein) and dissociation of Gβγ (Ste4 and Ste18) from the Gα subunit (Gpa1). The Ste5 RING domain binds to the free Gβγ complex (54, 55), triggering rapid recruitment of the scaffold to the membrane. Stabilization and discrete localization of Ste5 at the plasma membrane also require the interaction of its PM domain (an amphipathic helix) (14, 25) and a cryptic pleckstrin homology (PH) domain with the lipid bilayer or anchored phosphoinositides (13). A region on Ste5 that overlaps with the PH domain binds to the MAPKKK Ste11 (5, 56) and, upon pathway activation, colocalizes the MAPKKK Ste11 with its activator, Ste20 [a MAPKKKK, similar to the p21-activated kinase (PAK)], which is localized to the membrane in a preactivated state. Phosphorylation of the MAPKKK Ste11 by Ste20 initiates the MAPK cascade. The MAPKK Ste7 is assembled into the mating signaling complex through the VWA domain of Ste5 (PDB ID 3FZE), and can be efficiently phosphorylated by the colocalized and activated MAPKKK Ste11. The minimal VWA also functions as a coactivator that permits Ste7 activation of the MAPK Fus3, which is tethered to Ste7 via docking motifs (PDB ID 2B9H) (18, 57) (see Fig. 4C). (C) The Ste5 scaffold also takes part in higher-order regulatory processes. Phosphorylation of the PM helix by Cdk blocks Ste5 membrane binding (25), thus preventing activation of the mating response at specific stages of the cell cycle. The Fus3-binding domain (Fus3-BD; PDB ID 2F49) appears to be important for regulatory feedback phosphorylation of Ste5 by Fus3, rather than for core signal transmission through the MAPK cascade (27). Phosphorylation of at least four sites on the Ste5 scaffold is dependent on recruitment of Fus3 though the Fus3-BD. These regulatory phosphorylation sites on the scaffold inhibit pathway activity and are thought to help shape the ultrasensitive cell morphology response (shmooing) that occurs during mating. Mutation of the Fus3-BD does not prevent mating but rather leads to a much more graded shmooing response when stimulated by α-factor (27, 28, 39). Thus, this regulatory interaction may shape this switch-like cell-fate decision. Structures not denoted by PDB numbers in (B) and (C) were created using homology models.

  3. Fig. 2

    Scaffold proteins can mediate pathway regulation and feedback to shape complex signaling responses. (A) Scaffold proteins are analogous to circuit boards—modular platforms that wire together components and direct the flow of information—and can program complex signaling behaviors. (B) Scaffold proteins function to wire pathway input and output through alternative possible routes. (C) Scaffold proteins can mediate branching of pathways to multiple outputs. (D) Scaffold proteins are themselves the targets of regulation. In T cell signaling, activation of the T cell receptor causes phosphorylation of the LAT and Slp76 scaffolds by Zap70, and phosphorylation-dependent recruitment of substrates leads to phospholipase C–γ (PLCγ) activation and PIP2 hydrolysis. (E) Scaffold proteins can be the target of feedback phosphorylation that tunes pathway responses. Feedback phosphorylation of the KSR scaffold by activated ERK blocks Raf (MAPKKK) binding and attenuates MEK activation, thereby decreasing pathway output. [Plot adapted from (24)]

  4. Fig. 3

    Benefits and costs of scaffold tethering mechanisms. (A) By colocalizing enzyme and substrate, scaffold proteins can lower the entropic cost of signaling interactions; the loss of independent translational and rotational degrees of freedom is paid through binding interactions with the scaffold. The size of the advantage gained depends on the flexibility of the scaffold structure. (B) By restricting the conformational freedom of interacting proteins, scaffolds can orient these molecules to enhance the rate of signal transfer. The rigid cullin scaffold proteins tether E2 ubiquitin-conjugating enzymes and their substrates. If the cullin backbone is made flexible by mutation, the rate of substrate ubiquitination is greatly decreased. (C) Tethering has potential drawbacks: At high concentrations, scaffolds may titrate enzyme and substrate away from one another. (D) Increased affinities can restrict substrate release and diffusion throughout the cell, potentially limiting signal amplification and spatial redistribution (e.g., nuclear localization).

  5. Fig. 4

    Allosteric regulation by scaffold proteins. (A) Scaffolds can allosterically modulate the conformation of enzymes and substrates to gate information flow. (B) In MAPK ERK signaling, KSR can directly bind to the MAPKKK Raf and influence its activity toward the MAPKK MEK. The kinase-homology domain of KSR dimerizes with Raf, altering the conformation of the C-helix on Raf so that its kinase domain becomes catalytically active (thereby allowing Raf to phosphorylate MEK). (C) The VWA domain of Ste5 promotes phosphorylation of the MAPK Fus3 by the MAPKK Ste7. The scaffold may unlock the activation loop of the MAPK Fus3 to make it a better substrate for MAPKK Ste7.

  6. Fig. 5

    Scaffold proteins are modular and can be used as platforms for redirecting information in evolution and engineering. (A) Pathogens can use scaffold-like proteins to rewire host signaling responses. The YopM scaffold from Y. pestis forces the interaction of the host Rsk1 and Prk2 kinases. The inappropriate activation is necessary for virulence. Viral scaffold proteins, such as HIV Vif, can target antiviral host proteins, such as the cytidine deaminase APOBEC3G, for degradation by targeting them to cullin-E2 ubiquitin ligases. (B) Engineered scaffolds can direct new cell signaling behaviors. A chimera of the Ste5 and Pbs2 yeast MAPK scaffold proteins can redirect mating pathway input to osmolarity pathway output. (C) Synthetic feedback loops can be engineered by controlling recruitment of positive and negative effectors to the Ste5 MAPK scaffold protein. Such loops can be used to precisely shape the dynamics and dose response of the yeast mating MAPK pathway to produce a wide range of signaling behaviors. (D) Natural metabolic pathways are often organized into multienzyme complexes that function like an assembly line to enhance the rate and yield of metabolite production. Engineered scaffold proteins can link together novel combinations of metabolic enzymes to more efficiently synthesize desired chemical products. [Adapted from (53)]

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