PubMed · 7634063
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
L N Johnson. 1994. Control by protein phosphorylation.. https://doi.org/10.1038/nsb1094-657
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here we reasoned that engineered allostery, a technique that has been applied to design light- and chemically gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase Förster resonance energy transfer biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. The resulting synthetic ERK-responsive transcription factor responds with comparable sensitivity to the c-fos promoter and reveals spatial ERK signaling patterns in mammalian developmental organoids. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.
Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.
The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.