Ordered phosphorylation governs oscillation of a three-protein circadian clock

Abstract

The simple circadian oscillator found in cyanobacteria can be reconstituted in vitro using three proteins-KaiA, KaiB, and KaiC. The total phosphorylation level of KaiC oscillates with a circadian period, but the mechanism underlying its sustained oscillation remains unclear. We have shown that four forms of KaiC differing in their phosphorylation state appear in an ordered pattern arising from the intrinsic autokinase and autophosphatase rates of KaiC and their modulation by KaiA. Kinetic and biochemical data indicate that one of these phosphoforms inhibits the activity of KaiA through interaction with KaiB, providing the crucial feedback that sustains oscillation. A mathematical model constrained by experimental data quantitatively reproduces the circadian period and the distinctive dynamics of the four phosphoforms.

Fig. 1

Phosphorylation of KaiC is cyclically ordered. (A) Decomposition of total KaiC phosphorylation (“Total”) into its constituent phosphoforms, measured by SDS-PAGE (used throughout this study unless noted otherwise). The percentage of U-KaiC (not shown) is equal to 100% - Total. See also fig. S10. (B) Comparison of phosphoform distributions measured by SDS-PAGE (dotted lines, from A) and by mass spectrometry (solid symbols). (C) The initial phosphoform distribution of KaiC determines the subsequent dynamics. We prepared KaiC enriched in T-KaiC (solid circles) by incubating unphosphorylated KaiC with epitope-tagged KaiA for 2.25 h, and then removing KaiA by immunoprecipitation. We prepared KaiC enriched in S-KaiC (open squares) by incubating unphosphorylated KaiC with epitope-tagged KaiA for 18 h, removing KaiA, and then allowing KaiC to autodephosphorylate for 5.5 h. In both cases, circadian oscillations were then initiated by adding KaiB, incubating for 1.5 h, then reintroducing KaiA (28). Pie charts show the KaiC phosphoform distribution at the time of KaiA readdition.

Fig. 2

KaiC phosphoform kinetics in partial reactions. (A) KaiC phosphorylation in the presence of KaiA. A least-squares fit (solid lines) to a four-state linear model (Fig. 2C) is shown. (B) Autonomous dephosphorylation of KaiC. Phosphorylated KaiC was prepared by incubation with KaiA, which was then removed by immunoprecipitation (28), initiating dephosphorylation. A least-squares fit (solid lines) to the same four-state model is shown, with phosphorylation disallowed. (C) Reaction diagram for the four-state model with first-order kinetics.

Fig. 3

KaiB suppresses KaiA activity in an S-KaiC dependent manner. (A) Global KaiA activity varies during the circadian cycle. Dephosphorylated His₆-KaiC (triangles, circles, diamonds) was added at 10% of the concentration of untagged KaiC (squares) at the indicated times. (B) Phosphorylation activity is rapidly restored by the addition of a five-fold excess of KaiA during the dephosphorylation phase (triangles), which continues in a control without excess KaiA (squares). (C) KaiB interaction with KaiC and KaiA scales with the abundance of S-KaiC. The right-hand axis shows the normalized amount of KaiC and KaiA that coimmunoprecipitate with KaiB-FLAG in an oscillating reaction; the left-hand axis shows the corresponding phosphoform distribution. (D) KaiB does not affect phosphorylation until S-KaiC is abundant. Dephosphorylated KaiC was incubated with KaiA, and KaiB was introduced into the reaction at various time points (triangles, circles, diamonds) and compared to a control without added KaiB (squares). Pie charts show the KaiC phosphoform distribution at the time of KaiB addition.

Fig. 4

A model for circadian oscillation driven by multisite KaiC phosphorylation. (A) KaiA activity alters the first-order rate constants for interconversion of KaiC phosphoforms. Lines emanating from KaiA ending in an arrowhead (black) or bar (gray) indicate stimulation or repression, respectively, of the transition towards the indicated form of KaiC. We show only the dominant effects (see table S2 and fig. S11). S-KaiC inactivates KaiA via KaiB. The interconversion rates from phosphoform X to Y, k_XY, depend hyperbolically on the concentration A(S) of active KaiA monomers, which in turn depends on S-KaiC through its inhibitory activity. See the supporting online text for the complete equations of the dynamical model. (B) Numerical integration of the model reproduces circadian oscillation of KaiC phosphorylation.