Intramolecular regulation of phosphorylation status of the circadian clock protein KaiC

Abstract

Background: KaiC, a central clock protein in cyanobacteria, undergoes circadian oscillations between hypophosphorylated and hyperphosphorylated forms in vivo and in vitro. Structural analyses of KaiC crystals have identified threonine and serine residues in KaiC at three residues (T426, S431, and T432) as potential sites at which KaiC is phosphorylated; mutation of any of these three sites to alanine abolishes rhythmicity, revealing an essential clock role for each residue separately and for KaiC phosphorylation in general. Mass spectrometry studies confirmed that the S431 and T432 residues are key phosphorylation sites, however, the role of the threonine residue at position 426 was not clear from the mass spectrometry measurements.

Methodology and principal findings: Mutational approaches and biochemical analyses of KaiC support a key role for T426 in control of the KaiC phosphorylation status in vivo and in vitro and demonstrates that alternative amino acids at residue 426 dramatically affect KaiC's properties in vivo and in vitro, especially genetic dominance/recessive relationships, KaiC dephosphorylation, and the formation of complexes of KaiC with KaiA and KaiB. These mutations alter key circadian properties, including period, amplitude, robustness, and temperature compensation. Crystallographic analyses indicate that the T426 site is phosphorylatible under some conditions, and in vitro phosphorylation assays of KaiC demonstrate labile phosphorylation of KaiC when the primary S431 and T432 sites are blocked.

Conclusions and significance: T426 is a crucial site that regulates KaiC phosphorylation status in vivo and in vitro and these studies underscore the importance of KaiC phosphorylation status in the essential cyanobacterial circadian functions. The regulatory roles of these phosphorylation sites--including T426--within KaiC enhance our understanding of the molecular mechanism underlying circadian rhythm generation in cyanobacteria.

Figure 1. Mutations at residue 426 abolish circadian rhythms of luminescence in vivo.

This figure summarizes the effects of expressing a single copy of KaiC (either wild-type or mutated version) in cyanobacterial cells. (A) Effect of single mutations at T426 and double mutations at S431/T432 on the kaiBCp-driven luminescence rhythm and expression levels. The luminescence rhythms were obtained from strains expressing wild-type KaiC (TST) or mutant KaiCs (aST, nST, eST, or Tae) in constant light (LL). Three replicates for each case are shown in different colors. (B) T426 mutations alter KaiC phosphorylation profiles and abolish KaiC phosphorylation rhythms. The cultures expressing wild type or mutant KaiCs were harvested at 15 h or 27 h in LL and extracts were analyzed by immunoblot. P-KaiC denotes hyperphosphorylated KaiC bands. (C) Ratios (mean±SD) of the P-KaiC:total KaiC ratio from densitometry of data in panel B. *  =  p<0.05; **  =  p<0.01 (by Student's t-test).

Figure 2. Co-expression reveals genetic relationships of KaiC^TST with mutant versions of KaiC in vivo.

This figure summarizes the effects of co-expressing KaiC^TST (WT) with another KaiC (either wild-type or mutated version) under the control of trcp in the absence of IPTG induction. (A) Luminescence reporter strain with the endogenous kaiABC cluster and also expressing an additional copy of (i) wild-type KaiC (WT/TST), (ii) T426 single mutant KaiCs (WT/aST, WT/nST, or WT/eST), or (iii) S431/T432 double mutant KaiC (WT/Tae). Three replicates for each combination are shown in different colors. (B) Comparison of periods of the luminescence rhythms in LL at 30°C between wild-type strain (WT only) and various KaiC-coexpressing strains. Error bars are standard deviation (SD). *, p<0.05; **, p<0.01 (by Student's t-test). AR  =  arhythmia.

Figure 3. Effect of co-expression of KaiCs in the wild-type strain on the KaiC phosphorylation patterns in vivo at two phases.

(A) Immunoblot analysis of KaiC in strains co-expressing KaiCs mutated at residue 426 with wild-type KaiC at 15 h or 27 h in LL: WT/TST, WT/aST, WT/nST, or WT/eST. (B) Ratios of the P-KaiC/total KaiC from densitometry of (A). *, p<0.05; **, p<0.01 (Student's t-test; error bars  =  SD). (C) KaiC expression profiles at 24 h or 36 h in LL in strains expressing wild-type KaiC^TST or the S431/T432 double mutant KaiC^Tae alone or co-expressing KaiC^Tae with wild-type KaiC^TST (TST & Tae). Lower panel is a shorter exposure of the blot to show the double bands of KaiC^Tae mobility. (D) Ratios of the P-KaiC:total KaiC from densitometry of panel C.

Figure 4. ³²P-labeled phosphorylation of wild-type or mutant KaiCs.

Purified wild-type KaiC (TST) or mutant KaiCs (aST, nST, Tae, Taa, aae, or aaa) were incubated at 30°C for 24 hours in the reaction buffer containing [γ-³²P]ATP. (A) Before loading, the samples were heated at 100°C. (B) Before loading, the samples were heated to 72°C (left portion of panel B) for 10 m, or loaded without heating above room temperature (right portion of panel B). Following SDS-PAGE, ³²P-labeled KaiC was detected by autoradiography.

Figure 5. T426 mutations affect the rate of KaiC dephosphorylation and the formation of Kai complexes in vitro.

(A) Dephosphorylation rate of wild-type KaiC (KaiC^TST) and T426 mutant KaiCs (KaiC^aST and KaiC^nST). Purified KaiC proteins in the hyperphosphorylated state were incubated at 30°C with or without KaiA and/or KaiB for up to 12 h. (B) Densitometry of the KaiC phosphorylation/dephosphorylation profiles from panel A. (C) & (D) Detection of Kai complexes. Purified KaiC^TST, KaiC^aST or KaiC^nST were incubated with KaiB (panel C) or KaiA & KaiB (panel D) at 30°C for 8 h. The formation of KaiBC complex (panel C) and/or KaiBC/ABC complexes (panel D) were then identified by native gel analyses. Positions of KaiA, KaiB, KaiC and KaiBC/ABC complexes on native gel are noted.