Protein phosphatase PHLPP1 controls the light-induced resetting of the circadian clock

Abstract

The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) differentially attenuates Akt, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. PHLPP1 is expressed in the mammalian central clock, the suprachiasmatic nucleus, where it oscillates in a circadian fashion. To explore the role of PHLPP1 in vivo, we have generated mice with a targeted deletion of the PHLPP1 gene. Here we show that PHLPP1-null mice, although displaying normal circadian rhythmicity, have a drastically impaired capacity to stabilize the circadian period after light-induced resetting, producing a large phase shift after light resetting. Our findings reveal that PHLPP1 exerts a previously unappreciated role in circadian control, governing the consolidation of circadian periodicity after resetting.

Fig. 1.

Generation of PHLPP1 ^−/− mice. (A) Targeting strategy to disrupt the PHLPP1 gene. Shown are the wild-type allele, the targeting vector, the targeted (floxed) allele, and the deleted allele generated by Cre-mediated recombination of the floxed allele. The DNA probe used for screening the Southern blots is marked by a dotted box, and the PCR primers used for genotyping are indicated by small arrows. (B) Southern blot analysis of SacI-digested DNA from embryonic stem cells used to generate chimeric mice. The probe labels an 11-kb fragment in the wild-type cells and a 7-kb fragment in cells in which homologous recombination has occurred. (C) Genotyping PCR results from a cross between heterozygous PHLPP1 mutant mice. The PHLPP1 WT (+) allele gives a 264-bp PCR product whereas the deleted allele (−) produces a 486-bp fragment. (D) Lack of PHLPP1 transcript expression in PHLPP1^−/− mice. Total RNA isolated from PHLPP1 ^+/+ and PHLPP1^−/− mouse brain was used as templates for the RT-PCR analysis. The PHLPP1-specific primers used were located in exons 16 and 17, respectively. The hypoxanthine phosphoribosyltransferase 1 (HPRT) primers were used as controls for RT-PCR reactions. For a negative control, the cDNA template was omitted in the reaction (the lane labeled “neg”). (E) Western blot of PHLPP1 protein in brain lysates derived from PHLPP1^+/+ and PHLPP1^−/− mice. β-Actin serves as a loading control. (F) Western blot of PHLPP2 protein in liver and kidney whole cell lysates derived from PHLPP1^+/+ and PHLPP1^−/− mice. β-Actin serves as a loading control.

Fig. 2.

Differential light-induced phase shifts in PHLPP1-null mice. Double-plotted activity records of wild-type mice (PHLPP1^+/+) and PHLPP1-deficient mice (PHLPP1^−/−). Mice were entrained in 12:12 light–dark (LD) cycles and then placed in constant darkness (DD) from the light off (ZT12), on day 1. After 50 h in DD (CT14, day 3), no light pulse (no light; Top), or a 30-min light pulse (short light; Middle) was administered to PHLPP1^+/+ and PHLPP1^−/− mice. A separate group of mice were entrained and moved to constant darkness following 8 h of light prolongation (long light; Bottom) on the last day (day 1) of LD cycle. Locomotor activities were monitored by infrared sensors and are expressed in the histogram. Periods of darkness are indicated by gray backgrounds. Short-light pulses are denoted by asterisks. Red regression lines estimate the phase shift from days 10 to 21. Yellow regression lines estimate the phase shifts occurring several days after light pulses (short-light task = days 4–10; long-light task = days 2–10; lines are shown for phase shifted PHLPP1^−/− only).

Fig. 3.

Light task effects on the phase and circadian period of PHLPP1^−/− mice. (A) Quantification of short-light task-induced changes in activity rhythm phases [no light; ^+/+ (n = 6): ^−/− (n = 6), short light; ^+/+ (n = 8]): ^−/− (n = 8)] from days 4 to 10 (E, early phase) and days 10 to 21 (L, late phase). Extrapolated activity onsets of early phase and late phase are indicated by open circles (PHLPP1^+/+) and filled circles (PHLPP1^−/−) (mean ± SEM). *P < 0.005 (Fisher’s PLSD). (B) Circadian period during days 4–10 (E, early phase) and from days 10 to 21 (L, late phase) are indicated by open bars (PHLPP1^+/+) and filled bars (PHLPP1^−/−) (mean ± SEM). *P < 0.05 (Welch’s t test), †P < 0.01 (paired t test). (C) Acute expression of mPer1 and mPer2 in the SCN by short-light tasks. Upper: quantified values of mPer1 [^+/+ (n = 3), ^−/− (n = 3)] and mPer2 [^+/+ (n = 4), ^−/− (n = 4)]. Short-light induced RNA levels of PHLPP1^+/+ are adjusted to 100. Normalized RNA levels are indicated by open bars (PHLPP1^+/+) and filled bars (PHLPP1^−/−) (mean ± SEM). After short-light exposure (CT14–CT14.5, 200 lx) mice were returned to DD and killed 30 min later (CT15) for mPer1 and 60 min later (CT15.5) for mPer2. (Lower) representative films (Scale bar, 0.5 mm.) (D) mPer2 expression rhythms in the SCN after short-light tasks. Upper: quantified values of mPer2 [^+/+ (n = 3), ^−/− (n = 3)]. Peak level of RNA in PHLPP1^+/+ mice is adjusted to 100. Normalized RNA levels are indicated by open circles (PHLPP1^+/+) and filled circles (PHLPP1^−/−) (mean ± SEM). After short-light exposure (CT14–CT14.5, 200 lx) mice were returned to DD and killed 12–32 h later from the short-light onset. (Lower) representative films (Scale bar, 0.5 mm.) (E) Quantification of long-light task-induced changes in activity rhythm phases [no light; ^+/+ (n = 6): ^−/− (n = 6), long light; ^+/+ (n = 14): ^−/− (n = 14)] days 2–10 (E, early phase) and days 10–21 (L, late phase). Extrapolated activity onsets of the early phase and late phase are indicated by open circles (PHLPP1^+/+) and filled circles (PHLPP1^−/−) (mean ± SEM). *P < 0.0005 (Fisher’s PLSD). (F) Circadian periods of activities days 2–10 (E, early phase) and from days 10 to 21 (L, late phase) are indicated by open bars (PHLPP1^+/+) and filled bars (PHLPP1^−/−) (mean ± SEM). *P < 0.05 (Welch’s t test), †P < 0.005 (paired t test).

Fig. 4.

Model depicting how PHLPP1 affects the circadian system. Light pulse-induced phase shift of circadian rhythms and period change. Light pulse directly (A) or via clock resetting (B) controls circadian period after the phase shift. Irregular and delayed circadian period change in PHLPP1^−/− affects the magnitude of resetting (C).