Circadian activation of the mitogen-activated protein kinase MAK-1 facilitates rhythms in clock-controlled genes in Neurospora crassa

Abstract

The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus Neurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in Saccharomyces cerevisiae and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from Neurospora cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ~500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e., ccg-1 and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.

Fig 1

Detection of MAK-1 and MAK-2. (A) Western blot showing that P-MAK-1 and P-MAK-2 are detectable using an anti-phospho-p44/42 antibody. Proteins were isolated from the indicated strains, and the stained membrane is shown as a loading control. (B) Western blot showing that total MAK-2 protein, but not total MAK-1 protein, is detectable using the anti-p44/42 antibody. Proteins were isolated from the indicated strains, and the stained membrane is shown as a loading control. No specific bands were observed for the wild-type (WT) or Δmak-2 strain that were absent in the Δmak-1 strain, indicating that the antibody does not recognize MAK-1. Lanes 2, 4, and 6 were treated with λ-phosphatase in an attempt to increase the resolution of the bands, while lanes 1, 3, and 5 were left untreated.

Fig 2

Circadian regulation of MAK-1 and MAK-2 phosphorylation. (A) The accumulation of P-MAK-1 (left) and P-MAK-2 (right) is shown in representative Western blots of total protein from the indicated strains grown in a standard circadian time course in constant dark (DD) and harvested every 4 h. The blots were probed with an anti-phospho-p44/42 antibody. The stained membranes were used as loading controls. (B) Plots of normalized P-MAK-1 and P-MAK-2 data from panel A. The WT data (solid black line) were better fit to a sine wave (dotted black line) (for P-MAK-1, P < 0.001; for P-MAK-2, P < 0.0001), whereas the Δfrq data (solid gray line) were better fit to a line (dotted gray line) (values are means ± standard errors of the means [SEM]; n = 3). (C) The levels of P-MAK-1 and P-MAK-2 are not altered in clock mutant strains. Western blotting was performed on total proteins harvested from the specified strains grown in DD for 16 h, and the membrane was probed with an anti-phospho-p44/42 antibody. The stained membrane is shown as a loading control. The data are plotted on the right. The levels of P-MAK-1 and P-MAK-2 in WT cells were set to 1 (values are means ± SEM; n = 3). (D) Representative Western blot showing the levels of P-MAK-1 at 25°C and after 10, 30, or 60 min at 42°C. Stained membranes are shown as a loading control. This experiment was performed 3 times with similar results.

Fig 3

The total level of MAK-1 is not clock controlled. Western blotting was performed on total protein from MAK-1::LUC cells grown in a circadian time course (see the legend to Fig. 2). The blots were probed with anti-LUC or anti-phospho-p44/42. The stained membrane was used as a loading control. Plots of MAK-1::LUC protein levels and luciferase activity are shown below the blots (values are means ± SEM; n = 3).

Fig 4

The FWO remains functional in the absence of MAK-1. (A) Western blots probed with anti-FRQ, showing both the levels and phosphorylation state of FRQ protein in WT and Δmak-1 cells grown in a circadian time course (see the legend to Fig. 2). The stained membrane was used as a loading control. (B) Northern blots probed for ccg-15 expression from WT and Δmak-1 strains grown in DD and harvested every 4 h. rRNA was used as a loading control. Consistent results were obtained from two independent experiments.

Fig 5

MAK-1 is required for regulation of target genes. (A) Representative Northern blots of total RNAs from the indicated strains, validating the MAK-1-dependent regulation of 12 of 14 genes identified by microarray analysis. The membranes were probed with the indicated genes. The known or predicted functions or domains of the genes are shown below the gene names. rRNA was used as a loading control. (B) Plots of mRNA levels of the indicated genes in Δmak-1 cells, normalized to rRNA (see panel A) (values are means ± SEM; n = 3), relative to the levels in WT cells. The level of mRNA in WT cells in each blot was set to 1. Twelve of 14 genes tested showed significantly different expression levels between the WT and Δmak-1 strains (for NCU06871, P ≤ 0.02; for NCU04352, P ≤ 0.01; for NCU02044, P ≤ 0.05; for NCU08923, P ≤ 0.04; for acw-5, P ≤ 0.02; for hsp88, P ≤ 0.02; for NCU03980, P ≤ 0.05; for NCU02075, P ≤ 0.03; for crp-4, P ≤ 0.004; for ccg-1, P ≤ 0.01; for NCU07465, P ≤ 0.02; for acw-2, P ≤ 0.04; for NCU05429, P ≤ 0.39; and for NCU07242, P ≤ 0.21). (C) Northern blots probed for NCU06871 and acw-2 expression from WT and Δmak-1 strains grown in DD for the indicated times. rRNA is shown as a loading control.

Fig 6

MAK-1 is necessary for robust rhythmic accumulation of downstream target ccgs. (A) Representative Northern blots of ccg-1 mRNA from WT and Δmak-1 strains grown in a circadian time course (see the legend to Fig. 2). rRNA is shown as a loading control. A 24-h exposure was used to detect ccg-1 mRNA in Δmak-1 cells. The data are plotted on the right (values are means ± SEM; n = 3). The WT data are represented by a solid black line, and the Δmak-1 data by a solid gray line. ccg-1 data from WT cells were fit to a sine wave (dotted black line) (P < 0.02), whereas ccg-1 data from the Δmak-1 strain were better fit to a line (dotted black line). (B) Representative Northern blots of NCU04352 mRNA from the indicated strains, labeled and plotted as described for panel A. The data for the Δfrq strain are represented with a dotted gray line. NCU04352 data from WT cells were fit to a sine wave (dotted black line) (P < 0.0001), whereas NCU04352 data from Δmak-1 and Δfrq cells were better fit to a line (dotted black line). (C) Representative Northern blots of NCU07465 mRNA from the indicated strains, labeled and plotted as described for panel A. NCU07465 data from WT cells were fit to a sine wave (dotted black line) (P < 0.0001), whereas for Δmak-1 and Δfrq cells, NCU07465 data were better fit to a line (dotted black line).