The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway

Abstract

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

Figure 1

Mcs4 and Wak1 are not required for sexual differentiation. (A) Cells lacking mcs4 or wak1 enter the G₁ phase efficiently in nitrogen-limiting medium. Log phase cultures of wild-type (WT) (PR109), wak1::ura4 (Δwak1) (JM1436), or mcs4::his7 (Δmcs4) (JM 1468) cells growing in EMM medium at 30°C were analyzed for DNA content before (0 h) and after (12 or 24 h) incubation in the same medium lacking NH₄Cl. (B) Mating efficiency of cells lacking Wak1 or Mcs4. Homothallic cultures of wild-type (WT) (JY878), atf1::ura4 (Δαtf1) (NT147), sty1::ura4 (Δsty1) (JM1263), wis1::ura4 (Δwis1) (JM1260), wak1::ura4 (Δwak1) (JM1505), or mcs4::ura4 (Δmcs4) (JM1355) cells were grown to log phase in liquid EMM and then transferred to the same medium lacking NH₄Cl for 24 h at 30°C. Mating efficiency was assessed microscopically.

Figure 2

Win1 interacts with components of the StyI MAPK pathway. (A) Win1–1 is suppressed by wak1 but not mcs4. win1–1 cdc25–22 cells (JM1354) were transformed either with a control plasmid pREP41 (Control), pREP41-wis1 (pWis1), pREP41-wak1 (pWak1), or pREP41-mcs4 (pMcs4), and gene expression was regulated via the thiamine-repressible nmt1 promoter. Transformants were grown and streaked to minimal medium lacking thiamine and leucine and cultured for 3 d at either 25°C (left hand plate) or 33°C (right hand plate). (B) Loss of win1 suppresses overexpression of wis1. Wild-type (PR109), win1–1 (win1–1) (JM1413), wak1::ura4 (Δwak1) (JM 1436), or sty1::ura4 (Δsty1) (JM 1160) cells were transformed with pREP1-wis1. Transformants were selected on minimal medium lacking leucine containing 10 μM thiamine and then streaked onto the same medium (+ thiamine) or the same medium lacking thiamine (−thiamine), and growth of the cells was monitored after 3 d at 30°C.

Figure 3

Win1 is required for stress-induced gene expression. Expression of the Pyp2 MAPK phosphatase is attenuated in cells lacking win1. Top panel, log phase cultures growing in YEPD at 30°C of either wild-type (WT), (PR109), win1–1 (win1–1) (JM1413), wak1::ura4 (Δwak1) (JM 1436), or wak1::ura4 win1–1 (Δwak1 win1–1) (JM1504) cells were incubated in the same medium containing 0.6 M KCl, 1 mM H₂O₂, 10 μg/ml anisomycin or shifted to 42°C for the times indicated. Total RNA was extracted, and equal quantities were separated by electrophoresis and then probed using DNA specific to the pyp2 gene. Middle panel, induction of catalase (ctt1) is attenuated in cells lacking win1–1. Log phase cultures of wild type (WT) (PR109), win1–1 (win1–1) (JM1413), wak1::ura4 (Δwak1) (JM 1436), or wak1::ura4 win1–1 (Δwak1 win1–1) (JM1504) cells growing in YEPD were incubated in the same medium containing either 10 μg/ml anisomycin or 1 mM H₂O₂ for the times indicated. In this experiment total RNA was extracted as described previously and probed using DNA specific to the ctt1 gene. Bottom panel, blots were reprobed with a cdc2-specific probe to verify equal loading of RNA.

Figure 4

Win1 is required for stress-induced activation of the Sty1 MAPK. (A) Tyrosine phosphorylation of StyI in cells lacking win1. Log phase cultures of wild-type (WT) (JM1520) or win1–1 (win1–1) (MW 1539) cells bearing an integrated and epitope-tagged version of Sty1 were incubated in medium containing 0.6 M KCl for the times indicated. Approximately 2 × 10⁸ cells were harvested and lysed at each time point, and the Sty1 protein was precipitated using Ni⁺⁺-NTA agarose. Precipitates were probed by Western blot for the presence of phosphotyrosine (α-pTyr) or the HA epitope tag (α-HA). (B) Activation of Sty1 in cells lacking win1. Log phase cultures of either wild-type (WT) (PR109) or win1–1 (win1–1) (JM1413) cells growing in YEPD at 30°C were shifted to 42°C for the times indicated. Cell extracts were prepared as above, and Sty1 was precipitated with GST-Atf1 prebound to glutathione beads. Precipitates were washed extensively and incubated in the presence of [γ-³²P]-ATP for 20 min at 30°C. Phosphorylation of Atf1 was visualized after SDS-PAGE and autoradiography.

Figure 5

Win1 and Wak1 act in concert to control stress resistance. Wild-type (WT) (PR109), sty1::ura4 (Δsty1) (JM 1160), wis1::ura4 (Δwis1) (JM 544), wak1::ura4 (Δwak1) (JM 1436), win1–1 (win1–1) (JM1413), or wak1::ura4 win1–1 (Δwak1 win1–1)(JM 1504) cells were grown on YEPD and then streaked to the same medium at 30°C (top left plate) or to the same medium containing 1.5 M sorbitol at 30°C (top right plate) or to YEPD at 37°C (bottom left plate) and incubated for 2 d.

Figure 6

Distinct roles for Wak1 and Win1 in the control of the StyI MAPK. (A) Win1–1 are partially defective in G₁ arrest. Log phase cultures of win1–1 cells (JM1413) growing in EMM medium at 30°C were analyzed for DNA content before (0 h) and after (12 or 24 h) incubation in the same medium lacking NH₄Cl. (B) Win1 is required for efficient sexual conjugation. Homothallic cultures of wild-type (open squares) (JY878), wak1::ura4 (closed squares) (JM1505), win1–1 (open circles) (ED623), wak1::ura4 win1–1 (closed circles) (JM1509), sty1::ura4 (open triangles) (JM1263), and wis1::ura4 (closed triangles) (JM1260) cells were grown to log phase in liquid EMM and transferred to the same medium lacking NH₄Cl for the times indicated at 30°C, and mating efficiency was assessed microscopically. (C) Win1 but not Wak1 is required for long-term viability in stationary phase. Log phase (2 × 10⁶ cells/ml) cultures of wild-type (open squares) (PR109), wis1::ura4 (closed triangles) (JM 544), wak1::ura4 (closed squares) (JM 1436), or win1–1 (open circles) (JM1413) were grown in YEPD at 30°C until the cultures reached stationary phase. At the times indicated, equal numbers of cells from these cultures were plated to fresh YEPD plates in triplicate, and viability was assessed by colony formation after an additional 3 d incubation at 30°C.

Figure 7

Evidence for a Wak1- and Win1- independent pathway controlling Wis1. (A) Wis1 suppresses the mating deficiency of a Δwak1 win1–1 strain. The homothallic strain Wak1::ura4 win1–1 leu1–32 h⁹⁰ (Δwak1 win1–1) (JM 1509) was transformed either with a control plasmid pREP41 (Cont.) or with pREP41-wis1 (pWis1) or pREP41-wak1 (pWak1) (as above). Transformants were grown to log phase at 30°C in liquid EMM lacking leucine and transferred to the same medium lacking NH₄Cl for 48 h, and mating efficiency was assessed microscopically. (B) Wis1 suppresses the temperature sensitivity of a Δwak1 win1–1 strain. Wak1::ura4 win1–1 leu1–32 (Δwak1 win1–1) (JM 1504) cells were transformed with a control plasmid pREP41 (Cont.) or either pREP41-wis1 (pWis1) or pREP41-wak1 (pWak1) in which the wis1 and wak1 genes were expressed from the thiamine-repressible nmt1 promoter. Transformants were streaked on minimal medium lacking thiamine and leucine, and colony formation was monitored after 3 d incubation at either 30°C (left hand plate) or 37°C (right hand plate).

Figure 8

A model for the role of Win1 in controlling the fission yeast stress-activated Sty1 MAPK. We propose that Win1 controls the activity of Wis1 MAPKK in parallel with the Wak1 MAPKKK and that the Mcs4 response regulator acts upstream of both Wak1 and Win1. We also tentatively suggest that the Wis1 MAPKK may be controlled by an additional Wak1- and Win1-independent pathway.