TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species - PubMed (original) (raw)
TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species
Brad J Niles et al. Mol Biol Cell. 2014.
Abstract
The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells.
© 2014 Niles and Powers. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Figures
FIGURE 1:
Ypk1-dependent ROS perturbs actin cytoskeleton organization. (A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS yap1Δ (PLY1527), Ypk1-AS act1C374A (PLY1588), and Ypk1-AS Act1-WT (PLY1626) were grown in SCD-Ura medium, with 20 mM NAC as noted, and then all strains were treated for 1 h with 0.5 μM 2,3-DMB-PPI, and with 1 mM H2O2 where noted, and then fixed and labeled for actin with rhodamine-phalloidin. Scale bar, 5 μm. (B) Quantification of actin polarization for the same strains as in A, with at least 100 budded cells counted for each sample. (C) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1-AS act1C374A (PLY1588) were grown as in A and incubated for the last 30 min with 10 μM DCF. Quantification represents percentage of 200–300 cells labeled with DCF, including the SD from at least three experiments. p values were calculated using Student's t test; **p ≤ 0.01. (D) torc2-ts (PLY1134) transformed with empty vector (pPL187) or YPK1D242A (pPL240) were grown overnight at 25°C, with 20 mM NAC where noted, and then shifted to 30°C as noted for 1 h. Cells were fixed and labeled for actin with rhodamine-phalloidin as in A and quantified as in B.
FIGURE 2:
Ypk1 regulates actin polarization by suppression of ROS from multiple sources. (A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS rho0 (PLY1528), Ypk1-AS tpk3Δ (PLY1529), Ypk1-AS fpk1Δ (PLY1533), Ypk1-AS dnf1Δdnf2Δdnf3Δ (PLY1534), and Ypk1-AS fpk1Δ rho0 (PLY1536) were grown, fixed, and labeled for actin with rhodamine-phalloidin as in Figure 1A and quantified as in Figure 1B. (B) Act1-WT + Fpk1 3A KD (PLY1629), Act1-WT + Fpk1 3A (PLY1630), and act1 C374A + Fpk1 3A (PLY1631) were grown in SCD-Ura-Leu medium, with 20 mM NAC as noted, and then incubated with DCF as in Figure 1C or (C) fixed and labeled for actin and quantified as in A. (D) WT (PLY062), fpk1Δ (PLY1440), act1 C374A (PLY1628), and Act1-WT (PLY1627) were grown in SCD, or in SCD + MES as noted, and treated with 1.25 μM myriocin (Myr) for 1 h as noted, fixed, and labeled for actin and quantified as in A.
FIGURE 3:
Regulation of ROS by Pkc1 is downstream of Ypk1 and Fpk1. (A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS + PKC1R398P (1531), Ypk1-AS rho0 + PKC1R398P (PLY1532), Ypk1-AS fpk1Δ (PLY1533), and Ypk1-AS fpk1Δ + PKC1R398P (PLY1538) were grown in either SCD-Ura or SCD-Ura/-Leu medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. ROS was determined and quantified as in Figure 1C. p values were calculated using Student's t test; *p between 0.05 and 0.01; **p ≤ 0.01. (B) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in A, with at least 100 cells counted for each sample. (C) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1-AS fpk1Δ (PLY1533) were grown in either SCD-Ura or SCD-Ura + 50 mM MES, pH 6.2, and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Cells were harvested and lysed, and the resulting protein extracts were resolved by SDS–PAGE and immunoblotted with anti–phospho-p44/42 MAPK (for p-Mpk1), anti-Mpk1, and anti-G6PDH antibodies. Quantification below the blot describes the difference relative to Ypk1-WT after normalizing to the anti-p44/p42 MAPK signal. (D) WT (PLY062) and fpk1Δ (PLY1440) were grown in SCD medium and treated with 1.25 μM myriocin (Myr) for 1 h as noted and then processed as in C. (E) WT (PLY062) and WT + PKC1R398P (PLY1550) were grown in SCD or SCD-Leu medium and treated as in D. ROS was detected and quantified as in Figure 1C. p values were calculated using Student's t test; **p ≤ 0.01. (F) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in E, with least 100 cells counted for each sample.
FIGURE 4:
Regulation of Pkc1/MAPK activity requires Fpk1- and sphingolipid-dependent PM localization of Rom2. (A) Ypk1-WT rom2Δ + ROM2-GFP (PLY1563), Ypk1-AS rom2Δ + ROM2-GFP (PLY1564), Ypk1-AS lcb4Δ rom2Δ + ROM2-GFP (PLY1566), and Ypk1-AS fpk1Δ rom2Δ + ROM2-GFP (PLY1565) cells were grown in SCD-Ura/-Leu medium, with 4 μM PHS or 20 mM NAC where noted, and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Single focal plane images were collected by confocal microscopy. Quantification represents percentage of small-budded cells labeled with GFP, with 30–50 cells counted for each sample. Scale bar, 5 μm. (B) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), and Ypk1-AS fpk1Δ rom2Δ (PLY1561) were grown in SCD-Ura medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. Cells were harvested and lysed, and the resulting protein extracts were resolved by SDS/PAGE and immunoblotted with anti-phospho-p44/42 MAPK (for p-Mpk1), anti-Mpk1, and anti-G6PDH antibodies. Quantification below the blot describes the difference relative to Ypk1-WT after normalizing to the anti-p44/p42 MAPK signal. (C) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1-AS + Rom2-HA (PLY1568) were grown in SCD-Ura medium, treated with 0.5 μM 2,3-DMB-PPI for 1 h, and then processed as in B. (D) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), Ypk1-AS fpk1Δ rom2Δ (PLY1561), and Ypk1-AS + ROM2-HA (PLY1568) were grown in SCD-Ura medium and treated with 0.5 μM 2,3-DMB-PPI for 1 h. ROS was determined and quantified as in Figure 1C. p values were calculated using Student's t test; **p ≤ 0.01. (E) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in D, with at least 100 cells counted for each sample.
FIGURE 5:
Pkc1/MAPK regulates ROS through cyclin C protein stability. (A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS + Bck1-20 (PLY1585), Ypk1-AS cnc1Δ (PLY1586), and Ypk1-AS cdk8Δ (PLY1587) were grown in either SCD-Ura or SCD-Ura/-Leu and treated with 0.5 μM 2,3-DMB-PPI for 1 h. ROS was determined and quantified as in Figure 1C. p values were calculated using Student's t test; **p ≤ 0.01. (B) Quantification of actin polarization after fixing and rhodamine-phalloidin labeling in the same strains as in A, with at least 100 cells counted for each sample. (C) WT (PLY062), mpk1Δ (PLY517), Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), and Ypk1-AS lcb4Δ (PLY1556) cells all expressing myc-tagged cyclin C (pRL101) were grown in 0.5-l cultures of SCD-Leu medium, treated with 0.5 μM 2,3-DMB-PPI for 1 h, and treated with 0.2M H2O2 where noted. Total protein lysates and myc immunoprecipitates were resolved by SDS–PAGE and immunoblotted with anti-myc and anti-G6PDH antibodies. Quantification below the blot describes the difference relative to its control after normalizing to the anti-G6PDH signal.
FIGURE 6:
Model for TORC2/Ypk1-dependent regulation of ROS and actin polarization. See the text for details.
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