MCB Accepted Manuscript Posted Online 26 January 2015 Mol. Cell. Biol. doi:10.1128/MCB.01118-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Revised version: MCB01118-14

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Methylglyoxal Activates Target of Rapamycin Complex 2—Protein Kinase C

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Signaling Pathway in Saccharomyces cerevisiae

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Wataru Nomura, Yoshiharu Inoue*

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Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

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Running title: Methylglyoxal activates TORC2 signaling

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* To whom correspondence should be addressed. E-mail: [email protected].

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This manuscript contains 1296 words for the Materials and Methods section, and 4395

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words for the Introduction, Results, and Discussion sections.

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ABSTRACT

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Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We here showed that

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methylglyoxal activated the Pkc1-Mpk1 MAP kinase cascade in a target of rapamycin

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complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae.

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We demonstrated that TORC2 phosphorylated Pkc1 at Thr1125 and Ser1143. Methylglyoxal

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enhanced the phosphorylation of Pkc1 at Ser1143, which transmitted the signal to the

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downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of

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Pkc1T1125 affected the phosphorylation of Pkc1 at Ser1143, in addition to its protein levels.

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Methylglyoxal activated mammalian TORC2 signaling, which in turn, phosphorylated Akt

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at Ser473. Our results suggest that methylglyoxal is a conserved initiator of TORC2

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signaling among eukaryotes.

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INTRODUCTION

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All biological activities in organisms must be accompanied by an ample supply

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of nutrients, which warrants energy production to meet the demand for biochemical

38

reactions in cells. Therefore, the machinery for sensing and transducing nutritional

39

conditions inside and outside of the cell plays crucial roles. Nutrients and hormones are

40

pivotal signal initiation molecules that regulate cellular energy metabolism in animals.

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Although nutrients are essential for living cells, several adverse byproducts are formed

42

during energy metabolism, such as reactive oxygen species (ROS) (1). ROS are

2

43

inevitably formed by oxygen respiration in accordance with ATP production, and cause

44

oxidative stress, which damages cellular components (2).

45

We have been focusing on methylglyoxal (MG) and its role as a metabolic

46

stressor. MG (CH3COCHO) is a typical 2-oxoaldehyde, the main source of which in the

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cell is glycolysis, a ubiquitous energy-producing pathway (3, 4). Since MG is highly

48

reactive because of its two carbonyl groups in one molecule, it reacts with DNA/RNA

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and proteins to make adducts that change their functions, which sometimes leads to

50

several diseases, such as diabetes and Alzheimer disease (5-7). MG and other carbonyl

51

compounds produce various carbonyl compound-modified proteins through a process

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that is sometimes referred to as carbonyl stress. Carbohydrate-derived carbonyl

53

compounds produce advanced glycation end products (AGEs) via spontaneous reactions

54

with the amino groups of proteins and carbonyl compounds. However, even though the

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reactivity of MG with proteins is >20,000-fold higher than that of glucose, the formation

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of AGEs has been shown to occur on a time scale of days, weeks, or longer (8, 9). The

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harmful effects of MG on cellular functions have been attributed to MG-derived AGEs

58

(4-9). We previously reported that MG caused acute cellular responses in yeasts (10, 11).

59

For example, the exogenous addition of MG activated yeast AP-1-like transcription

60

factors (Yap1 in Saccharomyces cerevisiae, and Pap1 in Schizosaccharomyces pombe)

61

through the reversible modification of their critical Cys residues within 45 min (10, 11).

62

MG was also found to enhance the influx of extracellular Ca2+ into the cell, thereby

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activating the Ca2+/calcineurin system in S. cerevisiae (12). In the present study, we

3

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found that MG enhanced the target of rapamycin complex 2 (TORC2)-protein kinase C

65

(Pkc1) signaling pathway in S. cerevisiae.

66

The target of rapamycin (TOR) signaling network is evolutionally conserved

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among eukaryotes, and is involved in diverse cellular activities (13, 14). TOR is a

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Ser/Thr kinase that forms two distinct protein kinase complexes, TOR complex 1

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(TORC1) and TOR complex 2 (TORC2) (15). TORC phosphorylates protein kinases

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involved in the AGC (named initially for cAMP- and cGMP-dependent protein kinases

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and protein kinase C) kinase family (16). S. cerevisiae TORC1 has been shown to

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phosphorylate Sch9, whereas TORC2 phosphorylates Ypk1 and Ypk2 (17-19), both of

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which belong to the AGC kinase family. A previous study demonstrated that TOR

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complexes phosphorylate conserved Thr/Ser residues within the turn motif (TM) and

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hydrophobic motif (HM) of AGC kinase (16). PKC1 is the only gene that encodes protein

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kinase C in S. cerevisiae (20, 21), and Pkc1 structurally belongs to the AGC kinase family.

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Thr/Ser residues in the TM and HM were also found to be conserved in Pkc1 (Thr1125 in

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TM, and Ser1143 in HM). Genetic interactions between PKC1 and some genes (TOR2,

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AVO1, and AVO3), the products of which are involved in the assembly of TORC2, have

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been reported previously (15, 22-24); however, it still remains unclear whether Pkc1 is a

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direct target of TORC2 (25).

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TORC1 is involved in events concerning cell growth such as protein synthesis,

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while TORC2 plays a role in actin organization (14). In the present study, we demonstrated

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that the organization of actin was perturbed by an MG treatment, thereby inhibiting the

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polarized cell growth of S. cerevisiae. Since TORC2 is involved in actin organization, we 4

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investigated the relationship between MG and TORC2 signaling. We showed that Thr1125

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and Ser1143 in Pkc1 were phosphorylated by TORC2 both in vivo and in vitro. We also

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found that MG enhanced TORC2-Pkc1 signaling, and the downstream Mpk1 MAP kinase

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cascade was subsequently activated following the treatment of yeast cells with MG.

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Furthermore, we showed that MG activated TORC2 signaling in mammalian cells. A

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physiological trigger that has been shown to activate TORC2 signaling is insulin (and

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insulin-like growth factor) (26-28); however, lower eukaryotes, such as yeast, do not have a

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hormonal system. Therefore, it remains unclear whether the ubiquitous physiological

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molecules that activate TORC2 exist among eukaryotes. Our results suggest that MG is a

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common initiator of TORC2 signaling.

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MATERIALS AND METHODS

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Media. The media used were SD (2% glucose, 0.67% yeast nitrogen base without amino

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acids) and SC/Gal (2% galactose, 3% glycerol, 0.67% yeast nitrogen base without amino

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acids). Appropriate amino acids and bases were added to SD or SC/Gal medium as

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necessary. Cells were cultured at 28ºC unless otherwise stated.

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Strains. The yeast strains and PCR primers used are summarized in Tables S1 and S2. The

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deletion allele of MID2 with KanMX or his5+ was amplified by PCR with the following

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primer sets: mid2-F and mid2-R from S. cerevisiae BY4741-based deletion mutants (29),

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and the corresponding loci of S. cerevisiae YPH250 were disrupted using PCR products. To

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construct wsc1∆ and mpk1∆, the wsc1∆::CgLEU2 allele of YOC2573 (30) and 5

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mpk1∆::HIS3 allele of TNP46 (31) were amplified by PCR with WSC1F and WSC1R, and

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MPK1FSalI and MPK1REcoRI, respectively. The disruption of MKK1, MKK2, and BCK1

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was performed as described previously (32).

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Plasmids. The plasmids used are summarized in Table S3. Details for the construction of

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plasmids were described in the supplementary materials.

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Actin staining. Cells were cultured in SD medium until the A610 reached 0.3-0.5. MG was

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then added. Cells were fixed with formaldehyde (final concentration, 4%) for 1 h. After

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fixation, the cells were harvested, washed twice with PBS (pH 7.4), and suspended in 30 µl

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of PBS. Rhodamine-phalloidin (Molecular Probes) was added to the cell suspension to a

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final concentration of 33 units/ml (1.1 µM), and the cell suspension was then incubated at

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4ºC in the dark overnight. Cells were collected by centrifugation, washed twice with PBS,

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and the distribution of actin was observed using a fluorescence microscope.

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Immunoprecipitation. Cells were cultured in SD medium until the A610 reached 0.5. Cells

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were then collected, washed with a 0.85% NaCl solution, and suspended in lysis buffer B

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[50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 0.5% Tween 20] containing 2.5

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mM phenylmethylsulfonyl fluoride, 50 mM NaF, and protease inhibitor cocktail (Nacalai

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Tesque). Cells were disrupted with glass beads using Beads Smash 12 (Wakenyaku), and

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cell homogenates were centrifuged at 700 x g for 10 min at 4ºC to remove cell debris. The

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protein concentrations in the cell extracts were determined using the DC protein assay 6

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(Bio-Rad Laboratories). The Avo3-13myc, Pkc1-3HA, or Pkc1-3FLAG protein was

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immunoprecipitated from the cell extracts (1.5 or 1 mg protein) by an incubation with

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anti-c-Myc antibodies coupled with agarose resin (Nacalai Tesque), anti-HA antibodies

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coupled with agarose resin (MBL), or anti-FLAG antibody-conjugated resin (Sigma) for 2

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h at 4ºC in lysis buffer B. After being incubated, the agarose resins were precipitated by

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centrifugation, washed four times with lysis buffer B, and suspended in SDS-PAGE sample

136

buffer. SDS-PAGE was then performed, followed by Western blotting.

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Bacterial expression and purification of Ypk2. E. coli BL21 (DE3) cells carrying

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pET-15b-Ypk2 were grown in LB medium containing ampicillin at 37ºC until the A610

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reached 0.3, and isopropyl-thio-β-D-galactopyranoside was added to a final concentration

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of 0.2 mM. Cells were incubated at 25ºC overnight, and then disrupted by sonication at 4ºC

142

in 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 mM NaCl and 10 mM

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imidazole. Cell homogenates were centrifuged to remove cell debris, cell extracts were

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applied to a HisTrapTM HP column (GE Healthcare) equilibrated with sonication buffer,

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and the column was washed with the same buffer. The Proteins adsorbed were eluted with

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20 mM sodium phosphate buffer (pH 7.4) containing 0.5 mM NaCl and 250 mM imidazole.

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Fractions containing Ypk2 were dialyzed against 20 mM Hepes-KOH buffer (pH 7.4)

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containing 150 mM NaCl and 2 mM dithiothreitol.

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In vitro protein kinase assay. An in vitro protein kinase assay was performed for TORC2,

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as described previously (17) with some modifications. Briefly, TORC2 was 7

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immunopurified using myc-tagged Avo3 instead of HA-tagged Tor2, which was used in a

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previous study (17). Immunopurified TORC2 was incubated with 5 µg of Pkc1 peptides

154

(Pkc1WT:

155

APPTLAPLPSVLTTSQQEEFRGFAFMPDDL) or 4 µg of recombinant Ypk2 protein.

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Synthetic Pkc1 peptides were produced by the GenScript Corporation (Piscataway, NJ,

157

USA). The reaction was initiated by adding [-32P]ATP (10 µCi). After being incubated for

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30 min at 30ºC, the reaction was terminated by the addition of SDS-PAGE sample buffer,

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and samples were then incubated for 5 min at 65ºC. Samples were subjected to

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Tricine-SDS-PAGE (33) or SDS-PAGE, and phosphorylated peptides were detected by

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autoradiography.

APPTLTPLPSVLTTSQQEEFRGFSFMPDDL,

Pkc1T1125A/S1143A;

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Western blotting of Mpk1. Cells were collected and washed with lysis buffer A [50 mM

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Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM sodium

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pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 20

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mM NaF, and 2 µg each of pepstatin A and leupeptin per ml], and cell pellets were frozen

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with liquid nitrogen. Cell pellets were suspended in lysis buffer A, and agitated with glass

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beads using Beads Smash 21 (Wakenyaku). Cell homogenates were centrifuged for 5 min at

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15000 x g, 4ºC, and the protein concentration was determined in clear supernatants using

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the DC protein assay (Bio-Rad Laboratories). Samples were subjected to SDS-PAGE, and

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the separated proteins were transferred to a PVDF membrane (Millipore). Blots were

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incubated with appropriate dilutions of the primary antibodies [anti-phospho-p44/42 MAP

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kinase (Thr202/Tyr204) (Cell Signaling) and anti-Mpk1 (yC-20: sc-6803; Santa Cruz 8

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Biotechnology)]. Immunoreactive bands were visualized with a BCIP-NBT solution kit for

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alkaline phosphatase (Nacalai Tesque) or Immobilon Western chemiluminescent HRP

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substrate (Millipore) using a LAS-4000 mini imaging system (FUJIFILM).

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Western blotting of Pkc1. Total protein extracts were prepared using the trichloroacetic

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acid extraction method (12). The protein concentrations of the samples were determined

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using the RC DC protein assay kit (Bio-Rad Laboratories). The antibodies used for Western

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blotting were anti-Pkc1 antibodies.

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Anti-Pkc1, anti-phospho-Pkc1 at Thr1125 (p-T1125), and anti-phospho-Pkc1 at Ser1143

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(p-S1143) antibodies. Anti-Pkc1 polyclonal antibodies were raised by immunizing rabbits

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with a peptide corresponding to the amino acid residue numbers of 470-488 in Pkc1.

186

Antisera were used to detect Pkc1 in Western blotting. Anti-phospho-Pkc1 polyclonal

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antibodies were raised by immunizing rabbits with a phospho-peptide of Pkc1 containing

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Thr1125 in the turn motif (NH2-C+APPTL(pT)PLPSVLTTSQQEE-COOH) or Ser1143 in the

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hydrophobic motif (NH2-C+EFRGF(pS)FMPD-COOH). Antisera were loaded onto a

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column packed with the resin conjugated with each of the phosphorylated peptides. After

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the adsorbed fractions were recovered, they were passed through a column packed with the

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resin conjugated with each of the unphosphorylated peptides. The fractions were recovered,

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and used as anti-phospho-Pkc1 at Thr1125 (p-T1125) or anti-phospho-Pkc1 at Ser1143

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(p-S1143) antibodies.

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Culture conditions for mammalian cells. Mouse 3T3-L1 cells were maintained in the

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maintenance medium (10% FBS and 10 mg/ml penicillin/streptomycin in DMEM) at 37ºC

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with 5% CO2. Preadipocytes were differentiated to adipocytes by the established protocol

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(34) as described briefly below. 3T3-L1 preadipocytes were grown for 2 days until

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post-confluence, and medium was switched to a differentiation medium consisting of the

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maintenance medium supplemented with 0.25 µM dexamethasone, 10 µg/ml insulin, and

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0.5 mM 3-isobutyl-1-methylxanthine. After 48 h, the medium was replaced with a

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post-differentiation medium consisting of the maintenance medium supplemented with 5

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µg/ml insulin. Medium was exchanged every 2 days with a fresh post-differentiation

205

medium until cells were ready for experimentation.

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3T3-L1 adipocytes were incubated in the maintenance medium for 20 h in

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DMEM for Western blotting, and then starved of serum for 5 h in DMEM. Cells were lysed

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with SDS-PAGE sample buffer, and the protein concentrations of the samples were

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determined using the RC DC protein assay kit (Bio-Rad Laboratories). The antibodies used

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for Western blotting were anti-Akt antibodies (Cell Signaling) to detect the bulk protein

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levels of Akt, anti-phospho-Akt (Ser473) antibodies (Cell Signaling) to detect the

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phosphorylation of Ser473 in the HM of Akt, and anti-phospho-PKC/II (Thr638/641)

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antibodies (Cell Signaling) to detect the phosphorylation of Thr450 in the TM of Akt (35).

214

Immunoreactive bands were visualized with a kit (Immobilon Western chemiluminescent

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HRP substrate, Millipore) using an LAS-4000 mini imaging system (FUJIFILM).

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RESULTS 10

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MG perturbs actin organization. We previously demonstrated that a moderate

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concentration of MG reversibly inhibited the growth of S. cerevisiae cells without

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decreasing cell viability (12). S. cerevisiae exhibits polarized growth, and actin is known to

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play crucial roles in determining cellular polarity (36). To gain an insight into the

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MG-induced inhibition of growth, we examined the organization of actin. Actin patches

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accumulated in the buds of logarithmically growing cells; however, actin patches were

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almost completely (84 - 91%) and uniformly dispersed in both the mother cell and bud 30 -

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60 min after the MG treatment (Fig. 1A). The depolarization of actin patches by the MG

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treatment was reversible, i.e., the patches returned to the bud 30 min after the removal of

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MG from the medium (Fig. 1A).

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PKC1R398P is a constitutively active allele of PKC1 that has been shown to

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suppress the depolarization of actin patches in a rho1-2 temperature-sensitive mutant at

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nonpermissive temperatures when PKC1R398P was expressed using its own promoter in a

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single copy plasmid (23, 37). Therefore, we investigated whether the depolarization of actin

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patches in the presence of MG is alleviated by the expression of PKC1R398P. As shown in

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Fig. 1B, Pkc1R398P partially repressed the depolarization of actin patches in cells treated

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with MG.

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Pkc1-Mpk1 MAP kinase cascade mutants are sensitive to MG. The organization of actin

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was previously shown to be regulated by the Pkc1-Mpk1 MAP kinase cascade (38, 39). To

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verify whether MG influences the growth of mutants defective in the Pkc1-Mpk1 MAP

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kinase cascade, we conducted spot assay experiments on SD agar plates containing MG. 11

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pkc1∆ cells are not able to grow without sorbitol (38) because synthesis of the cell wall is

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defective in this mutant and cell lysis occurs without an osmoprotectant. Therefore, spot

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assays were performed using SD agar plates containing 1 M sorbitol. As shown in Fig. 1C,

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the pkc1∆ mutant exhibited susceptibility to MG. Similarly, bck1∆, mkk1∆mkk2∆, and

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mpk1∆ mutants, defective in the MAP kinase cascade, also showed susceptibility to MG

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(Fig. 1D). Wild-type cells displayed an increased tolerance toward MG when PKC1R398P

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was expressed from its own promoter using a single copy plasmid (Fig. 1E).

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MG activates the Mpk1 MAP kinase cascade. Since mutants defective in the Pkc1-Mpk1

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MAP kinase cascade were sensitive to MG (Fig. 1C and D), we speculated that MG may

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activate this signaling pathway to combat MG-induced cellular stress. We monitored the

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phosphorylation levels of Mpk1. As shown in Fig. 2A, Mpk1 was phosphorylated

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following the treatment with MG, whereas no phosphorylated band was detected in pkc1∆

253

cells or in any of the Mpk1 MAP kinase cascade mutants (Fig. 2B and C). These results

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indicate that MG activates the Pkc1-Mpk1 MAP kinase cascade.

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The membrane-integrated proteins Wsc1 and Mid2 act as sensors for the heat

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shock-induced activation of the Mpk1 MAP kinase cascade (see Fig. 8) (38-40). To explore

257

whether these proteins function as sensors for MG-triggered stress, we disrupted WSC1 and

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MID2, and determined the phosphorylation of Mpk1 in the resultant mutants. However, as

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shown in Fig. 2D, Mpk1 was phosphorylated following the treatment with MG in wsc1∆,

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mid2∆, and wsc1∆mid2∆ cells. Subsequently, susceptibility to MG was not increased by the

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deletion of WSC1 and/or MID2 (Fig. 2E). Hence, the MG-induced activation of the 12

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Pkc1-Mpk1 MAP kinase cascade occurred irrespective of the Wsc1/Mid2 pathway. Besides

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Wsc1/Mid2, Wsc2 and Mtl1 have also been reported to act as upstream sensors for the

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Pkc1-Mpk1 pathway (41, 42). Therefore, we determined whether these proteins are

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involved in the MG-induced phosphorylation of Mpk1. As shown in Fig. S1A, the

266

phosphorylation of Mpk1 occurred in wsc2∆ and mtl1∆ mutants following the treatment

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with MG, and these mutants did not subsequently exhibit increased sensitivity to MG (Fig.

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S1B).

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Yeast TORC2 phosphorylates Pkc1. Previous studies suggested that genetic interactions

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may occur between TORC2 and the Pkc1-Mpk1 signaling pathway (22-24, 43). For

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example, the temperature sensitivity of a tor2ts mutant was suppressed by the

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overexpression of RHO1, PKC1, and MPK1 (22, 23, 43). Pkc1 belongs to the AGC kinase

274

family, and AGC kinases are generally the targets of TOR kinases (16). Potential

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phosphorylation sites (Thr1125 in TM, and Ser1143 in HM) are conserved in Pkc1 (Fig. 3A)

276

(16, 35, 44); however, it currently remains unknown whether yeast TORC2 directly

277

phosphorylates Pkc1. To gain an insight into the MG-induced activation of the Pkc1-Mpk1

278

MAP kinase cascade, we investigated whether Pkc1 is a substrate of TORC2.

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TORC2 consists of Tor2, Avo1, Avo2, Avo3, Bit61, and Lst8. Tor2 and Lst8 have

280

also been shown to be involved in TORC1 (14, 15). We carried out the immunopurification

281

of TORC2 from yeast cells that simultaneously expressed Avo3-myc, Tor2-HA, and

282

Bit61-FLAG. The immunoprecipitation of Avo3-myc was performed using the anti-myc

283

monoclonal antibody-conjugated resin, and we verified that Tor2-HA and Bit61-FLAG 13

284

were co-immunoprecipitated with Avo3-myc (Fig. 3B). Since Tor2 is involved not only in

285

TORC2, but also in TORC1 (15), we performed immunoprecipitation with myc-tagged

286

Avo3, which was previously shown to be specifically involved in TORC2 (18, 45), in order

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to reduce the probability of the contamination of TORC1. We then determined whether

288

immunopurified TORC2 actually possessed TORC2 activity. Since the substrate of yeast

289

TORC2 has been identified as Ypk2 (17), we carried out an in vitro kinase assay using a

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6xHis-tagged Ypk2 protein purified from recombinant E. coli cells as a substrate. As shown

291

in Fig. 3C, immunopurified TORC2 was able to phosphorylate 6xHis-Ypk2. Therefore, the

292

in vitro kinase assay system that we constructed was confirmed to work properly.

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Using this assay system, we conducted an in vitro kinase assay to determine

294

whether Pkc1 is phosphorylated by TORC2. As shown in Fig. 3D, the wild-type Pkc1

295

peptide (the C-terminal region of Pkc1 [amino acid residue no. 1120-1149] including

296

Thr1125, Ser1143, and some other Ser and Thr residues) was phosphorylated, while the

297

Pkc1T1125A/S1143A peptide, an Ala-substituted variant of Thr1125 and Ser1143, was not. In

298

addition, TORC2 containing the kinase-dead Tor2 did not phosphorylate Pkc1WT peptide

299

(Fig. S2). These results indicate that TORC2 phosphorylates Pkc1 in vitro.

300

We then raised anti-phospho-Pkc1T1125 and anti-phospho-Pkc1S1143 antibodies to

301

examine whether Pkc1 is a target of TORC2 in yeast cells. To verify the specificity of these

302

antibodies, Ala-substituted Pkc1 variants at Thr1125 (Pkc1T1125A) and Ser1143 (Pkc1S1143A)

303

were constructed. As shown in Fig. 3E, each antibody specifically crossed with the

304

respective phosphorylation sites. To determine whether these amino acid residues are

305

phosphorylated by TORC2, we used a strain carrying the AVO1 gene, which encodes an 14

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essential component of TORC2, driven by the GAL1 promoter (45), and shifted the cell

307

from a galactose medium to a glucose medium to stop the expression of AVO1, which

308

impaired TORC2 activity. The impairment of TORC2 activity in yeast cells in the glucose

309

medium was verified by observing the depolarization of actin (Fig. S3). As shown in Fig.

310

3F, Thr1125 and Ser1143 were phosphorylated in the basal state in both wild-type and

311

GAL1p-AVO1 cells in the galactose medium; however, the phosphorylation levels of these

312

amino acids decreased in GAL1p-AVO1 cells in the glucose medium. These results indicate

313

that Pkc1 is phosphorylated in a TORC2-depepdent manner in yeast cells.

314 315

Involvement of Rho1 in the phosphorylation of Pkc1 at Thr1125 and Ser1143. Rho1 is a

316

small G protein that crucially involved in the heat shock-triggered phosphorylation of

317

Mpk1 (37, 38, also see Fig. 8). Rho1 also physically interacts with Pkc1 (37, 38). Therefore,

318

we investigated whether Rho1 is involved in TORC2-Pkc1 signaling. Since RHO1 is an

319

essential gene, we used temperature-sensitive rho1 mutants. Saka et al. isolated several

320

rho1ts mutants, of which rho1-2, rho1-3, and rho1-5 were impaired in heat shock-induced

321

phosphorylation of Mpk1 (46). These findings prompted us to examine Pkc1

322

phosphorylation levels in these mutants. As shown in Fig. 4A, the basal phosphorylation

323

levels of Thr1125 and Ser1143 of Pkc1 were decreased in the temperature-sensitive mutants of

324

RHO1 at nonpermissive temperatures.

325

We then constructed Pkc1 mutants defective in the domains for the physical

326

interaction with Rho1. Two domains in the N-terminal region of Pkc1, the homology region

327

1 (HR1) domain and Cys-rich C1 domain, have been shown to contribute to the physical 15

328

interactions with Rho1 (37, 47, 48) (Fig. 3A). Previous studies reported that a Pkc1∆HR1

329

mutant was deleted in the HR1 domain, and also that a Pkc14C/S mutant was a Cys-to-Ser

330

substituted variant of the four Cys residues (Cys434, Cys437, Cys514, and Cys517) necessary

331

for the zinc-finger motif in the C1 domain (47-49). The basal phosphorylation levels of

332

Thr1125 and Ser1143 were markedly decreased in the Pkc1∆HR1 mutant (Fig. 4B). The

333

phosphorylation level of Ser1143 was also markedly decreased in the Pkc14C/S mutant,

334

whereas the phosphorylation level of Thr1125 was similar to that of wild-type Pkc1 (Pkc1WT)

335

(Fig. 4B). The MG-induced phosphorylation of Mpk1 did not occur in either mutant (Fig.

336

4C). Additionally, overproduction of the C1 domain, which may have competed with

337

endogenous Pkc1, inhibited the MG-induced phosphorylation of Mpk1, whereas

338

overproduction of the C1 domain with Cys-to-Ser substituted mutation (C14C/S) did not (Fig.

339

4D).

340 341

MG facilitates TORC2 signaling in yeast. We demonstrated that MG activated the

342

Pkc1-Mpk1 MAP kinase cascade. To assess whether TORC2 is involved in this signaling

343

pathway triggered by MG, we first monitored the phosphorylation of Mpk1 using a

344

tor2-21ts mutant. Although Tor2 was shown to be involved in both TORC1 and TORC2, the

345

tor2-21ts allele was specifically defective in TORC2 function (22). The MG-induced

346

phosphorylation of Mpk1 was observed at 28ºC in tor2-21ts cells, whereas its level was

347

greatly reduced at a nonpermissive temperature (Fig. S4A). The MG-induced

348

phosphorylation of Mpk1 also rarely occurred in cells defective in TORC2 activity (Fig.

349

5A), which was achieved by transferring cells carrying the essential components of TORC2 16

350

(AVO1 or AVO3) with the GAL1 promoter from the galactose medium to the glucose

351

medium. The same result was obtained when cells with GAL1 promoter-driven TOR2 were

352

used (Fig. 5A). We confirmed that TORC2 function was impaired under these conditions by

353

observing the depolarization of actin (Fig. S4B).

354

Since Thr1125 and Ser1143 in Pkc1 were the direct phosphorylation sites of TORC2

355

(Fig. 3), we investigated whether these amino acids are necessary for the MG-induced

356

phosphorylation of Mpk1. As shown in Fig. 5B, Mpk1 was slightly phosphorylated in

357

PKC1S1143A cells, but not in PKC1T1125A or PKC1T1125A/S1143A cells following the treatment

358

with MG. These results indicate that Thr1125 and Ser1143 in Pkc1 are involved in the

359

MG-induced activation of the Mpk1 MAP kinase cascade.

360

We next determined whether MG enhances the phosphorylation of Pkc1. As

361

shown in Fig. 5C, the phosphorylation levels of Thr1125 in the TM of Pkc1 remained

362

unchanged following the treatment with MG. Meanwhile, the phosphorylation levels of

363

Ser1143 in HM were increased by the MG treatment (Fig. 5D). To examine whether MG

364

directly affects the activity of TORC2 to increase the phosphorylation level of Pck1 at

365

Ser1143, we added MG in a mixture of the in vitro kinase assay. However, the

366

phosphorylation level of Pkc1Ser1143 was substantially unchanged in the presence of MG

367

(Fig. S2). Next, to verify that increase in the phosphorylation level of Pkc1 at Ser1143 in

368

MG-treated cells is dependent on TORC2, cells carrying the AVO1 gene driven by the

369

GAL1 promoter were shifted from the galactose medium to the glucose medium in order to

370

reduce the activity of TORC2 (Fig. S5), after which these cells were treated with MG. As

371

shown in Fig. 5E, the phosphorylation levels of Ser1143 in TORC2-impaired cells were not 17

372

increased by the treatment with MG. These results implied that MG may have facilitated

373

TORC2 signaling thereby increasing the phosphorylation level of Pkc1 at Ser1143, which

374

transmitted the signal downstream to the Mpk1 MAP kinase cascade.

375 376

The phosphorylation status of Pkc1 at Thr1125 affects its protein levels. Although

377

Thr1125 in the TM of Pkc1 did not respond to MG (Fig. 5C), the basal phosphorylation of

378

this site appeared to be necessary for transmitting the signal to the Mpk1 MAP kinase

379

cascade (Fig. 5B). However, it currently remains unclear why the phosphorylation of Mpk1

380

did not occur in Pkc1T1125A cells following the treatment with MG in spite of Ser1143 being

381

intact. Previous studies reported that the phosphorylation of Thr450 in the TM of Akt secured

382

its stability in mammalian cells (35, 50). To verify whether the substitution of Thr1125 in TM

383

to Ala affects the stability of Pkc1, whereby the MG-triggered signal is not adequately

384

transmitted to the Mpk1 MAP kinase cascade, we determined Pkc1 protein levels. We

385

raised anti-Pkc1 antibodies to monitor Pkc1 protein levels. Although some non-specific

386

bands appeared in the blots, the Pkc1 antibodies raised were able to detect Pkc1 proteins in

387

cell-free extracts for the Western blotting analysis (Fig. S6). As shown in Fig. 5F, the steady

388

state levels of the Pkc1T1125A protein were markedly lower than those of the Pkc1WT protein.

389

This may be one of the reasons why the MG-induced phosphorylation of Mpk1 did not

390

occur in Pkc1T1125A cells (Fig. 5B).

391 392

Role of the phosphorylation of Pkc1 at Thr1125 and Ser1143 under MG stressed

393

conditions. To explore its physiological relevance to the TORC2-dependent 18

394

phosphorylation of Pkc1, we investigated the effects of the substitution of the

395

phosphorylation sites of Pkc1 on growth and sensitivity to MG. The phosphorylation sites

396

of Pkc1 were found to be irrelevant to the growth of yeast cells. The pkc1∆ mutant is unable

397

to grow in the absence of an osmoprotectant such as sorbitol because cell wall biogenesis is

398

defective (51, 52). However, we found that the Pkc1T1125A/S1143A mutant was able to grow in

399

the medium without sorbitol (Fig. 6A). Pkc1 mutants defective in the HR1 and C1 domains,

400

in which the phosphorylation levels of Thr1125 and Ser1143 were reduced (Fig. 4B), were also

401

able to grow without sorbitol (Fig. 6B). These results suggest that the phosphorylation of

402

Thr1125 and Ser1143 in Pkc1 is dispensable for the growth of yeast cells under normal osmotic

403

conditions. However, the substitution of Thr1125 and/or Ser1143 to Ala enhanced the

404

susceptibility of cells to MG (Fig. 6A).

405

The growth defect in TORC2-knockdown cells was partially suppressed by

406

multicopy PKC1WT, PKC1T1125A, or PKC1S1143A without sorbitol in the medium (Fig. 6C).

407

Since the addition of sorbitol to a medium was previously shown to suppress the growth

408

defect in TORC2-knockdown cells (22), suppression of the growth defect in

409

TORC2-impaired cells by multicopy PKC1 may be attributed to the recovery of cell wall

410

integrity. However, the growth defect in TORC2-knockdown cells was not suppressed by

411

multicopy PKC1T1125A/S1143A. To explore the physiological relationship between TORC2 and

412

Pkc1, we examined the organization of actin. As shown in Fig. S7, the proportion of cells

413

with depolarized actin increased when TORC2 was impaired (cells with depolarized actin:

414

wild-type cells, 16.9%; TORC2-impaired cells, 59.8%); however, it reverted to nearly

415

wild-type levels (29.3%) when PKC1 was expressed in a multicopy vector. However, 19

416

multicopy PKC1T1125A/S1143A was not able to recover the polarization of actin. This may have

417

been because of a failure in the growth-recovery of TORC2-knockdown cells carrying

418

multicopy PKC1T1125A/S1143A.

419 420

MG activates mTORC2 signaling in mammalian cells. We demonstrated that MG

421

functioned as a signal initiator for TORC2 signaling using the budding yeast S. cerevisiae as

422

a model organism. To determine whether this is also the case in higher eukaryotes, we

423

measured the phosphorylation levels of Akt, a substrate of mammalian TORC2 (mTORC2)

424

(Fig. 7A), using mouse 3T3-L1 adipocytes. The phosphorylation level of Ser473 in the HM

425

of Akt increased in adipocytes following the treatment with MG (Fig. 7B). Meanwhile,

426

Thr450 in the TM of Akt was constitutively phosphorylated, and its level remained

427

unchanged with the MG treatment (Fig. 7B). To verify the MG-induced phosphorylation of

428

AktSer473 was dependent upon mTORC2, we used Torin 1 to inhibit mTOR. Rapamycin

429

specifically inhibits TORC1, while Torin 1 acts on both mTORC1 and mTORC2 (53).

430

MG-induced phosphorylation of Ser473 was completely inhibited in the presence of Torin 1,

431

however, rapamycin did not (Fig. 7C). In mouse adipocytes, the phosphorylation of Akt at

432

Ser473 was previously shown to depend on mTORC2 (54). Therefore, these results suggest

433

that MG may function as a signal initiator of mTORC2 signaling in mammalian cells,

434

similar to yeast cells.

435 436 437

DISCUSSION 20

438

In the present study, we demonstrated that MG activated the Pkc1-Mpk1 MAP kinase

439

cascade in S. cerevisiae. The activation of this kinase cascade appeared to be necessary for

440

yeast cells to combat MG-induced stress. An important feature of our results is that TORC2

441

was involved in activating the Pkc1-Mpk1 signaling pathway triggered by MG. We herein

442

demonstrated for the first time that Pkc1 was a substrate of TORC2, i.e., TORC2

443

phosphorylated Thr1125 in the TM and Ser1143 in the HM of Pkc1. Genetic studies previously

444

reported a relationship between Tor2 and the Pkc1-Mpk1 pathway; i.e., PKC1 and MPK1

445

have been isolated as multicopy suppressors that rescue growth defects in the tor2-21ts

446

mutant at nonpermissive temperatures (22, 23, 43). Since the tor2-21ts mutant was only

447

defective in TORC2 at nonpermissive temperatures (22), TORC2 appeared to communicate

448

with the Pkc1-Mpk1 pathway. However, the heat shock-induced activation of Mpk1 was

449

previously shown to be controlled independently of Tor2 (23, 55). We demonstrated that

450

TORC2-Pkc1 signaling was involved in the MG-induced activation of the Mpk1 MAP

451

kinase cascade (a schematic model is shown in Fig. 8). However, since the MG-induced

452

phosphorylation of Mpk1 occurred in wsc1∆mid2∆ cells as well as in mutants defective in

453

other sensor proteins (Wsc2 or Mtl1), MG appeared to trigger TORC2-Pkc1 signaling via

454

different routes from the known pathway for the heat shock-induced activation of the

455

Pkc1-Mpk1 MAP kinase cascade (Fig. 8).

456

We have previously demonstrated that MG treatment triggers other cellular

457

responses including HOG (high osmolarity glycerol) pathway and Ca2+/calcineurin

458

signaling pathway (10, 12). The cytoplasmic membrane proteins Sln1 and Sho1 played as

459

MG sensors in activation of HOG pathway, although the phosphorylation of Mpk1 21

460

following the treatment with MG occurred in mutants defective in both branches (data not

461

shown). On the other hand, it was reported that both Mpk1 and calcineurin were involved

462

in the regulatory mechanism of G2/M delay by high dose of Ca2+ (56). However, the

463

MG-induced phosphorylation of Mpk1 occurred in the presence of Ca2+ chelator EGTA

464

(data not shown), under conditions by which we have previously shown that the uptake of

465

Ca2+ into the cell was inhibited and subsequently activation of Ca2+/calcineurin signaling

466

did not occur (12). Collectively, the MG-triggered stimulation of TORC2-Pkc1 signaling

467

does not seem to crosstalk with other MG-induced cellular responses.

468

The phosphorylation levels of Ser1143 in the HM of Pkc1 were increased

469

following the treatment with MG, whereas those of Thr1125 in the TM were not (Fig. 5C and

470

D). Nevertheless, the MG-induced phosphorylation of Mpk1 did not occur in the Pkc1T1125A

471

mutant (Fig. 5B). In mammalian cells, the phosphorylation of Akt at Thr450 in the TM

472

secured the stability of the Akt protein per se (35, 50). We showed that Pkc1 protein levels

473

were markedly decreased when Thr1125 in the TM of Pkc1 was changed to Ala (Fig. 5F).

474

Therefore, the phosphorylation of this site appears to affect the stability of Pkc1 in S.

475

cerevisiae cells. In addition, intriguingly, we noticed that the phosphorylation level of

476

Ser1143 in Pkc1T1125A was significantly lower than that in Pkc1WT (Fig. 3E). This may be

477

another reason why the phosphorylation of Mpk1 did not occur in Pkc1T1125A cells

478

following the treatment with MG. Since not only the Pkc1 protein levels but also

479

phosphorylation levels of Sre1143 were lower in Pkc1T1125A, the MG-triggered signal was not

480

adequately transmitted to the Mpk1 MAP kinase cascade in Pkc1T1125A cells. Therefore, the

481

maintenance of a certain level of phosphorylation at both Thr1125 and Ser1143 appeared to 22

482

secure the transmittance of the signal downstream to the Mpk1 MAP kinase cascade. To the

483

best of our knowledge, this is the first study to demonstrate that the phosphorylation status

484

of TM affected the phosphorylation levels of HM in AGC kinases.

485

The heat shock-induced activation of Mpk1 occurs through the activation of

486

Rho1 (38, 39) (Fig. 8). We showed that the basal phosphorylation levels of Pkc1 were

487

decreased by a deficiency in the Pkc1 domains (HR1 and C1 domains) needed for the

488

physical interaction with Rho1 (37, 47-49) (Fig. 4B). Previous studies reported that TORC2

489

was predominantly located in punctate structures adjacent to the cytoplasmic membrane in

490

S. cerevisiae cells (57, 58). Since TORC2 exists beneath the cytoplasmic membrane, its

491

substrate (Pkc1) also has to be present near the cytoplasmic membrane in which the enzyme

492

(TORC2) exists. Rho1 is associated with the plasma membrane (38, 59); therefore, the

493

physical interaction between Rho1 and Pkc1 may guarantee contact between TORC2 and

494

Pkc1. In addition to the physical interaction with Rho1, the C1 domain of Pkc1 has been

495

suggested to bind to lipids (49, 55). A previous study using a multicopy Pkc1-GFP showed

496

that Pkc1 localized to the bud cortex or bud neck (60). In addition, the C1 domain of Pkc1

497

was shown to be sufficient for its cell peripheral localization (60); therefore, the C1 domain

498

of Pkc1 may contribute to TORC2-Pkc1 signaling not only by interacting with Rho1, but

499

also by binding to lipids in the plasma membrane, thereby allowing Pkc1 to interact with

500

TORC2 beneath the cytoplasmic membrane. Overexpression of the C1 domain may

501

compete with endogenous Pkc1 to interfere with the interaction with Rho1, thereby

502

inhibiting the MG-induced phosphorylation of Mpk1 (Fig. 4D). A possible explanation for

503

these results is that both the HR1 and C1 domains are necessary for Pkc1 to be present near 23

504

the cytoplasmic membrane through a physical interaction with Rho1, whereby TORC2

505

phosphorylates Pkc1.

506

The physiological significance of the MG-induced activation of TORC2-Pkc1

507

signaling in yeast cells is the prevention of physiological disorders caused by MG. We here

508

demonstrated that MG also facilitated mTORC2 signaling in mammalian cells. Although

509

the mechanism by which MG activates TORC2 currently remains unknown, a study is now

510

being conducted to elucidate the mechanism underlying the sensing of MG and facilitation

511

of TORC2 signaling using yeast as a model organism, and also to determine the common

512

physiological significance of MG-induced TORC2 signaling in eukaryotes.

513 514

ACKNOWLEDGMENTS

515

We thank Drs. T. Kawada, N. Takahashi, T. Goto, Y. Kim, S. Izawa, and K. Maeta for their

516

technical support and helpful discussions. We are grateful to Drs. M. N. Hall, D. E. Levin, T.

517

Miyakawa, J. Thorner, Y. Ohya, J. J. Heinisch, M. Y. Chen, M. S. Cyert, J. R. Broach, K.

518

Irie, and the National Bio-Resource Project (NBRP) of the MEXT, Japan for providing the

519

plasmids and yeast strains.

520 521

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58. Sturgill TW, Cohen A, Diefenbacher M, Trautwein M, Martin DE, Hall MN.

681

2008. TOR1 and TOR2 have distinct locations in live cells. Eukaryot. Cell

682

7:1819-1830.

683

59. Inoue SB, Qadota H, Arisawa M, Watanabe T, Ohya Y. 1999. Prenylation of

684

Rho1p is required for activation of yeast 1, 3--glucan synthase. J. Biol. Chem.

685

274:38119-24.

686

60. Denis V, Cyert MS. 2005. Molecular analysis reveals localization of Saccharomyces

687

cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the

688

nucleus and mitotic spindle. Eukaryot. Cell 4:36-45.

689

Figure legends

690

FIG 1 MG depolarizes the actin cytoskeleton. (A) Cells (YPH250) were cultured in SD

691

medium until A610=0.3, and then treated with 10 mM MG for 60 min. Cells were stained for

692

actin with rhodamine-phalloidin and observed using a fluorescence microscope. The

693

proportion of cells with depolarized actin was determined by counting cells in which actin

694

had not accumulated in the bud. Approximately >200 cells were counted in each

695

experiment. (B) Cells (YPH250) carrying YCp50 (vector) or YCp50-PKC1R398P were

696

cultured in SD medium until A610=0.3, and were then treated with 10 mM MG for the 32

697

prescribed time as indicated in the figure. Cells were stained for actin with

698

rhodamine-phalloidin. The numbers (%) in each picture indicate the proportion of cells

699

with depolarized actin. (C) Wild-type (DL100) and pkc1∆ (DL376) cells were cultured in

700

SD medium containing 0.5 M sorbitol until the log phase of growth, and diluted serially

701

(1:10) with 0.5 M sorbitol solution. An aliquot (4 µl) of each cell suspension was spotted

702

onto SD agar plates containing 0.5 M sorbitol with or without MG. (D) Cells (YPH250)

703

defective in the components of the Mpk1 MAP kinase cascade (bck1∆, mkk1∆mkk2∆, and

704

mpk1∆) were cultured in SD medium until the log phase of growth, diluted serially (1:10)

705

with 0.85% NaCl solution, and 4 µl of each cell suspension was then spotted onto SD agar

706

plates containing MG. (E) Cells (YPH250) carrying YCp50 (vector) or YCp50-PKC1R398P

707

were cultured in SD medium until the log phase of growth. The cell suspension was diluted

708

as described in (D), and 4 µl of each suspension was spotted onto SD agar plates containing

709

MG.

710 711

FIG 2 Effects of MG on activation of the Mpk1 MAPK cascade. (A) Cells (YPH250) were

712

cultured in SD medium until A610=0.3, and 10 mM MG was added. The phosphorylation of

713

Mpk1 (p-Mpk1) and the Mpk1 protein (Mpk1) were determined after an incubation for the

714

prescribed time. (B) Wild-type (DL100) and pkc1∆ (DL376) cells were cultured in SD

715

medium containing 1 M sorbitol until A610=0.3-0.5, and were treated with 10 mM MG for

716

the prescribed time. (C) Cells (YPH250) defective in the components of the Mpk1 MAP

717

kinase cascade (bck1∆, mkk1∆mkk2∆, and mpk1∆) were cultured in SD medium containing

718

1 M sorbitol until A610=0.3-0.5, and 10 mM MG was added. (D) Wild-type (YPH250), 33

719

wsc1∆, mid2∆, and wsc1∆mid2∆ cells were cultured in SD medium until A610=0.3-0.5,

720

treated with 10 mM MG for 30 min, and the phosphorylation of Mpk1 was then determined.

721

(E) The cells of each mutant in the YPH250 background were cultured in SD medium until

722

the log phase of growth, diluted serially (1:10) with a 0.85% NaCl solution, and 4 µl of each

723

cell suspension was then spotted onto SD agar plates containing MG.

724 725

FIG 3 TORC2 phosphorylates Pkc1. (A) A diagram of Pkc1. The HR-1 domain, C1 domain,

726

and C2 domain represent homology region 1, the cysteine-rich domain, and lipid-binding

727

domain, respectively. AGC kinases contain conserved Ser/Thr in turn motif (TM) and

728

hydrophobic motif (HM), which are phosphorylated by TOR kinases. Pkc1 also contains

729

Thr1125 in TM and Ser1143 in HM, both are potential phosphorylation sites of TORC2;

730

however, TORC2-dependent phosphorylation of these sites has not yet been biochemically

731

confirmed. (B) TORC2 components were immunoprecipitated using anti-myc antibodies

732

from wild-type (YPH250) cells with or without Avo3-myc carrying HA-Tor2, and

733

Bit61-FLAG. Each TORC2 component was determined using respective antibodies for

734

each Tag. (C) Immunoprecipitated TORC2 containing Avo3-myc from wild-type

735

(YPH250) cells was incubated with [γ-32P]ATP and recombinant Ypk2. The

736

phosphorylation of Ypk2 was detected by autoradiography. (D) The Pkc1 peptide of a

737

wild-type (WT) or Ala-substituted variant (T1125A/S1143A) was incubated with

738

immunopurified TORC2 and [-32P]ATP. After Tricine-SDS-PAGE, the phosphorylation of

739

each Pkc1 peptide was detected by autoradiography. (E) Cells (YPH250) carrying

740

PKC1-3FLAG (WT), PKC1T1125A-3FLAG (T1125A), or PKC1S1143A-3FLAG (S1143A) were 34

741

cultured in SD medium until A610=0.4-0.6. After the immunoprecipitation of FLAG-tagged

742

Pkc1 using the anti-FLAG-antibody-conjugated resin, samples were subjected to

743

SDS-PAGE followed by Western blotting using anti-phospho Pkc1T1125 antibodies [p-Pkc1

744

(p-T1125)], anti-phospho Pkc1S1143 antibodies [p-Pkc1 (p-S1143)], or an anti-FLAG

745

monoclonal antibody (Sigma). (F) Wild-type (TB50a) and GAL1p-AVO1 (RL25-1c) cells

746

carrying pFL39-PKC1-3HA were cultured overnight in SC/Gal medium (Gal). After being

747

transferred to SD medium (Glc), cells were cultured for 17 h to impair TORC2 activity.

748

After the immunoprecipitation of HA-tagged Pkc1 using the anti-HA-antibody-conjugated

749

resin, samples were subjected to SDS-PAGE followed by Western blotting using

750

anti-phospho Pkc1T1125 antibodies [p-Pkc1 (p-T1125)], anti-phospho Pkc1S1143 antibodies

751

[p-Pkc1 (p-S1143)], or the anti-HA monoclonal antibody (Cell Signaling).

752 753

FIG 4 The role of Rho1 in MG-triggered TORC2-Pkc1 signaling. (A) RHO1 (YOC764),

754

rho1-2

755

pFL39-PKC1-3HA were cultured in SD medium at 25ºC until A610=0.3-0.5, and then

756

shifted to 37ºC for 3 h. After the immunoprecipitation of HA-tagged Pkc1 using the

757

anti-HA-antibody-conjugated resin, samples were subjected to SDS-PAGE followed by

758

Western blotting using anti-phospho Pkc1T1125 antibodies (p-Pkc1 (p-T1125)), anti-phospho

759

Pkc1S1143 antibodies (p-Pkc1 (p-S1143)), or an anti-HA monoclonal antibody (Cell

760

Signaling).

761

pFL39-PKC1∆HR1-3HA (∆HR1), or pFL39-PKC14C/S-3HA (4C/S) were cultured in SD

762

medium until A610=0.4-0.6. Immunoprecipitation and Western blotting were performed as

(YOC752),

(B)

rho1-3

Cells

(YOC729),

(YPH250)

35

and

rho1-5

carrying

(YOC755)

cells

pFL39-PKC1-3HA

carrying

(WT),

763

described in (A). (C) pkc1∆ (DL376) cells carrying pFL39-PKC1-3HA (WT),

764

pFL39-PKC1∆HR1-3HA (∆HR1), or pFL39-PKC14C/S-3HA (4C/S) were cultured in SD

765

medium until A610=0.3-0.5, and 10 mM MG was added. The phosphorylation of Mpk1 was

766

determined after an incubation for 30 min. (D) Cells (YPH250) carrying the vector

767

(pRS426), ADH1p-PKC1C1 (C1WT), or ADH1p-PKC1C14C/S (C14C/S) were cultured in SD

768

medium until A610=0.3-0.5, and 10 mM MG was added. The phosphorylation of Mpk1 was

769

determined after an incubation for 30 min.

770 771

FIG 5 MG functions as an initiator of the TORC2-Pkc1 signaling pathway. (A)

772

GAL1p-TOR2 (JM340), GAL1p-AVO1 (RL25-1c), and GAL1p-AVO3 (RS61-5b) cells were

773

cultured overnight in SC/Gal medium. After being transferred to SD medium, cells were

774

cultured for 16 h (GAL1p-TOR2), 9 h (GAL1p-AVO1), or 10 h (GAL1p-AVO3) to impair

775

TORC2 activity, and were then treated with 10 mM MG for 30 min. (B) pkc1∆ (DL376)

776

cells carrying YEp352-PKC1 (pFR22), YEp352-PKC1T1125A, YEp352-PKC1S1143A

777

(pFR74), or YEp352-PKC1T1125A/S1143A were cultured in SD medium until A610=0.3-0.5, and

778

were then treated with 10 mM MG for the prescribed time. (C) Cells (YPH250) carrying

779

pFL39-PKC1-3HA were cultured in SD medium until A610=0.4-0.6, and 10 mM MG was

780

added.

781

anti-HA-antibody-conjugated resin, samples were subjected to SDS-PAGE followed by

782

Western blotting using anti-phospho Pkc1T1125 antibodies (p-T1125) or an anti-HA

783

monoclonal

784

YCplac111-PKC1-3HA were cultured in SD medium. Immunoprecipitation and Western

After

the

antibody

immunoprecipitation

(Cell

Signaling).

36

of

(D)

HA-tagged

Cells

Pkc1

(YPH250)

using

the

carrying

785

blotting were performed as described in (C), except for the usage of anti-phospho Pkc1S1143

786

antibodies (p-S1143). (E) Wild-type (TB50a) cells and GAL1p-AVO1 (RL25-1c) cells

787

carrying YCplac111-PKC1-3HA were cultured overnight in SC/Gal medium. After being

788

transferred to SD medium, cells were cultured for 15 h to impair TORC2 activity, and were

789

then treated with 10 mM MG for 60 min. Immunoprecipitation and Western blotting were

790

performed as described in (C). (F) pkc1∆ (DL376) cells carrying YEp352-PKC1 (pFR22),

791

YEp352-PKC1T1125A, or YEp352-PKC1S1143A (pFR74) were cultured in SD medium until

792

A610=0.4-0.6. DL376 cells carrying the vector (YEp352) were cultured in SD medium

793

containing 1 M sorbitol. Cell extracts were subjected to SDS-PAGE followed by Western

794

blotting to determine the protein levels of Pkc1 using anti-Pkc1 antibodies. As the loading

795

control, Pgk1 protein levels were determined using an anti-Pgk1 monoclonal antibody

796

(Molecular Probes). An asterisk indicates a nonspecific band.

797 798

FIG 6 Physiological significance of the phosphorylation states of Pkc1 at Thr1125 and

799

Ser1143 on the growth of yeast cells. (A) pkc1∆ cells (DL376) carrying various PKC1 alleles

800

(WT, PKC1WT; T1125A, PKC1T1125A; S1143A, PKC1S1143A; and T1125A/S1143A,

801

PKC1T1125A/S1143A) with YCp50 (CEN-type plasmid) were cultured in SD medium until the

802

log phase of growth. Cells were then diluted serially (1:10) with a 0.85% NaCl solution, and

803

4 µl of each cell suspension was spotted onto SD agar plates containing MG. Cells were

804

grown at 28ºC for 3 days. (B) pkc1∆ cells (DL376) carrying various PKC1 alleles (WT,

805

PKC1WT; ∆HR1, PKC1∆HR1; 4C/S, PKC14C/S) with pFL39 (CEN-type plasmid) were

806

streaked on SD agar plates with or without 1 M sorbitol, and cultured at 28ºC for 3 days. (C) 37

807

Cells with GAL1 promoter-driven AVO1 (RL25-1C) carrying various PKC1 alleles (WT,

808

PKC1WT;

809

PKC1T1125A/S1143A) with YEp352 (2µ-type plasmids) were cultured in the SC/Gal medium

810

until the log phase of growth. Cells were then diluted serially (1:10) with 0.85% NaCl

811

solution, and 4 µl of each cell suspension was spotted onto SC/Gal (Gal) or SD (Glc) agar

812

plates. Cells were cultured at 28ºC for 3 days.

T1125A,

PKC1T1125A;

S1143A,

PKC1S1143A;

and

T1125A/S1143A,

813 814

FIG 7 Effects of MG on mTORC2 signaling. (A) A diagram of Akt. The PH domain

815

represents the pleckstrin homology domain. (B) Mouse 3T3-L1 adipocytes were treated

816

with 5 mM MG. After being incubated for the prescribed time, the phosphorylation of

817

Thr450 in the turn motif (p-Akt TM) and Ser473 in the hydrophobic motif (p-Akt HM) of Akt

818

was determined using respective specific antibodies. (C) Mouse 3T3-L1 adipocytes were

819

pre-incubated with 250 nM Torin 1 (Torin) or 20 nM rapamycin (Rap) for 60 min before the

820

treatment with 5 mM MG for 60 min.

821 822

FIG 8 A schematic model for the integration of TORC2 into the Pkc1-Mpk1 MAP kinase

823

cascade in S. cerevisiae. Details are described in the text. Briefly, heat shock stress was

824

sensed by the stress sensor proteins Wsc1 and Mid2 on the cytoplasmic membrane, and the

825

heat shock signal was transmitted to Rom2 (guanine-nucleotide exchange factor for Rho1),

826

which in turn activated Rho1. GTP-bound Rho1 interacted with Pkc1, and Pkc1 activated

827

the Mpk1 MAP kinase cascade [the so-called cell wall integrity (CWI) pathway]. TORC2

828

phosphorylated Pkc1 at Thr1125 in the turn motif and at Ser1143 in the hydrophobic motif, and 38

829

the phosphorylation of these amino acid residues was necessary for transmitting the MG

830

signal downstream to the Mpk1 MAP kinase cascade. The phosphorylation level of Ser1143

831

in Pkc1 increased following the treatment with MG.

39

FIG 1

A

0

15

Actin

30

60

MG

15

30

(min)

52%

25%

wash

BF Cells with depolarized actin

23%

29 %

B

84%

C

MG 0

30

91%

60

(min)

MG (mM) 0

4

6

WT pkc1∆

vector 20%

85%

93%

D

PKC1R398P

0 18%

49%

E

55%

MG (mM) 0 vector PKC1R398P

MG (mM)

8

WT bck1∆ mkk1∆mkk2∆ mpk1∆

4

6

FIG 2

A 0

MG

15

30

45

60 90 120 150 180 （min）

p-Mpk1 Mpk1

B

WT MG

pkc1∆

0 15 30 45 60 90 0 15 30 45 60 90 (min)

p-Mpk1 Mpk1

C

mkk1∆ mkk2∆ mpk1∆ 0 30 60 0 30 60 0 30 60 0 30 60 (min) WT

MG

bck1∆

p-Mpk1 Mpk1

D WT

wsc1∆

mid2∆

wsc1∆ mid2∆

MG p-Mpk1 Mpk1

E

MG (mM) 0 WT wsc1∆ mid2∆ wsc1∆mid2∆

4

6

FIG 3

A TORC2

Thr1125

Pkc1

HR1

H2N

C2

C1

Ser1143

Kinase domain

COOH Turn motif Hydrophobic motif

B

C Avo3-myc

+

IP: myc

+

Avo3-myc

HA-Tor2

Ypk2

Kinase assay

Ypk2

CBB staining

Bit61-FLAG Avo3-myc anti-myc HA-Tor2

input

Bit61-FLAG Avo3-myc

D

T1125A Pkc1 WT WT S1143A Avo3-myc Kinase assay anti-myc

E

F Pkc1

p-Pkc1 (p-T1125)

Gal GAL1pWT AVO1

WT T1125A S1143A p-Pkc1 (p-T1125) p-Pkc1 (p-S1143)

p-Pkc1 (p-S1143) anti-HA anti-FLAG

Glc WT

GAL1pAVO1

FIG 4

A

B RHO1 rho1-2 rho1-3 rho1-5

p-Pkc1 (p-T1125)

Pkc1 WT ∆HR1 4C/S p-Pkc1 (p-T1125)

p-Pkc1 (p-S1143) p-Pkc1 (p-S1143) anti-HA anti-HA

C

D Pkc1

WT

∆HR1

vector

4C/S

MG

MG

p-Mpk1

p-Mpk1

Mpk1

Mpk1

C1WT

C14C/S

FIG 5

A WT

GAL1pTOR2

GAL1pWT AVO1

GAL1pWT AVO3

MG p-Mpk1 Mpk1

B Pkc1

WT

MG (min)

0 30 60

T1125A

S1143A

T1125A S1143A

0 30 60

0 30 60

0 30 60

p-Mpk1 Mpk1

C

D MG

MG p-Pkc1 (p-T1125)

p-Pkc1 (p-S1143)

anti-HA

anti-HA

E WT

GAL1p -AVO1

Pkc1

F

vector WT T1125A S1143A

MG p-Pkc1 (p-S1143)

anti-Pkc1

Pkc1

*

anti-HA anti-Pgk1

FIG 6

A

MG (mM) 0

4

6

WT T1125A Pkc1 S1143A T1125A/S1143A

B w/ Sorbitol

w/o Sorbitol

WT ∆HR1 vector

4C/S

C

GAL1p-AVO1 Gal vector

Pkc1

WT T1125A S1143A T1125A/S1143A

Glc

FIG 7

A

mTORC2

Thr450

Akt

H2N

PH

Kinase domain

Ser473 COOH

Turn motif Hydrophobic motif

B

3T3-L1 adipocytes MG

0

15

30 60 90 (min)

phospho-Akt TM phospho-Akt HM Akt

C

3T3-L1 adipocytes Rap Torin MG phospho-Akt HM Akt

+ + + + +

FIG 8

Heat shock stress Wsc1

Mid2 Rho1

T1125 P

Pkc1

Rom2

S1143 P

Bck1 Mpk1 MAPK cascade

Plasma membrane

MG

Mkk1/2 Mpk1

CWI pathway

P

TORC2