Role of phosphatidylserine in the activation of Rho1-related Pkc1 signaling in Saccharomyces cerevisiae

doi:10.1016/j.cellsig.2017.01.002

Cellular Signalling

Volume 31, February 2017, Pages 146-153

https://doi.org/10.1016/j.cellsig.2017.01.002 Get rights and content

Highlights

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The C1 domain of yeast Pkc1 has an ability to interact with phosphatidylserine.
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Phosphatidylserine is necessary for the activation of Mpk1 MAP kinase cascade.
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Phosphatidylserine is involved in the interaction between Rho1 and Pkc1.

Abstract

Protein kinase C (PKC) belongs to a family of serine/threonine kinases and is evolutionary conserved among eukaryotes. It contains several functional domains, with the C1 domain being identified as a membrane-targeting module. Diacylglycerol (DAG) and phorbol esters bind to the C1 domain to enhance its kinase activity. The C1 domain is conserved in PKC (Pkc1) in the budding yeast Saccharomyces cerevisiae; however, its kinase activity does not respond to DAG. Although the C1 domain of Pkc1 physically interacts with the small GTPase Rho1, the interaction between C1 domain and lipids has not yet been characterized. We herein provide evidence to show the physical interaction between the C1 domain of Pkc1 and phosphatidylserine (PS), but not DAG. The stress-induced activation of Pkc1 signaling was abolished in a cho1 mutant, which was defective in PS synthase. The deletion of CHO1 perturbed the appropriate localization of Pkc1 at the bud tip, and impaired the physical interaction between Pkc1 and GTP-bound Rho1 in vivo. Our results suggest that PS is necessary for Pkc1 signaling due to its role in regulating the localization of Pkc1 as well as the physical interaction between Rho1 and Pkc1.

Introduction

Cells are continuously exposed to changes in environmental conditions, and, thus, are equipped with a number of different machineries to sense and respond to various extracellular environments. Signal transduction pathways are important for transmitting extracellular stress signals to cells in order to adapt to changes in the extracellular environment. Protein kinase C (PKC) belongs to a family of serine/threonine kinases [1], which are involved in several critical signal transduction pathways, and is broadly found in eukaryotes [2].

To date, at least 10 isozymes of PKCs and three PKC-related kinases have been identified in mammals [2], [3]. PKC isozymes have been classified into three groups based on differences in their domain structures, i.e., conventional PKCs (cPKCs), novel PKCs (nPKCs), and atypical PKCs (aPKCs). The conserved region 1 (C1) domain is common among the PKC isozymes. The C1 domains of cPKCs and nPKCs consist of a tandem repeat of a cysteine-rich motif that forms a zinc finger structure, termed C1a and C1b [2], [3]. A single copy of the motif is present in the C1 domain of aPKCs. The C1 domain is regarded as a membrane-targeting module that binds to diacylglycerol (DAG) and phorbol 12-myristate 13-acetate (PMA) [2], [3]. Since DAG and PMA activate cPKCs and nPKCs, the C1 domain is considered to be involved in the activation of PKCs [2]. Besides the activation of kinase activity, the binding of the C1 domain to DAG plays an important role in targeting PKCs to the cytoplasmic membrane [4].

In the budding yeast Saccharomyces cerevisiae, PKC1 is the sole gene that codes for PKC. The gene product (Pkc1) is regarded as an archetype of PKC because it possesses every functional domain of PKC, including the C1 domain, in one molecule [5], [6]. Pkc1 plays an important role in the regulation of polarized growth and stress responses, particularly in the control of the cell wall integrity (CWI) signaling pathway, which involves the remodeling of cell walls and expression of stress response genes [7], [8]. The activation of the CWI pathway is induced by heat shock stress and cell wall stresses such as treatments with cell wall-perturbing agents (zymolyase, Calcofluor white, or Congo red) [7], [9], [10]. The Mpk1 mitogen-activated protein kinase (MAPK) cascade, in which Bck1 is a MAPK kinase kinase, Mkk1 and Mkk2 are redundant MAPK kinases, and Mpk1 is a MAPK, consists of the CWI pathway [7], [8]. Transcription factors such as Rlm1 [11], [12] and Swi4/Swi6 [7] function downstream of the Mpk1 MAPK cascade, thereby facilitating the expression of genes involved in cell wall synthesis and stress responses [7], [8], [13]. Pkc1 is an upstream module for the Mpk1 MAPK cascade, and phosphorylates and activates Bck1 [9]. In heat shock stress responses, the membrane proteins Wsc1 and Mid2 function as heat shock stress sensors, activating the small GTPase Rho1 through the actions of Rom1 and Rom2 [14], which are guanine nucleotide exchange factors (GEFs) downstream of Wsc1/Mid2, and heat shock signals are subsequently transmitted to the Pkc1-Mpk1 MAPK cascade [7], [8].

Unlike mammalian PKCs, yeast Pkc1 does not require DAG or PMA for its kinase activity [7], [15], [16]; however, the C1 domain is conserved in Pkc1 [5], [6]. Previous studies reported that the C1 domain of Pkc1 interacted with GTP-bound Rho1 in order to activate Pkc1 [17], [18], and homology region 1 (HR1), which is an N-terminal domain of Pkc1, is also involved in the interaction between GTP-bound Rho1 and Pkc1 [19]. Therefore, the C1 domain of Pkc1 appears to contribute to the activation of Pkc1 through a physical interaction with Rho1; however, it currently remains unclear whether the C1 domain of Pkc1 has the potential to bind to DAG and other phospholipids.

In the present study, we showed that the C1 domain of Pkc1 bound to phosphatidylserine (PS), but not to DAG. We also found that the deletion of CHO1, encoding PS synthase, impaired the stress-inducible activation of the Pkc1-Mpk1 MAPK cascade as well as the appropriate localization of Pkc1 in S. cerevisiae cells. We demonstrated that PS was involved in the interaction between GTP-bound Rho1 and Pkc1 in vivo. Our results provide an insight into the physiological role of PS in yeast cells in terms of the activation of Pkc1 signaling through its role in regulating the cellular localization of Pkc1 as well as the physical interaction between Rho1 and Pkc1.

Section snippets

Medium

The medium used was synthetic dextrose (SD) (2% glucose, 0.67% yeast nitrogen base without amino acids). Appropriate amino acids and bases were added as necessary. Ethanolamine (1 mM) was added to the medium to support the growth of a mutant defective in CHO1.

Chemicals

Methylglyoxal and ethanolamine were purchased from Sigma. Hoechst 33342 was obtained from Invitrogen.

Strains

The S. cerevisiae strains used were YPH250 (MATa trp1-∆ 1 his3-∆ 200 leu2-∆ 1 lys2-801 ade2-101 ura3-52) and its isogenic cho1::TRP1 mutant,

The C1 domain of Pkc1 is necessary for the membrane targeting of Pkc1

The C1 domain of mammalian PKCs binds to DAG, which is important not only for facilitating PKC kinase activity, but also targeting PKC to the cytoplasmic membrane [3], [30], [31]. In contrast to mammalian PKCs, yeast Pkc1 does not require DAG for its kinase activity [7], [15], [16]; nevertheless, Pkc1 was observed in the cytoplasmic membrane, i.e. microscopic studies showed that GFP-tagged Pkc1 with a multicopy vector preferentially localized to the bud tip and bud neck [22]. We confirmed the

Discussion

Mammalian PKC isoforms and their related kinases play distinct physiological roles in a wide variety of biological aspects. On the other hand, in the budding yeast S. cerevisiae, a sole PKC or Pkc1 is involved in a number of biological processes, such as signal transduction [6], [7], [39], cell wall organization [7], [39], cell polarity [7], [40], P-body assembly [41], the cell cycle [8], [42], gene expression [8], [12], [43], [44], nucleic acid synthesis [45], and septin organization [46].

Funding

This work was partially supported by a Grant-in-Aid for JSPS Fellows (W. N.) from the Japan Society for the Promotion of Science (JSPS) [grant number JP12J00522], and by the Uehara Memorial Foundation (Y. I.).

Author contributions

W. Nomura and Y. Inoue designed experiments, and W. Nomura and Y. Ito performed experiments. W. Nomura and Y. Inoue analyzed the data and wrote the manuscript.

Acknowledgements

We are grateful to Drs. D.E. Levin, Y. Ohya, J.J. Heinisch, M.S. Cyert, and S. Grinstein for providing the plasmids and yeast strains.

References (58)

F. Colón-González et al.
C1 domains exposed: from diacylglycerol binding to protein-protein interactions
Biochim. Biophys. Acta
(2006)
J.J. Heinisch
Baker's yeast as a tool for the development of antifungal kinase inhibitors--targeting protein kinase C and the cell integrity pathway
Biochim. Biophys. Acta
(2005)
K.Y. Kim et al.
Yeast Mpk1 mitogen-activated protein kinase activates transcription through Swi4/Swi6 by a noncatalytic mechanism that requires upstream signal
Mol. Cell. Biol.
(2008)
B. Antonsson et al.
Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product
J. Biol. Chem.
(1994)
M. Watanabe et al.
Saccharomyces cerevisiae PKC1 encodes a protein kinase C (PKC) homolog with a substrate specificity similar to that of mammalian PKC
J. Biol. Chem.
(1994)
Y. Kamada et al.
Activation of yeast protein kinase C by Rho1 GTPase
J. Biol. Chem.
(1996)
J.J. Jacoby et al.
Mutants affected in the putative diacylglycerol binding site of yeast protein kinase C
FEBS Lett.
(1997)
S. Corbalán-García et al.
Protein kinase C regulatory domains: the art of decoding many different signals in membranes
Biochim. Biophys. Acta
(2006)
A.C. Newton
Regulation of protein kinase C
Curr. Opin. Cell Biol.
(1997)
T. Strahl et al.
Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae
Biochim. Biophys. Acta
(2007)

F.H. Santiago-Tirado et al.

PI4P and Rab inputs collaborate in myosin-V-dependent transport of secretory compartments in yeast

Dev. Cell

(2011)

G. Luo et al.

Nutrients and the Pkh1/2 and Pkc1 protein kinases control mRNA decay and P-body assembly in yeast

J. Biol. Chem.

(2011)

D.E. Levin et al.

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle

Cell

(1990)

W.L. Yang et al.

Regulation of yeast CTP synthetase activity by protein kinase C

J. Biol. Chem.

(1996)

M.G. Kazanietz et al.

Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids

J. Biol. Chem.

(1995)

L. Bittova et al.

Roles of ionic residues of the C1 domain in protein kinase C-α activation and the origin of phosphatidylserine specificity

J. Biol. Chem.

(2001)

G. Zhang et al.

Crystal structure of the Cys2 activator-binding domain of protein kinase C δ in complex with phorbol ester

Cell

(1995)

M.D. Stewart et al.

Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains

J. Biol. Chem.

(2014)

X. Zhang et al.

Cdc42 interacts with the exocyst and regulates polarized secretion

J. Biol. Chem.

(2001)

F. Vilella et al.

Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress

J. Biol. Chem.

(2005)

Y. Nishizuka

Protein kinase C and lipid signaling for sustained cellular responses

FASEB J.

(1995)

C. Rosse et al.

PKC and the control of localized signal dynamics

Nat. Rev. Mol. Cell Biol.

(2010)

S.F. Steinberg

Structural basis of protein kinase C isoform function

Physiol. Rev.

(2008)

H. Mellor et al.

The extended protein kinase C superfamily

Biochem. J.

(1998)

D.E. Levin

Cell wall integrity signaling in Saccharomyces cerevisiae

Microbiol. Mol. Biol. Rev.

(2005)

D.E. Levin

Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway

Genetics

(2011)

Y. Kamada et al.

The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response

Genes Dev.

(1995)

H. de Nobel et al.

Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance

Microbiology

(2000)

E. Dodou et al.

The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway

Mol. Cell. Biol.

(1997)

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Role of RhoGAP Rgd1 in Pkc1 signaling-related actin repolarization under heat shock stress in Saccharomyces cerevisiae
2021, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
The heat shock stress-induced activation of Pkc1 is known to be mediated through its physical interaction with the GTP-bound form of Rho1, for which the C1 domain of Pkc1 is crucial [7,10,11,34]. The substitution of 4 cysteine residues (Cys442, Cys445, Cys512, and Cys515) in the C1 domain with serines (PKC14C/S) impairs this physical interaction with Rho1 [34,35]. The heat shock stress-induced phosphorylation of Mpk1 was impaired in the PKC14C/S mutant, where the increase in the Rgd1 phosphorylation level was also significantly attenuated (Fig. 2B).
A serine/threonine kinase Pkc1 is the sole protein kinase C in the budding yeast Saccharomyces cerevisiae, and plays an important role in the regulation of polarized growth and stress responses such as those due to heat shock. Exposure of cells to high temperature transiently arrests polarized growth and leads to depolarization of the actin cytoskeleton, followed by actin repolarization during adaptation to heat shock stress. Actin repolarization is ensured by the activation of Pkc1 signaling; however, the molecular mechanisms underlying this phenomenon remain poorly understood.
Using an overexpression construct of a constitutively active mutant of Pkc1 (Pkc1^R398P), we explored the Pkc1 target molecules involved in actin repolarization.
PKC1^R398P overexpression as well as heat shock stress increased the phosphorylation levels of Rho GTPase-activating protein (RhoGAP) Rgd1. Rgd1 was found to contribute to Pkc1-signaling-related actin repolarization during adaptation to heat shock stress in a GAP activity-independent manner, with Ser148 in Rgd1 playing a crucial role. Furthermore, Rgd1 was involved in the maintenance of phosphorylation status of the mitogen-activated protein (MAP) kinase Mpk1, a downstream effector of Pkc1, under heat shock stress.
Rgd1 is a target of Pkc1 signaling under conditions of heat shock stress, and required for the normal process of actin repolarization during adaptation to heat shock stress.
Our results provide insights into the molecular mechanism underlying Pkc1-mediated modulation of actin repolarization under heat shock stress.
Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase
2018, Advances in Biological Regulation
The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UAS_INO), a cis-acting element for the phosphatidate-controlled Henry (Ino2–Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme.
Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae
2017, Journal of Biological Chemistry
Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and this 2-oxoaldehyde has been implicated in some diseases including diabetes. However, the physiological significance of MG for cellular functions is yet to be fully elucidated. We previously reported that MG activates the Mpk1 (MAPK) cascade in the yeast Saccharomyces cerevisiae. To gain further insights into the cellular functions and responses to MG, we herein screened yeast-deletion mutant collections for susceptibility to MG. We found that mutants defective in the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) are more susceptible to MG. PtdIns(3,5)P₂ levels increased following MG treatment, and vacuolar morphology concomitantly changed to a single swollen shape. MG activated the Pkc1–Mpk1 MAPK cascade in which a small GTPase Rho1 plays a crucial role, and the MG-induced phosphorylation of Mpk1 was impaired in mutants defective in the PtdIns(3,5)P₂ biosynthetic pathway. Of note, heat shock-induced stress also provoked Mpk1 phosphorylation in a Rho1-dependent manner; however, PtdIns(3,5)P₂ was dispensable for the heat shock-stimulated activation of this signaling pathway. Our results suggest that PtdIns(3,5)P₂ is specifically involved in the MG-induced activation of the Mpk1 MAPK cascade and in the cellular adaptation to MG-induced stress.
Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis
2017, Journal of Biological Chemistry
Citation Excerpt :
The cho1Δ mutant lacks the ability to synthesize PS (21, 22) and thus requires the supplementation of choline or ethanolamine to synthesize PC or PE by the Kennedy pathway (1, 2). Studies with cells lacking Cho1 have revealed that PS is required for protein kinase C function (23, 24), tryptophan transport (25), vacuole function and morphogenesis (26), and direction of endocytic proteins to the plasma membrane (27). Cho1 and Pah1 are regulated for their functions in lipid metabolism by genetic mechanisms.
The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP–diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1. Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UAS_INO), a cis-acting element for the phosphatidate-controlled Henry (Ino2–Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UAS_INO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.
Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol
2017, Journal of Lipid Research
Citation Excerpt :
In any event, when a lipid metabolic protein was used as substrate, its maximum phosphorylation by Pkc1 was dependent on PS and DAG and, in particular, on the phospholipid. In a protein-lipid overlay assay, the GST-C1 (lipid binding domain of Pkc1) has been shown to interact with PS, but not with DAG (62). Here, we demonstrated that Pkc1 binds to liposomes in a PS-dependent manner, and the protein-lipid interaction occurs in the absence of Rho1 GTPase.
Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo.
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¹: Present address: Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.

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Role of phosphatidylserine in the activation of Rho1-related Pkc1 signaling in Saccharomyces cerevisiae

Highlights

Abstract

Introduction

Section snippets

Medium

Chemicals

Strains

The C1 domain of Pkc1 is necessary for the membrane targeting of Pkc1

Discussion

Funding

Author contributions

Acknowledgements

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Mol. Cell. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta

Curr. Opin. Cell Biol.

Biochim. Biophys. Acta

Dev. Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Protein kinase C and lipid signaling for sustained cellular responses

FASEB J.

PKC and the control of localized signal dynamics

Nat. Rev. Mol. Cell Biol.

Structural basis of protein kinase C isoform function

Physiol. Rev.

The extended protein kinase C superfamily

Biochem. J.

Cell wall integrity signaling in Saccharomyces cerevisiae

Microbiol. Mol. Biol. Rev.

Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway

Genetics

The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response

Genes Dev.

Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZ expression, glucanase resistance and thermotolerance

Microbiology

The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway

Mol. Cell. Biol.