Role of phosphatidylserine in the activation of Rho1-related Pkc1 signaling in Saccharomyces cerevisiae
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)
- et al.
C1 domains exposed: from diacylglycerol binding to protein-protein interactions
Biochim. Biophys. Acta
(2006) 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)- 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) - et al.
Protein kinase C in yeast. Characteristics of the Saccharomyces cerevisiae PKC1 gene product
J. Biol. Chem.
(1994) - 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) - et al.
Activation of yeast protein kinase C by Rho1 GTPase
J. Biol. Chem.
(1996) - et al.
Mutants affected in the putative diacylglycerol binding site of yeast protein kinase C
FEBS Lett.
(1997) - et al.
Protein kinase C regulatory domains: the art of decoding many different signals in membranes
Biochim. Biophys. Acta
(2006) Regulation of protein kinase C
Curr. Opin. Cell Biol.
(1997)- et al.
Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae
Biochim. Biophys. Acta
(2007)
PI4P and Rab inputs collaborate in myosin-V-dependent transport of secretory compartments in yeast
Dev. Cell
Nutrients and the Pkh1/2 and Pkc1 protein kinases control mRNA decay and P-body assembly in yeast
J. Biol. Chem.
A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle
Cell
Regulation of yeast CTP synthetase activity by protein kinase C
J. Biol. Chem.
Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids
J. Biol. Chem.
Roles of ionic residues of the C1 domain in protein kinase C-α activation and the origin of phosphatidylserine specificity
J. Biol. Chem.
Crystal structure of the Cys2 activator-binding domain of protein kinase C δ in complex with phorbol ester
Cell
Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains
J. Biol. Chem.
Cdc42 interacts with the exocyst and regulates polarized secretion
J. Biol. Chem.
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.
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.
Cited by (17)
Role of RhoGAP Rgd1 in Pkc1 signaling-related actin repolarization under heat shock stress in Saccharomyces cerevisiae
2021, Biochimica et Biophysica Acta - General SubjectsCitation 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).
Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase
2018, Advances in Biological RegulationYeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis
2017, Journal of Biological ChemistryCitation 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.
Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol
2017, Journal of Lipid ResearchCitation 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.
- 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.