Cross-regulation between CDK and MAPK control cellular fate

Abstract

Background: Commitment to a new cell cycle is controlled by a number of cellular signals. Mitogen-activated protein kinase (MAPK) pathways, which transduce multiple extracellular cues, have been shown to be interconnected with the cell cycle and can modulate its progression.

Methods: In budding yeast, we have introduced fluorescent biosensors that monitor in real time the signaling activity of the MAPKs Fus3 and Kss1 and the cyclin-dependent kinase (CDK) in individual cells. We have quantified in hundreds of live single cells the interplay between the MAPKs regulating the mating response and the CDK controlling cell cycle progression.

Results: Different patterns of MAPK activity dynamics could be identified by clustering cells based on their CDK activity, denoting the tight relationship between these two cellular signals. Our data suggest that beyond the already well-established mechanisms of regulation between the MAPK and the CDK, additional mechanisms remain to be identified.

Conclusion: A tight interplay between MAPK pathways and the cell cycle is essential to control cellular proliferation and cell fate decisions.

Author summary

Quantitative measurements of signal transduction in single cells can reveal how cells integrate various cues in order to select a cellular fate. In this study, we have monitored the interplay between the mating response and the cell cycle in budding yeast. By clustering single cell traces, based on their cell cycle stage, we can observe different signaling activity patterns in the mating pathway.

Graphical abstract

Keywords

MAPK signaling / cell cycle / yeast mating / single cell analysis / fluorescent biosensors

References

[1] Roux, P. P. and Blenis, J. (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev., 68, 320–344

[2] Chen, R. E. and Thorner, J. (2007) Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1773, 1311–1340

[3] Strickfaden, S. C., Winters, M. J., Ben-Ari, G., Lamson, R. E., Tyers, M. and Pryciak, P. M. (2007) A mechanism for cell-cycle regulation of MAP kinase signaling in a yeast differentiation pathway. Cell, 128, 519–531

[4] Nagiec, M. J. and Dohlman, H. G. (2012) Checkpoints in a yeast differentiation pathway coordinate signaling during hyperosmotic stress. PLoS Genet., 8, e1002437

[5] Clement, S. T., Dixit, G. and Dohlman, H. G. (2013) Regulation of yeast G protein signaling by the kinases that activate the AMPK homolog Snf1. Sci. Signal., 6, ra78

[6] Saito, H. (2010) Regulation of cross-talk in yeast MAPK signaling pathways. Curr. Opin. Microbiol., 13, 677–683

[7] Sharifian, H., Lampert, F., Stojanovski, K., Regot, S., Vaga, S., Buser, R., Lee, S. S., Koeppl, H., Posas, F., Pelet, S., (2015) Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade. Integr. Biol ., 7, 412–422

[8] Oehlen, L. J. and Cross, F. R. (1994) G1 cyclins CLN1 and CLN2 repress the mating factor response pathway at Start in the yeast cell cycle. Genes Dev., 8, 1058–1070

[9] Peter, M. and Herskowitz, I. (1994) Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Far1. Science, 265, 1228–1231

[10] Wassmann, K. and Ammerer, G. (1997) Overexpression of the G1-cyclin gene CLN2 represses the mating pathway in Saccharomyces cerevisiae at the level of the MEKK Ste11. J. Biol. Chem., 272, 13180–13188

[11] Escoté, X., Zapater, M., Clotet, J. and Posas, F. (2004) Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol., 6, 997–1002

[12] Clotet, J. and Posas, F. (2007) Control of cell cycle in response to osmostress: lessons from yeast. Methods Enzymol., 428, 63–76

[13] Bardwell, L. (2005) A walk-through of the yeast mating pheromone response pathway. Peptides, 26, 339–350

[14] Atay, O. and Skotheim, J. M. (2017) Spatial and temporal signal processing and decision making by MAPK pathways. J. Cell Biol., 216, 317–330

[15] Peter, M., Gartner, A., Horecka, J., Ammerer, G. and Herskowitz, I. (1993) FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell, 73, 747–760

[16] McKinney, J. D., Chang, F., Heintz, N. and Cross, F. R. (1993) Negative regulation of FAR1 at the Start of the yeast cell cycle. Genes Dev., 7, 833–843

[17] Repetto, M. V., Winters, M. J., Bush, A., Reiter, W., Hollenstein, D. M., Ammerer, G., Pryciak, P. M. and Colman-Lerner, A. (2018) CDK and MAPK synergistically regulate signaling dynamics via a shared multi-site phosphorylation region on the scaffold protein Ste5. Mol. Cell, 69, 938–952.e6

[18] Durandau, E., Aymoz, D. and Pelet, S. (2015) Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors. BMC Biol., 13, 55

[19] Conlon, P., Gelin-Licht, R., Ganesan, A., Zhang, J. and Levchenko, A. (2016) Single-cell dynamics and variability of MAPK activity in a yeast differentiation pathway. Proc. Natl. Acad. Sci. USA, 113, E5896–E5905

[20] Doncic, A., Atay, O., Valk, E., Grande, A., Bush, A., Vasen, G., Colman-Lerner, A., Loog, M. and Skotheim, J. M. (2015) Compartmentalization of a bistable switch enables memory to cross a feedback-driven transition. Cell, 160, 1182–1195

[21] Reményi, A., Good, M. C., Bhattacharyya, R. P. and Lim, W. A. (2005) The role of docking interactions in mediating signaling input, output, and discrimination in the yeast MAPK network. Mol. Cell, 20, 951–962

[22] Pelet, S., Dechant, R., Lee, S. S., van Drogen, F. and Peter, M. (2012) An integrated image analysis platform to quantify signal transduction in single cells. Integr. Biol., 4, 1274–1282

[23] Bean, J. M., Siggia, E. D. and Cross, F. R. (2006) Coherence and timing of cell cycle start examined at single-cell resolution. Mol. Cell, 21, 3–14

[24] Doncic, A., Falleur-Fettig, M. and Skotheim, J. M. (2011) Distinct interactions select and maintain a specific cell fate. Mol. Cell, 43, 528–539

[25] Costanzo, M., Nishikawa, J. L., Tang, X., Millman, J. S., Schub, O., Breitkreuz, K., Dewar, D., Rupes, I., Andrews, B. and Tyers, M. (2004) CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell, 117, 899–913

[26] de Bruin, R. A. M., McDonald, W. H., Kalashnikova, T. I., Yates, J. 3rd and Wittenberg, C. (2004) Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5. Cell, 117, 887–898

[27] Charvin, G., Cross, F. R. and Siggia, E. D. (2008) A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells. PLoS One, 3, e1468

[28] Pelet, S. (2017) Nuclear relocation of Kss1 contributes to the specificity of the mating response. Sci. Rep., 7, 43636

[29] Roy, A., Lu, C. F., Marykwas, D. L., Lipke, P. N. and Kurjan, J. (1991) The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin. Mol. Cell. Biol., 11, 4196–4206

[30] Oehlen, L. J., McKinney, J. D. and Cross, F. R. (1996) Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1. Mol. Cell. Biol., 16, 2830–2837

[31] Aymoz, D., Solé, C., Pierre, J.-J., Schmitt, M., de Nadal, E., Posas, F. and Pelet, S. (2018) Timing of gene expression in a cell-fate decision system. Mol. Syst. Biol., 14, e8024

[32] Hao, N., Yildirim, N., Wang, Y., Elston, T. C. and Dohlman, H. G. (2003) Regulators of G protein signaling and transient activation of signaling: experimental and computational analysis reveals negative and positive feedback controls on G protein activity. J. Biol. Chem., 278, 46506–46515

[33] Bhattacharyya, R. P., Reményi, A., Good, M. C., Bashor, C. J., Falick, A. M. and Lim, W. A. (2006) The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science, 311, 822–826

[34] Yu, R. C., Pesce, C. G., Colman-Lerner, A., Lok, L., Pincus, D., Serra, E., Holl, M., Benjamin, K., Gordon, A. and Brent, R. (2008) Negative feedback that improves information transmission in yeast signalling. Nature, 456, 755–761

[35] Nagiec, M. J., McCarter, P. C., Kelley, J. B., Dixit, G., Elston, T. C. and Dohlman, H. G. (2015) Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK. Mol. Biol. Cell, 26, 3359–3371

[36] Hartwell, L. H., Culotti, J., Pringle, J. R. and Reid, B. J. (1974) Genetic control of the cell division cycle in yeast: A model to account for the order of cell cycle events is deduced from the phenotypes of yeast mutants. Science, 183, 46–51

[37] Dirick, L., Böhm, T. and Nasmyth, K. (1995) Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J., 14, 4803–4813

[38] Chang, F. and Herskowitz, I. (1990) Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell, 63, 999–1011

[39] Tyers, M. and Futcher, B. (1993) Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes. Mol. Cell. Biol., 13, 5659–5669

[40] Bishop, A. C., Ubersax, J. A., Petsch, D. T., Matheos, D. P., Gray, N. S., Blethrow, J., Shimizu, E., Tsien, J. Z., Schultz, P. G., Rose, M. D., (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature, 407, 395–401

[41] Bhaduri, S. and Pryciak, P. M. (2011) Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes. Curr. Biol., 21, 1615–1623

[42] Shou, W., Seol, J. H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z. W., Jang, J., Shevchenko, A., Charbonneau, H. and Deshaies, R. J. (1999) Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell, 97, 233–244

[43] Visintin, R., Hwang, E. S. and Amon, A. (1999) Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature, 398, 818–823

[44] Mohl, D. A., Huddleston, M. J., Collingwood, T. S., Annan, R. S. and Deshaies, R. J. (2009) Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis. J. Cell Biol., 184, 527–539

[45] Tyers, M. (1996) The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start. Proc. Natl. Acad. Sci. USA, 93, 7772–7776

[46] Oehlen, L. J., Jeoung, D. I. and Cross, F. R. (1998) Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast. Mol. Gen. Genet., 258, 183–198

[47] Cherkasova, V., Lyons, D. M. and Elion, E. A. (1999) Fus3p and Kss1p control G1 arrest in Saccharomyces cerevisiae through a balance of distinct arrest and proliferative functions that operate in parallel with Far1p. Genetics, 151, 989–1004

[48] Oehlen, L. J. and Cross, F. R. (1998) Potential regulation of Ste20 function by the Cln1-Cdc28 and Cln2-Cdc28 cyclin-dependent protein kinases. J. Biol. Chem., 273, 25089–25097

[49] Wu, C., Leeuw, T., Leberer, E., Thomas, D. Y. and Whiteway, M. (1998) Cell cycle- and Cln2p-Cdc28p-dependent phosphorylation of the yeast Ste20p protein kinase. J. Biol. Chem., 273, 28107–28115

[50] Oda, Y., Huang, K., Cross, F. R., Cowburn, D. and Chait, B. T. (1999) Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA, 96, 6591–6596

[51] Meloche, S. and Pouysségur, J. (2007) The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene, 26, 3227–3239

[52] Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., (2002) Mutations of the BRAF gene in human cancer. Nature, 417, 949–954

[53] Marshall, C. J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell, 80, 179–185

[54] Pumiglia, K. M. and Decker, S. J. (1997) Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. USA, 94, 448–452

[55] Orford, K. W. and Scadden, D. T. (2008) Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat. Rev. Genet., 9, 115–128

[56] Wosika, V., Durandau, E., Varidel, C., Aymoz, D., Schmitt, M. and Pelet, S. (2016) New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast. Mol. Genet. Genomics, 291, 2231–2240

[57] Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. and Pringle, J. R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 14, 953–961

[58] Goldstein, A. L. and McCusker, J. H. (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast, 15, 1541–1553

[59] Edelstein, A., Amodaj, N., Hoover, K., Vale, R. (2010) Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol., 2:14.20.1–14.20.17

AUTHORS CONTRIBUTIONS

ED and SP conceived the study and wrote the manuscript. ED constructed the strains and performed the experiments with exogenous stimulation of pheromone. SP performed the mating assays experiments. ED analyzed the single cell data.

ACKNOWLEDGEMENTS

We thank all members of the Pelet and Martin labs for helpful discussions and comments on the manuscripts, Marta Schmitt and Clémence Varidel for technical assistance. This study was supported by Swiss National Science Foundation grants (PP00P3_139121) and the University of Lausanne.

COMPLIANCE WITH ETHICS GUIDELINES

The authors Eric Durandau and Serge Pelet declare that they have no conflict of interests or financial conflicts to disclose.

All procedures performed in studies were in accordance with the ethical standards of the institution or practice at which the studies were conducted, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

RIGHTS & PERMISSIONS

2021 The Author(s) 2021. Published by Higher Education Press