Abstract
Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1. To this end, we generated a genetically encoded FRET (Förster Resonance Energy Transfer)-based Cdc25C phosphorylation biosensor to observe Cdc25 spatiotemporal phosphorylation during cell cycle progression in live single cell assays. Because this approach proved to be powerful, we provide here guidelines for the development of biosensors for any phosphorylation site of interest.
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References
Schmitz MH, Held M, Janssens V et al (2010) Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells. Nat Cell Biol 12:886–893, doi: ncb2092 [pii] 10.1038/ncb2092
Blethrow JD, Glavy JS, Morgan DO, Shokat KM (2008) Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates. Proc Natl Acad Sci U S A 105:1442–1447. doi:10.1073/pnas.0708966105
Kettenbach AN, Schweppe DK, Faherty BK et al (2011) Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal 4:rs5. doi:10.1126/scisignal.2001497
Macurek L, Lindqvist A, Lim D et al (2008) Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455:119–123, doi: nature07185 [pii] 10.1038/nature07185
Seki A, Coppinger JA, Jang C-YY et al (2008) Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320:1655–1658. doi:10.1126/science.1157425
Gheghiani L, Loew D, Lombard B et al (2015) Plk1 pool activation in late G2 sets up commitment to mitosis
Lénárt P, Petronczki M, Steegmaier M et al (2007) The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol 17:304–315. doi:10.1016/j.cub.2006.12.046
Lane HA, Nigg EA (1996) Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol 135:1701–1713
Petronczki M, Glotzer M, Kraut N, Peters J-M (2007) Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle. Dev Cell 12:713–725. doi:10.1016/j.devcel.2007.03.013
Watanabe N, Arai H, Iwasaki J et al (2005) Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways. Proc Natl Acad Sci U S A 102:11663–11668
Roshak AK, Capper EA, Imburgia C et al (2000) The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase. Cell Signal 12:405–411, doi: S0898-6568(00)00080-2 [pii]
Kumagai A, Dunphy WG (1998) Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273:1377–1380
Pilji A, Wilmanns M, Schultz C et al (2011) Rapid development of genetically encoded FRET reporters. ACS Chem Biol 6:685–691. doi:10.1021/cb100402n
Fritz RD, Letzelter M, Reimann A et al (2013) A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci Signal 6:rs12. doi:10.1126/scisignal.2004135
Thestrup T, Litzlbauer J, Bartholomäus I et al (2014) Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods 11:175–182. doi:10.1038/nmeth.2773
Liu D, Davydenko O, Lampson MA (2012) Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing. J Cell Biol 198:491–499. doi:10.1083/jcb.201205090
Nguyen AW, Daugherty PS (2005) Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23:355–360. doi:10.1038/nbt1066
Golsteyn RM, Mundt KE, Fry AM, Nigg EA (1995) Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J Cell Biol 129:1617–1628
Tsien R, Harootunian A (1990) Practical design criteria for a dynamic ratio imaging system. Cell Calcium 11:93–109. doi:10.1016/0143-4160(90)90063-Z
Durocher D, Taylor IA, Sarbassova D et al (2000) The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Mol Cell 6:1169–1182
Violin JD, Zhang J, Tsien RY, Newton AC (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 161:899–909, doi: 10.1083/jcb.200302125 jcb.200302125 [pii]
Yoshizaki H, Ohba Y, Kurokawa K et al (2003) Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J Cell Biol 162:223–232, doi: 10.1083/jcb.200212049 jcb.200212049 [pii]
Gavet O, Pines J (2010) Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J Cell Biol 189:247–259. doi:10.1083/jcb.200909144
Agircan FG, Schiebel E (2014) Sensors at centrosomes reveal determinants of local separase activity. PLoS Genet 10(10), e1004672. doi:10.1371/journal.pgen.1004672
Fuller BG, Lampson MA, Foley EA et al (2008) Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature 453:1132–1136, doi: nature06923 [pii] 10.1038/nature06923
Liu D, Vader G, Vromans MJ et al (2009) Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates. Science 323:1350–1353. doi:10.1126/science.1167000
Belal ASF, Sell BR, Hoi H et al (2014) Optimization of a genetically encoded biosensor for cyclin B1-cyclin dependent kinase 1. Mol Biosyst 10:191–195. doi:10.1039/c3mb70402e
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Gheghiani, L., Gavet, O. (2016). Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression. In: Coutts, A., Weston, L. (eds) Cell Cycle Oscillators. Methods in Molecular Biology, vol 1342. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2957-3_8
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DOI: https://doi.org/10.1007/978-1-4939-2957-3_8
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