ERK1 activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos.
Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. We show that activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. We combine experiments on populations of cells with analysis at the single cell level, and develop a proxy assay for DNA synthesis in single embryos, using GFP-PCNA. We compare the effects of low molecular weight inhibitors with a recombinant approach targeting the same signalling pathway. We find that inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, prevents accumulation of GFP-PCNA in the zygote nucleus and that U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle.
Philipova
Rada
R
University of Newcastle upon Tyne, Institute of Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle NE2 4HH, UK.
Kisielewska
Jolanta
J
Lu
Pin
P
Larman
Mark
M
Huang
Jun-Yong
JY
Whitaker
Michael
M
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology* / Q000196:embryology / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005306:Fertilization; D005419:Flavonoids / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D008297:Male; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054639:Mitogen-Activated Protein Kinase Phosphatases; D009570:Nitriles / Q000494:pharmacology; D010063:Ovum / Q000187:drug effects / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D012617:Sea Urchins / Q000166:cytology / Q000196:embryology* / Q000201:enzymology*; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism; D029867:Xenopus Proteins / Q000494:pharmacology
D002070:Butadienes; D002843:Chromatin; D005419:Flavonoids; D009570:Nitriles; D018809:Proliferating Cell Nuclear Antigen; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C113580:U 0126; D029867:Xenopus Proteins; D000001:Calcimycin; D016590:Aphidicolin; D016203:CDC2 Protein Kinase; D048052:Mitogen-Activated Protein Kinase 3; C096688:DUSP1 protein, Xenopus; D054639:Mitogen-Activated Protein Kinase Phosphatases; D017027:Protein Tyrosine Phosphatases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one