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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02624

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Phosphorylation of IP₃R1 and the regulation of [Ca²⁺]_i responses at fertilization: a role for the MAP kinase pathway

Bora Lee^1,*, Elke Vermassen^2,*,, Sook-Young Yoon¹, Veerle Vanderheyden², Junya Ito¹, Dominique Alfandari¹, Humbert De Smedt², Jan B. Parys² and Rafael A. Fissore^1,

¹ Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01002, USA.
² Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N1, bus 802, B-3000 Leuven, Belgium.

View larger version (57K): [in a new window]   Fig. 1. IP3R1 is differentially phosphorylated in mouse oocytes, eggs and zygotes. (A) Immunoblots (IB) of oocyte lysates during different stages of oocyte maturation (GV, GVBD, MI and IVM/MII) were probed with the MPM2 antibody (upper panel) and, after stripping of the blot, with the IP3R1 antibody (lower panel). Quantification of IP3R1 MPM2 reactivity is shown in the graph below the IB panels. Data are presented as means±s.e.m., both here and throughout the manuscript. Fifty mouse oocytes were used per lane, both here and elsewhere in the study. (B) Kinase activity assays performed on lysates from five oocytes collected simultaneously with oocytes for western blotting; H1 and MBP phosphorylation indicate MPF and MAPK activity, respectively. The graph below shows quantification of kinase activities. Bars with an asterisk are significantly different from those without a symbol within kinase here and elsewhere. The presence of two vertical lines indicates that lanes were cut and pasted from the same blot/exposure during the preparation of the figure here and elsewhere in the manuscript. (C) Lysates from MII eggs and zygotes collected at 2PB, PN and Mit I were probed with MPM2 antibody (upper panel) and re-probed with IP3R1 antibody (lower panel), as described above. (D) Kinase activity assays performed at the same cell-cycle stages as for C. Bars with asterisks are significantly different from those without a symbol.

View larger version (48K): [in a new window]   Fig. 2. IP3R1 is differentially phosphorylated in Xenopus eggs and zygotes. (A) Western blotting performed on Xenopus egg extracts (approximately three eggs/lane) collected at MII and at interphase (Int) shows MPM2 (upper panel) and IP3R1 reactivity (lower panel). (B) Immunoprecipitation (IP) experiments performed on MII Xenopus egg extracts using preimmune serum (Preim.), anti-IP3R1 antibody, MPM2 antibody or beads alone, followed by SDS-PAGE and western blotting with the anti-IP3R1 antibody (upper panel) and the MPM2 antibody (lower panel).

View larger version (13K): [in a new window]   Fig. 3. MAPK phosphorylation motifs are present in the sequence of IP3Rs. The sequences of IP3R1, IP3R2 and IP3R3 of various species were examined for the presence of MAPK sites. Two highly conserved (S436 and T945) and one less conserved (S1765) MAPK consensus sites were found in IP3R1, but not in IP3R2 or IP3R3.

View larger version (19K): [in a new window]   Fig. 4. In vitro phosphorylation of IP3R1 and GST-IP3R1 fragments by activated ERK2 kinase. (A) Purified IP3R1 and IP3R3 (1 µg) or GST-IP3R1 purified fragments (0.5 µg) were phosphorylated in vitro in the presence of [-32P]ATP at 30°C using activated ERK2. Phosphorylated IP3Rs and GST-fusion fragments were detected using a phosphorimager. (B) Equal amounts of IP3Rs in reaction were determined with an anti-IP3R antibody that recognizes both isoforms equally. (C) Phosphorylation of IP3R1 domains expressed as GST-fusion proteins by MAPK/ERK2 performed and quantified as above. Arrowheads indicate the position of ERK2 and of the various GST-fusion proteins. The amino acids included within each of the domains and the predicted molecular weight for each domain are noted in Table 2. (D) ERK2 phosphorylation of IP3R1 domain 2 after in vitro mutagenesis of S421 and S436. Equal loading was ascertained using the anti-cytI3b1 antibody (data not shown). All results are typical of at least three experiments.

View larger version (61K): [in a new window]   Fig. 5. MAPK activity is required for IP3R1 MPM2 phosphorylation of mouse eggs. (A) IP3R1 MPM2 (upper panel) and IP3R1 (lower panel) reactivity in oocytes matured in vitro (IVM) in the presence of U0126 (25 µM), in its absence or in the presence of U0124. Quantification of IP3R1 MPM2 reactivity is shown in the graph. (B) MPF and MAPK activities were assayed in the presence or absence of U0126 at the same time points as in A and are quantified in the graph below; asterisks indicate statistical significance. (C) IP3R1 MPM2 (upper panel) and IP3R1 (lower panel) reactivity in PN-stage zygotes treated with OA (10 µM), OA+U0126 (75 µM) or OA+roscovitine (Ros; 75 µM). The graph shows quantification of MPM2 reactivity. (D) Effect of OA on MPF and MAPK activities in PN-stage zygotes and inhibition of its effects by U0126 but not by Ros (not shown). The graph shows quantification of the effect of OA and U0126 on MPF and MAPK activities. Bars with asterisks are significantly different from those without a symbol.

View larger version (26K): [in a new window]   Fig. 6. Absence of MAPK activity and IP3R1 MPM2 phosphorylation influences the ability of mouse eggs to mount oscillations and affects IP3R1 function. (A)[Ca2+]i responses induced by SrCl2 (10 mM) and (B) PLC (0.1 µg/µl) were compared in oocytes matured in the absence (left panels) or presence (right panels) of U0126 (25 µM). (C) The content of the Ca2+ stores was examined in oocytes maturated in the absence (left panel) or presence of U0126 (right panel) by monitoring [Ca2+]i responses induced by the addition of thapsigargin (first arrow; TG; 10 µM) in Ca2+-free media. Ca2+ influx was examined in the same oocytes after the addition of CaCl2 up to a final concentration of 5 mM (second arrow).

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