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

Abstract

2+] i ) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP 3 R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca 2+ release. IP 3 R1-mediated Ca 2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca 2+ ] i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP 3 R1 function in eggs. Using mouse and Xenopus eggs, we show that IP 3 R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP 3 R1 at at least one highly conserved site, and that its mutation abrogates IP 3 R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP 3 R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca 2+ ] i oscillations in response to agonists and show compromised IP 3 R1 function. These findings identify IP 3 R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP 3 R1 function in eggs that serves to optimize [Ca 2+ ] i release at fertilization. KEY WORDS: Fertilization, Ca 2+, IP3R1, Mouse, MAPK, Xenopus Development 133, 4355-4365 (2006) Jones and Whittingham, 1996). Given that during maturation and after activation/fertilization the changes in IP 3 R1 concentrations and content of the Ca 2+ stores are small (Brind et al., 2000;Iwasaki et al., 2002;Jellerette et al., 2000), it is likely that other mechanisms might regulate IP 3 R1 function in eggs.Phosphorylation has been shown to be an important regulatory mechanism of IP 3 R1 function (Bezprozvanny, 2005;Patterson et al., 2004a). Among the protein kinases that phosphorylate IP 3 R1 are: protein kinase A and protein kinase C (Ferris et al., 1991;Vermassen et al., 2004a); protein kinase G (Koga et al., 1994); Ca 2+ /calmodulindependent protein kinase II (Ferris et al., 1991;Zhu et al., 1996); the tyrosine kinases Fyn (Jayaraman et al., 1996) and Lyn (Yokoyama et al., 2002); Rho kinase (Singleton and Bourguignon, 2002); and, very recently, protein kinase B (Khan et al., 2006) (V.V., H.D.S. and J.B.P., unpublished). In most cases, IP 3 R1 phosphorylation by these kinases enhances Ca 2+ conductivity, but none of these kinases appears to be intimately associated with cell-cycle transitions. Most importantly, abrogation of their activities by pharmacological inhibitors does not affect IP 3 R1 function in eggs (Carroll and Swann, 1992;Smyth et al., 2002;Swann et al., 1989). However, a ...

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

“…The TRPP2 D509V and R740X mutants were made by site-directed mutagenesis by introducing GTT (V) to replace GAT (D) at codon 509, and TAG (Stop) to replace CGG (R) at codon 740, respectively, with wild-type pDONR221-TRPP2 as donor DNA. The different domains of the IP 3 R1 (domains 1 to 6), the complete ligand-binding domain (LBD, aa 1-604), and the suppressor domains (aa 1-225) of IP 3 R1 and IP 3 R3 were subcloned in the bacterial pGEX-6p2 vector with an N-terminal glutathione S-transferase (GST) tag, as previously described (28,29).…”

Ca2+ signaling differentiation during oocyte maturation

“…In contrast, in starfish eggs although the increase in Ca 2þ release was dependent on MPF activation, MPF does not directly phosphorylate the IP 3 R but rather mediates its effect through the actin cytoskeleton Santella et al, 2003). More recently, the MAPK cascade has been shown to be important for shaping Ca 2þ dynamics in mouse eggs, and this effect is potentially mediated by direct phosphorylation of the IP 3 R by MAPK (Lee et al, 2006a;Matson and Ducibella, 2007). Together, these results argue that the sensitization of IP 3 -dependent Ca 2þ release is due to an increase in IP 3 R affinity, which may be regulated by phosphorylation during oocyte maturation.…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II. Kinase Regulators and Substrates

“…IP3R1 is phosphorylated during both maturation and the first cell cycle mediated by M-phase kinases e.g. MAPK/ERK2 or polo-like kinase 1 and this is vital for IP3R function in optimum Ca 2+ release at fertilization in Xenopus, mouse and pig (Ito et al 2008;Ito et al 2010;Lee et al 2006;Sun et al 2009;Vanderheyden et al 2009). Type 1 IP3R is differentially distributed during human oocyte maturation through GV to MII stage and after fertilization in both peripheral and central in the zygotes and early 2-4-cell embryos and in perinuclear in the 6-8-cell embryos (Goud et al 1999).…”