Characterization of Lipovitellin 2 as a Tyrosine-Phosphorylated Protein in Oocytes, Eggs and Early Embryos ofXenopus laevis

Abstract

A tyrosine-phosphorylated protein of 33 kDa is shown to be present in the solubilized yolk fraction of Xenopus laevis oocytes, eggs, and early embryos. The phosphoprotein is lipovitellin 2 as demonstrated by immunoprecipitation and mass spectrometry studies, and is termed pp33/LV2. Sub-cellular fractionation and immunoblotting studies demonstrate that pp33/LV2 is stably present in the Triton X-100-resistant and SDS-soluble yolk fractions during oogenesis, oocyte maturation, and early embryogenesis. From after the swimming tadpole stages (stage 39∼), however, it becomes partly soluble to Triton X-100-containing buffer and all disappear thereafter (stage 48∼49). In vitro enzyme assays with the use of the tyrosine phosphatase LAR and the tyrosine kinase Src demonstrate the reversible nature of the tyrosine phosphorylation of pp33/LV2. Microinjection studies demonstrate that the solubilized yolk fractions, but not those immunodepleted of pp33/LV2 or those pretreated with LAR, inhibit progesterone- or insulin-induced oocyte maturation. A pp33/LV2-like protein seems to present in two Xenopus subspecies, one other frog species, and two fish species, but not in other amphibian species, such as newt and salamander. These results suggest that LV2, in its tyrosine-phosphorylated form, serves in a cellular function in a species-specific manner, but the mechanism is still unknown.

Gap junction protein Connexin-43 is a direct transcriptional regulator of N-cadherin in vivo

“…The mix was magnetically purified and prepared for western blot. Nuclear fraction isolation of X. laevis embryos was performed using differential centrifugation protocols with modifications 64 , 65 . Briefly, stage 18 X. laevis embryo lysates using a 22 G needle and 20 μL homogenization buffer (HB: 250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, phosphatase and protease inhibitors) per embryo.…”

Focused Proteomics on Egg Membrane Microdomains to Elucidate the Cellular and Molecular Mechanisms of Fertilization in the African Clawed Frog Xenopus laevis

“…Other targets of the activated xSrc include Shc (Aoto et al 1999 ), hnRNP K (Iwasaki et al 2008 ), lipovitellin 2 (Kushima et al 2011 ), and pp40, whose identity is not yet demonstrated (manuscript in preparation) (Fig. 14.1 ).…”

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

“…It is well known that phosvitin and pp25 are highly serine/threoninephosphorylated proteins that serve as an energy source of oogenesis and early embryogenesis. On the other hand, tyrosine phosphorylation of LV1 (Browaeys-Poly et al 2007) and LV2 (Kushima et al 2011) has recently been demonstrated in Xenopus. In particular, tyrosine phosphorylation of LV2 is unusually stable during oogenesis, oocyte maturation, and early embryogenesis until the removal of yolk-associated materials from swimming tadpole (Kushima et al 2011).…”

“…On the other hand, tyrosine phosphorylation of LV1 (Browaeys-Poly et al 2007) and LV2 (Kushima et al 2011) has recently been demonstrated in Xenopus. In particular, tyrosine phosphorylation of LV2 is unusually stable during oogenesis, oocyte maturation, and early embryogenesis until the removal of yolk-associated materials from swimming tadpole (Kushima et al 2011). Possible function of tyrosine-phosphorylated form of Xenopus LV1 and LV2 so far suggested is oocyte maturation (Browaeys-Poly et al 2007;Kushima et al 2011), although it's upstream (liver or oocyte) kinase and downstream cellular function is uncertain.…”