IDENTIFICATION OF TLE6 AS A PROTEIN KINASE A SUBSTRATE DURING MOUSE OOCYTE MATURATION

Abstract

It is well established that protein kinase A (PKA) activity is required for the maintenance of meiotic arrest. However, once oocyte maturation begins, PKA activity also is required for successful completion of meiosis. To determine the localization of oocyte proteins phosphorylated by PKA during maturation, we performed immunofluorescence studies using an antibody that recognizes a consensus PKA phosphorylation site [RRXp(S/T)]. PKA substrate proteins were located in the cytoplasm and at both MI and MII spindles, and colocalized with the condensed chromosomes and centrosomes. Using a proteomic approach, we identified several candidate PKA substrates in metaphase II-arrested eggs. One of these candidate proteins is Tle6 (transducin-like enhancer of split 6), a Groucho-related protein of apparent Mr 65 kD, that in neural cells interacts with and inhibits forkhead family transcriptional co-repressors. Shifts in the apparent Mr of Tle6 were consistent with phosphorylation during oocyte maturation and dephosphorylation between the 1-cell and 2-cell embryo stages. The maturation-associated Mr shift was not observed when oocytes underwent germinal vesicle breakdown in the presence of H89, a PKA inhibitor. By immunofluorescence analysis, Tle6 was located in the cytoplasm and cortex of immature and fully-grown oocytes, and at the MII spindle, a localization pattern similar to that of the PKA type I regulatory subunit. In preimplantation embryos, Tle6 was located in the cytoplasm, with enhanced staining at the outer portions of the embryos. By immunofluorescence and immunoblot analyses, the amount of Tle6 protein decreased significantly by the blastocyst stage. Based on the role of Tle6 in neural cells, we anticipate that Tle6 may function as a transcription factor regulator in the oocyte and early preimplantation embryo.