Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3-y subunit of the TCR. To investigate the role of CD3'y phosphorylation in PKC-mediated TCR downregulation, point mutated CD3,y cDNA was transfected into the CD3'y-negative T cell line JGN and CD3'y transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3-y was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3'y truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3'y was required for PKC-mediated TCR downregulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKCmediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucineor tyrosine-based motifs is proposed.

Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes, however the underlying molecular mechanism remains unclear. Here, using mice fed a high fat diet (HFD) as an obesity model, we first detected enhanced reactive oxygen species (ROS) content and reduced Sirt3 expression in HFD oocytes. We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes, with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model. Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase (SOD2K68) is dependent on Sirt3 deacetylase activity in oocytes, and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS. Moreover, we found that acetylation levels of SOD2K68 are increased by ~80% in HFD oocytes and importantly, that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes. Together, our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development, and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions.

Sirtuins have been widely reported to be involved in multiple biological processes; however, their function in oocyte meiosis has not been. Here, by confocal scanning and quantitative analysis, we show that specific depletion of Sirt2 in mouse oocytes results in spindle defects and chromosome disorganization (35.5±8.7 vs. 9.6±3.8% control; P<0.05), with impaired microtubule-kinetochore interaction. Moreover, knockdown and overexpression experiments reveal that Sirt2 modulates the acetylation status of histone H4K16 and α-tubulin in oocytes, which may in part mediate the defective phenotypes described above by influencing microtubule dynamics and kinetochore function. Finally, we find lower Sirt2 protein level in oocytes from aged mice by immunoblotting and that maternal age-associated meiotic defects can be ameliorated through overexpression of Sirt2 (33.2±5.1% old vs.12.7±5.2% old+Sirt2; P<0.05), providing support for the hypothesis that decreased Sirt2 is one of a number of factors contributing to oocyte age-dependent deficits. In summary, our data indicate a role for Sirt2 during oocyte meiosis and uncover a striking beneficial effect of increased Sirt2 expression on aged oocytes.

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