EXPRESSION OF PIPPIN PROTEIN AND CELL DIFFERENTIATION.

We previously described a CSD-containing protein that seemed to bind mRNAs encoding histone variants and was present both in the nucleus and in the cytoplasm of specific populations of brain cells. Since other CSD-containing proteins have the ability to interact both with RNA and chromatin, we investigated the possibility that PIPPin binds to chromatin and indeed found that about 50% of nuclear PIPPin cannot be extracted from nuclei with salt and is instead extracted with acid, together with histones. Interestingly, a major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. In order to study the functions of PIPPin in vivo, we analyzed its expression in different tissues and cell lines and even in tumor cells and found that, even if it is much more expressed in the brain respect to other tissues of the adult rat, it is also expressed in brain tumors and in cell lines as different as kidney NRK cells and PC12 cells. More interestingly, expression of the protein is strongly increased by treatments that induce differentiation, such as treatment of PC12 cells with NGF. We also found an increase of PIPPin protein expression in PC12 cells transfected with a plsmid encoding a novel longer isoform of the calmodulin-binding protein PEP-19, that we recently cloned and called LPI. Finally, we found that one of the mRNAs bound by PIPPin should be its mRNA itself. In parallel experiments, we found that a significant fraction of PIPPin is phosphorylated and that this post-translational modification seems to have an effect on PIPPin ability to bind RNA. Therefore, we produced, by site-directed mutagenesis, new DNA fragments, encoding mutated proteins, in which either one out of two serines or one threonine, each included in one out of three putative PKC recognition sites, had been replaced by amino acids not accepting phosphorylation. The coding region of the starting PIPPin DNA, as well as the different mutated DNA have been cloned into the pEGFP-N1 plasmid. Recombinant vectors have been then transferred into mammalian cell lines of different origin. We are now selecting clones expressing PIPPin at high levels, in order to analyze: i) the intracellular localization of the recombinant protein; ii) the effect of PIPPin overexpression on the expression of histone variants, such as the H1° linker histone and the H3.3 core histone; iii) the effect of the mutations on the previous events. Bono E. et al. (2007). Endocrinology 148: 252-257 Castiglia D et al. (1996) Biochem Biophys Res Commun 218: 390-394 Derrigo M. et al. (2000) Int J Mol Med. 5: 111-123. Nastasi T. et al (2000) NeuroReport 11: 2233-2236 Nastasi T. et al. (1999) J Biol Chem 274: 24087-24093 Sala A et al. (2007) Int J Mol Med 19: 501-509 Scaturro M et al. (1998) J Biol Chem 273: 22788-2279