Pippin protein expression changes during cell differentiation

PIPPin is a CSD-containing protein with the ability to interact both with mRNAs encoding histone variants and chromatin. A major fraction of chromatin-bound PIPPin is sumoylated and sumoylation seems to be controlled by thyroid hormones, both in vivo and in vitro. We studied its expression in different tissues and cell lines and even in tumor cells and found that, even if 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. The expression of the protein is strongly increased by treatments that induce differentiation, such as treatment of PC12 with NGF. We also found an increase of PIPPin protein expression in PC12 transfected with a plasmid encoding a novel longer isoform of the calmodulin-binding protein PEP-19, that we recently cloned and called LPI. We then produced, by site-directed mutagenesis, new DNA fragments, encoding mutated proteins, in which either one out of two serine or one threonine, each included in one out of three putative PKC recognition sites, had been replaced by amino acids not accepting phosphorylation.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.