Examining the role of phosphorylation of p19INK4d on its structure, ubiquitination and proteasomal degradation using chemical synthesis

The P19ink4d protein plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRB) tumor suppresser. Recently, it was found that phosphorylation of p19ink4d at Ser76 and Ser66 causes structure changes followed by ubiquitination and its degradation. Here we show the first total chemical synthesis of mono and doubly phosphorylated P19ink4d employing solid phase peptide synthesis and chemical ligation. The synthesized proteins were characterized by circular dichroism and ubiquitination assay to test the effect of phosphorylation on the thermal stability and ubiquitination of this protein. Our results provided further evidence of the role of phosphorylation on the fate of P19ink4d and revealed a unique role of stepwise phosphorylation on its ubiquitination.