Cancer is described as being the product of malfunctions within the regulation of the cell cycle, such that mutated cells are allowed to progress through the cell cycle and accumulate mutations. The cell cycle is constantly regulated using cyclin-dependent kinases (CDKs), which are key enzymes for advancing the transition of the cell cycle phases. p19^INK4d and other INK4 members are one family of proteins that regulates CDKs in the cell cycle by inhibiting CDK4/6 responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumor suppressor. p19^INK4d is the only INK4 member that undergoes high phosphorylation and rapid proteasomal degradation, which prompted the investigation of possible crosstalk. It was reported recently that stepwise phosphorylation of p19^INK4d at Ser66 then Ser76 causes structural changes, which leads to its ubiquitination and degradation, yet the exact contribution of each phosphorylation site remains unclear. To shed light on these sites, especially on the role of phosphorylation of p19^INK4d at Ser76, we employed chemical protein synthesis of p19^INK4d, a powerful tool to obtain homogenous proteins, especially in cases of not naturally encoded amino acids such as phosphoserines or other post-translational modifications.

We have achieved the first total chemical synthesis of unmodified, mono- and doubly- phosphorylated p19^INK4d using state of the art methods for chemical protein synthesis such as Fmoc-SPPS and native chemical ligation. The four synthetic p19^INK4d analogs were characterized by circular dichroism and biochemical methods to determine the effect of each phosphorylation site on stability by measuring the melting temperature of each analog and to explore the effect of phosphorylation position on ubiquitination. Our results provide a clear determination of p19^INK4d stability upon phosphorylation on different sites and reveal that phosphorylation on both Ser residues might be necessary for promoting ubiquitination of p19^INK4d.