Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4Cdt2-mediated proteolysis

Abstract

PCNA links Cdt1 and p21 for proteolysis by Cul4-DDB1-Cdt2 (CRL4Cdt2) in the S phase and after DNA damage in mammalian cells. However, other PCNA-interacting proteins, such as ligase I, are not targets of CRL4Cdt2. In this study, we created chimera constructs composed of Cdt1 and ligase I and examined how the proteolysis of PCNA-interacting proteins is regulated. Consistent with a recent report using the Xenopus egg system (Havens & Walter 2009), two amino acid elements are also required for degradation in HeLa cells: TD amino acid residues in the PIP box and the basic amino acid at +4 downstream of the PIP box. In addition, we demonstrate that a basic amino acid at +3 is also required for degradation and that an acidic amino acid residue following the basic amino acids abolishes the degradation. Electrostatic surface images suggest that the basic amino acid at +4 is involved in a contact with PCNA, while +3 position extending to opposite direction is important to create a positively charged surface. When all these required elements were introduced in ligase I peptide, the substituted form became degraded. Our results demonstrate that PCNA-dependent degron is strictly composed to avoid illegitimate destruction of PCNA-interacting proteins. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.