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Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4 Cdt2 ‐mediated proteolysis
Author(s) -
Michishita Masato,
Morimoto Aya,
Ishii Takashi,
Komori Hirofumi,
Shiomi Yasushi,
Higuchi Yoshiki,
Nishitani Hideo
Publication year - 2011
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2010.01464.x
Subject(s) - biology , proteolysis , box model , amino acid , downstream (manufacturing) , biochemistry , biophysics , enzyme , physics , operations management , atmospheric sciences , economics
PCNA links Cdt1 and p21 for proteolysis by Cul4‐DDB1‐Cdt2 (CRL4 Cdt2 ) in the S phase and after DNA damage in mammalian cells. However, other PCNA‐interacting proteins, such as ligase I, are not targets of CRL4 Cdt2 . 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.