title: Positively charged residues located downstream of PIP box, together with TD amino acids within PIP box, are important for CRL4(Cdt2) -mediated proteolysis.
abstract: 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.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Michishita
authors.forename: Aya
authors.initials: A
authors.lastname: Morimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Ishii
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Komori
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shiomi
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Hideo
authors.initials: H
authors.lastname: Nishitani
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004249:DNA Damage; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D016196:S Phase; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; C429530:DTL protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases