Direct characterization of E2-dependent target specificity and processivity using an artificial p27-linker-E2 ubiquitination system.
Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone.
Ryu
Kyoung-Seok
KS
Korea Basic Science Institute, Korea.
Choi
Yun-Seok
YS
Ko
Junsang
J
Kim
Seong-Ock
SO
Kim
Hyun Jung
HJ
Cheong
Hae-Kap
HK
Jeon
Young Ho
YH
Choi
Byong-Seok
BS
Cheong
Chaejoon
C
D064173:Anaphase-Promoting Complex-Cyclosome; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C577447:CDC34 protein, human; C471546:UBE2B protein, human; C097309:UBE2D2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008239:Lysine