A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination.
We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF(βTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(βTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(βTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.
Jordan
J
Kovacev
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.
Kenneth
K
Wu
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Donald E
DE
Spratt
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.
Robert A
RA
Chong
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.
Chan
C
Lee
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.
Jaladhi
J
Nayak
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Gary S
GS
Shaw
Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574; Xuzhou Medical College, Jiangsu Key Laboratory of Biological Cancer Therapy, Jiangsu 221002, China. Electronic address: zhen-qiang.pan@mssm.edu.
Zhen-Qiang
ZQ
Pan
D057809:HEK293 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D000075687:NEDD8 Protein; D000072000:NF-KappaB Inhibitor alpha; D010441:Peptide Chain Elongation, Translational; D025821:Polyubiquitin / Q000096:biosynthesis* / Q000235:genetics; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D020672:I-kappa B Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C000606204:NFKBIA protein, human; D014452:Ubiquitins; D051176:beta Catenin; D025821:Polyubiquitin; D000072000:NF-KappaB Inhibitor alpha; D044843:SKP Cullin F-Box Protein Ligases; D008239:Lysine
Catenin; Cdc34; E2 Conjugating Enzyme; E3 Ubiquitin Ligase; Enzymes; Post Translational Modification; Protein Degradation; SCF; UbcH5c; Ubiquitination