FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest.
During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCFFBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.
Department of Internal Medicine, Roy and Lucille A. Carver College of Medicine, Iowa City, USA.
Hsiang
H
Wen
Namhun
N
Kim
Ernesto J
EJ
Fuentes
Adam
A
Mallinger
Pedro
P
Gonzalez-Alegre
Kevin A
KA
Glenn
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000737:chemistry / Q000378:metabolism; D044783:F-Box Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*
D044842:Cullin Proteins; D044783:F-Box Proteins; D044786:S-Phase Kinase-Associated Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome