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A promiscuous ubiquitin ligase controls cell cycle through a unique mechanism
Author(s) -
Glenn Kevin Andrew,
Wen Hsiang M.,
Kim Namhun,
Mallinger Adam,
GonzalezAlegre Pedro
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb151
Subject(s) - cullin , ubiquitin ligase , ubiquitin , dna ligase , microbiology and biotechnology , cell cycle , f box protein , biology , immunoprecipitation , ubiquitin protein ligases , chemistry , biochemistry , cell , dna , gene
Objective Identify the cullin partner proteins for FBG1. Methods Western blots of proteins co‐immunoprecipatating with FBG1 identified partner proteins. Growth rates determined by replicate cell counting via a hemocytometer or Countess automated counter. Cell cycle analysis was by FACS. During cell proliferation, protein degradation is strictly regulated by two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F‐box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF complexes degrade proteins during the G1 phase, but as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. This study explores an unexpected interaction between APC‐2 and SCF FBG1 . We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Site‐directed mutagenesis shows that this interaction requires a D‐Box within FBG1. Unexpectedly, we demonstrate that co‐expression with FBG1 increases total APC2 levels. Finally, FACS analysis demonstrates that FBG1 induces S‐phase arrest, illustrating the functional consequences of this interaction. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent of its ubiquitin ligase function. Funding: V.A. RCD and Ellison New Scholars in Aging Award.