Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

Molecular Biology and Biochemistry

Lyapina, Svetlana Anatol'Evna ; Deshaies, Raymond Joseph

University:California Institute of Technology

Department:Biology

关键词: Molecular Biology and Biochemistry;   

Others  :  https://thesis.library.caltech.edu/8114/1/lyapina%202001.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication bycata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of severalevolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box proteinCDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue ofCDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts withhSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover,hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitinligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions aspart of an SCF ubiquitin ligase complex in human cells. However, purified human SCFcomplexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additionalfactors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate variousphysiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27,for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about theregulation of various SCF complexes. By using sequential immunoaffinity purification and massspectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCFcomplexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes.Subsequent work by others demonstrated that these proteins are essential for SCF activity. Wealso discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor ofphotomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction isevolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin basedubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. Thiswork sheds light onto an intricate connection that exists between signal transduction pathwaysand protein degradation machinery inside the cell and sets stage for gaining further insights intoregulation of protein degradation.