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

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 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 ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological 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 the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, 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. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

Signal transduction, regulation, and developmental logic of EGFR signalling in C. elegans

RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.

My research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.

In an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.

By comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.

Finally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.