Project A SCF: Components, organization and function
The SCF (Skp1•CUL1•F-box protein) E3 ubiquitin (Ub) ligase defines a large class of evolutionally conserved four-subunit protein complexes. It is composed of an invariable core that contains Skp1, CUL1 and ROC1 (also named Rbx1 or Hrt1), as well as one interchangeable subunit belonging to the F-box protein family that consists of over 70 members in humans. SCF mediates timely, proteolytic destruction of substrates by marking the targets for ubiquitination in a manner that typically depends on substrate modification such as phosphorylation, thereby regulating cell cycle progression, signal transduction, as well as other cellular and organismal processes.
The molecular framework for SCF action has been elucidated by biochemical experiments and X-ray crystallography. In SCF, CUL1 functions as a scaffold anchoring simultaneously an Skp1•F-box protein heterodimer and the ROC1 RING finger protein. This structural arrangement positions the substrate–bound F-box protein within the proximity of ROC1 that recruits an E2 Ub conjugating enzyme.
We are now investigating the mechanisms by which the bound substrate, SCF and the E2 engage in concerted interactions that commit catalysis, yielding a covalent linkage connecting a K48-linked polyubiquitin chain to a substrate lysine residue.
The molecular framework for SCF action has been elucidated by biochemical experiments and X-ray crystallography. In SCF, CUL1 functions as a scaffold anchoring simultaneously an Skp1•F-box protein heterodimer and the ROC1 RING finger protein. This structural arrangement positions the substrate–bound F-box protein within the proximity of ROC1 that recruits an E2 Ub conjugating enzyme.
We are now investigating the mechanisms by which the bound substrate, SCF and the E2 engage in concerted interactions that commit catalysis, yielding a covalent linkage connecting a K48-linked polyubiquitin chain to a substrate lysine residue.
Selected publications:
Umanskaya K, Radke S, Chander H, Monardo R, Xu X, Pan ZQ, O'connell MJ, Germain D. Skp2B stimulates mammary gland development by inhibiting the repressor of the estrogen receptor REA. Mol Cell Biol. 2007 Sep 4 (epub ahead of print)
Li Y, Gazdoiu S, Pan ZQ, Fuchs SY. Stability of homologue of Slimb F-box protein is regulated by availability of its substrate. J Biol Chem. 2004;279:11074-80.
Kim J, Kim JH, Lee SH, Kim DH, Kang HY, Bae SH, Pan ZQ, Seo YS. The novel human DNA helicase hFBH1 is an F-box protein. J Biol Chem. 2002;277:24530-7.
Chen A, Wu K, Fuchs SY, Tan P, Gomez C, Pan ZQ. The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation. J Biol Chem 2000; 275(20):15432-9.
Wu K, Fuchs SY, Chen A, Tan P, Gomez C, Ronai Z, Pan ZQ. The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation. Mol Cell Biol 2000; 20(4):1382-93.
Fuchs SY, Chen A, Xiong Y, Pan ZQ, Ronai Z. HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and beta-catenin. Oncogene. 1999;18:2039-46.
Tan P, Fuchs SY, Chen A, Wu K, Gomez C, Ronai Z, Pan ZQ. Recruitment of a ROC1:Cullin1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of IkBa. Mol Cell 1999; 3:527-533.
Project B Nedd8: An ubiquitin-like modifier that activates cullin-based E3s
Nedd8 is a small Ub-like protein that modifies nearly all members of the cullin protein family. Conjugation of Nedd8 to a cullin protein is executed in a biochemical reaction termed neddylation that mechanistically parallels ubiquitination. A large body of work generated over recent years has established an indispensable role for neddylation in the regulation of cell cycle and embryogenesis.
Using in vitro systems, several studies have shown that Nedd8 activated the SCF-mediated ubiquitination of IκB or p27 through its conjugation to CUL1. Using a reconstitution system with components isolated from bacteria, we demonstrated that the ROC1-CUL1-Nedd8 complex is significantly more active than the unmodified form in supporting the assembly of multi-Ub chains catalyzed by Cdc34.
We are now exploring a possibility that Nedd8 activates SCF by inducing conformational changes that alter cullin-ROC1 interactions.
Using in vitro systems, several studies have shown that Nedd8 activated the SCF-mediated ubiquitination of IκB or p27 through its conjugation to CUL1. Using a reconstitution system with components isolated from bacteria, we demonstrated that the ROC1-CUL1-Nedd8 complex is significantly more active than the unmodified form in supporting the assembly of multi-Ub chains catalyzed by Cdc34.
We are now exploring a possibility that Nedd8 activates SCF by inducing conformational changes that alter cullin-ROC1 interactions.
Selected publications:
Yamoah K, Wu K, Pan ZQ. In vitro cleavage of Nedd8 from cullin 1 by COP9 signalosome and deneddylase 1. Methods Enzymol. 2005;398:509-22.
Reverter D, Wu K, Erdene TG, Pan ZQ, Wilkinson KD, Lima CD. Structure of a complex between Nedd8 and the Ulp/Senp protease family member Den1. J Mol Biol. 2005;345:141-51.
Pan ZQ, Kentsis A, Dias DC, Yamoah K, Wu K. Nedd8 on Cullin: Building an Expressway to Protein Destruction. Oncogene 2004 3; 15(23):1985-97.
Wu K, Yamoah K, Dolios G, Gan-Erdene T, Tan P, Chen A, Lee CG, Wei N, Wilkinson KD, Wang R, Pan ZQ. DEN1 is a dual function protease capable of processing the C-terminus of Nedd8 deconjugating hyper-neddylated CUL1. J Biol Chem 2003; 278:28882-91.
Gan-Erdene T, Nagamalleswari K, Yin L, Wu K, Pan ZQ, Wilkinson KD. Identification and characterization of DEN1, a deneddylase of the ULP family. J Biol Chem. 2003;278:28892-900.
Wu K, Chen A, Tan P, Pan ZQ. The Nedd8-conjugated ROC1-CUL1 Core Ubiquitin Ligase Utilizes Nedd8 Charged Surface Residues For Efficient Polyubiquitin Chain Assembly Catalyzed By Cdc34. J Biol Chem 2002; 277:516-527.
Wu K, Chen A, Pan ZQ. Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. J Biol Chem 2000;32317-32324.
Project C The Cdc34 E2 conjugating enzyme
Cdc34 is the class II E2 conjugating enzyme capable of working with SCF and the VHL E3 for ubiquitination in vitro. The S. cerevisiae (sc) Cdc34, encoded by an essential gene, promotes the G1 to S-phase transition by mediating the Ub-dependent degradation of the Sic1 CDK inhibitor in a fashion depending on the SCFCdc4 E3. The human Cdc34 cDNA can functionally substitute for scCdc34, underscoring a functional conservation between the two orthologs.
How Cdc34 assembles an Ub chain onto a substrate remains poorly understood. We are investigating whether human Cdc34 functions in a dimeric form. We are also dissecting the enzymatic properties of human Cdc34 in coordinating the attacking lysine, the donor and the acceptor Ub for catalysis.
How Cdc34 assembles an Ub chain onto a substrate remains poorly understood. We are investigating whether human Cdc34 functions in a dimeric form. We are also dissecting the enzymatic properties of human Cdc34 in coordinating the attacking lysine, the donor and the acceptor Ub for catalysis.
Selected publications:
Gazdoiu S, Yamoah K, Wu K, Pan ZQ. Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to substrate and assembly of polyubiquitin chains. Mol Cell Biol. 2007 Aug 13; [Epub ahead of print]
Gazdoiu S, Yamoah K, Wu K, Escalante CR, Tappin I, Bermudez V, Aggarwal AK, Hurwitz J, Pan ZQ. Proximity-induced activation of human Cdc34 through heterologous dimerization. Proc Natl Acad Sci U S A. 2005;102:15053-8.
Project D Role of the CUL7 E3 ligase in growth control
CUL7 is an atypical cullin composed of nearly 1,700 amino acids, more than doubling the size of a canonical cullin. Our original work and those from other groups subsequently, have established that CUL7 assembles SCF-like E3 complex, which comprises Skp1, Fbw8 (also called Fbx29), and ROC1.
In collaboration with Dr. Cormier-Daire (University of Paris, France) we identified 25 Cul7 germline mutations in patients with the 3-M syndrome, a heredtiary disorder characterized by pre- and post-natal growth retardation. Of these mutations, 19 predict premature termination of translation, with a majority implicated for loss of the functional cullin domain. In addtion, a recently published study has identified 43 patients having short stature syndrome in 37 Yakut families with pre- and postnatal non-progressive growth failure. A common mutation in the CUL7 gene, 4582insT, was found in all these families, and this mutation is predicted to produce a truncated protein terminating at amino acid position 1553. Thus, the Yakut patients are similar to 3-M in clinical feature and in genetic defects.
Our laboratory was able to identify insulin receptor substrate 1 (IRS-1) as a proteolytic target of the CUL7 E3 ligase. IRS-1 is critical component of both the insulin and IGF-1 pathway by mediating receptor activation to the PI3K/Akt and Erk MAPK pathways, thereby regulating several metabolic and mitogenic effects.
These findings underscore the role of CUL7 as a novel growth regulator and will hopefully help to elucidate the pathogenesis of short stature syndromes with CUL7 mutations, which we are currently investigating.
In collaboration with Dr. Cormier-Daire (University of Paris, France) we identified 25 Cul7 germline mutations in patients with the 3-M syndrome, a heredtiary disorder characterized by pre- and post-natal growth retardation. Of these mutations, 19 predict premature termination of translation, with a majority implicated for loss of the functional cullin domain. In addtion, a recently published study has identified 43 patients having short stature syndrome in 37 Yakut families with pre- and postnatal non-progressive growth failure. A common mutation in the CUL7 gene, 4582insT, was found in all these families, and this mutation is predicted to produce a truncated protein terminating at amino acid position 1553. Thus, the Yakut patients are similar to 3-M in clinical feature and in genetic defects.
Our laboratory was able to identify insulin receptor substrate 1 (IRS-1) as a proteolytic target of the CUL7 E3 ligase. IRS-1 is critical component of both the insulin and IGF-1 pathway by mediating receptor activation to the PI3K/Akt and Erk MAPK pathways, thereby regulating several metabolic and mitogenic effects.
These findings underscore the role of CUL7 as a novel growth regulator and will hopefully help to elucidate the pathogenesis of short stature syndromes with CUL7 mutations, which we are currently investigating.
Selected publications:
Xu X / Sarikas A, Dias-Santagata DC, Dolios G, Lafontant PJ, Tsai SC, Zhu W, Nakajima H, Nakajima HO, Field LJ, Wang R, Pan ZQ. The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation. Mol Cell. 2008;30:403-414.
Huber C, Dias-Santagata D, Glaser A, O'Sullivan J, Brauner R, Wu K, Xu X, Pearce K, Wang R, Uzielli ML, Dagoneau N, Chemaitilly W, Superti-Furga A, Dos Santos H, Megarbane A, Morin G, Gillessen-Kaesbach G, Hennekam R, Van der Burgt I, Black GC, Clayton PE, Read A, Le Merrer M, Scambler PJ, Munnich A, Pan ZQ, Winter R, Cormier-Daire V. Identification of mutations in CUL7 in 3-M syndrome. Nat Genet. 2005;37:1119-24.
Dias DC, Dolios G, Wang R, Pan ZQ. CUL7: A DOC domain-containing cullin selectively binds Skp1.Fbx29 to form an SCF-like complex. Proc Natl Acad Sci U S A. 2002;99:16601-6.
