Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome

<p>Abstract</p> <p>Background</p> <p>Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.</p> <p>Results</p> <p>We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCF^Skp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins <it>in vivo</it>. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.</p> <p>Conclusion</p> <p>SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.</p

Activation of a Metabolic Gene Regulatory Network Downstream of mTOR Complex 1

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease

Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signaling to suppress tumorigenesis

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at T86 and T398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin/IGF-1-mediated, but also PDGF or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Lastly, a cancer patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-mTORC2 activation via bypassing this negative regulation. Together, our work reveals a Sin1 phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1/S6K/Sin1 signaling axis might cause aberrant hyper-activation of mTORC2/Akt that facilitates tumorigenesis

Disruption of the COP9 Signalosome Csn2 Subunit in Mice Causes Deficient Cell Proliferation, Accumulation of p53 and Cyclin E, and Early Embryonic Death

Csn2 (Trip15/Cops2/Alien) encodes the second subunit of the COP9 signalosome (CSN), an eight-subunit heteromeric complex homologous to the lid subcomplex of the 26S proteasome. CSN is a regulator of SCF (Skp1-cullin-F-box protein)ubiquitin ligases, mostly through the enzymatic activity that deconjugates the ubiquitin-like protein Nedd8 from the SCF Cul1 component. In addition, CSN associates with protein kinase activities targeting p53, c-Jun, and IκB for phosphorylation. Csn2 also interacts with and regulates a subset of nuclear hormone receptors and is considered a novel corepressor. We report that targeted disruption of Csn2 in mice caused arrest of embryo development at the peri-implantation stage. Csn2(−/−) blastocysts failed to outgrow in culture and exhibited a cell proliferation defect in inner cell mass, accompanied by a slight decrease in Oct4. In addition, lack of Csn2 disrupted the CSN complex and resulted in a drastic increase in cyclin E, supporting a role for CSN in cooperating with the SCF-ubiquitin-proteasome system to regulate protein turnover. Furthermore, Csn2(−/−) embryos contained elevated levels of p53 and p21, which may contribute to premature cell cycle arrest of the mutant

Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome-4

Les were blotted with antibodies against HA, Nedd8, Elongin B or CAND1. Nedd8 modified and un-modified Cul2 proteins are indicated. HA-Cul2 and its truncations as indicated were transfected into Flag-SAP130 stable cells (upper panel) or HEK293 cells along with Flag-CSN1 (bottom panel). Flag IP was performed and samples were blotted using anti-HA antibody. A diagram indicating the Cul2 truncation constructs and the interaction results from and .<p><b>Copyright information:</b></p><p>Taken from "Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome"</p><p>http://www.biomedcentral.com/1471-2091/9/1</p><p>BMC Biochemistry 2008;9():1-1.</p><p>Published online 3 Jan 2008</p><p>PMCID:PMC2265268.</p><p></p

Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome-1

empty vector (-) or HA-SAP130 (+) were analyzed by immunoblotting using the antibodies against cullins or SAP155, a component of SF3b RNA-splicing complex. Rabbit reticulocyte lysate (RRL) containing translated HA-SAP130 were incubated with recombinant GST-Nedd8, GST-Ub or GST (1 μg each). HA IP samples were analyzed by anti-Cul2 and anti-GST blots and autoradiogram (S). () HA-SAP130 was translated in RRL supplemented with GST or GST-Nedd8 (1 μg each). Anti-GST blot shows that free GST-Nedd8, like GST alone, was not associated with HA-SAP130. () HeLa Cells were transfected with siRNA reagent against CAND1 or laminin-A control. Input lysate or HA IP samples were blotted with anti-CAND1 or Cul2 antibodies. Note that there was weak non-specific binding of un-neddylated Cul2 to HA beads (lane 1).<p><b>Copyright information:</b></p><p>Taken from "Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome"</p><p>http://www.biomedcentral.com/1471-2091/9/1</p><p>BMC Biochemistry 2008;9():1-1.</p><p>Published online 3 Jan 2008</p><p>PMCID:PMC2265268.</p><p></p

Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome-2

Ed antibodies. An asterisk (*) indicates a non-specific band in Flag IP. () HA-SAP130 was expressed in HEK293 cells. Anti-HA IP samples were probed with indicated antibodies. Flag immunocomplex isolated from HEK293 cells expressing empty vector, Flag-SAP130 or Flag-CPSF160 were incubated with N-terminal biotin-labeled ubiquitin, E1 and E2 (UbcH5b) as indicated. Polyubiquitination was determined by the detection of biotin-Ub.<p><b>Copyright information:</b></p><p>Taken from "Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome"</p><p>http://www.biomedcentral.com/1471-2091/9/1</p><p>BMC Biochemistry 2008;9():1-1.</p><p>Published online 3 Jan 2008</p><p>PMCID:PMC2265268.</p><p></p

Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome-3

N428, HA-Cul1C450 and HA-Cul1C280 were transiently expressed in HEK293 cells. The HA-IP samples were blotted with indicated antibodies. HA-Cul1 and its truncations as indicated were transfected into Flag-SAP130 stable cells. Flag IP was performed and samples were probed with anti-HA antibody. HeLa cells were transfected with HA-Cul1 or vector. Cell extracts were incubated with anti-CSN2 antibody or pre-immune serum for 15 min at room temperature and then blotted for Cul1 or HA. Arrowheads indicate endogenous Cul1 and the lines on the right indicate overexpressed HA-Cul1.<p><b>Copyright information:</b></p><p>Taken from "Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome"</p><p>http://www.biomedcentral.com/1471-2091/9/1</p><p>BMC Biochemistry 2008;9():1-1.</p><p>Published online 3 Jan 2008</p><p>PMCID:PMC2265268.</p><p></p