Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

Abstract

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT Δ69-83 ), impaired 26S proteasomedependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways. S tudies with simian virus 40 (SV40), a member of the Polyomaviridae family of tumor viruses, have led to fundamental insights into molecular processes of cell transformation and oncogenesis (1, 2). SV40 encodes the large tumor antigen (LT) with the potential to transform cells in culture and induce tumors in rodents. The tumorigenic features of SV40 have been attributed to binding and deactivation of key tumor suppressor proteins of the host cell including p53 and members of the retinoblastoma (pRB) family (1-3). In addition, SV40 LT was shown to be physically associated with Cullin 7 (CUL7; also named p185 or p193) (4, 5) as well as insulin receptor substrate 1 (IRS1) (6). It has been proposed that the association of SV40 LT with either CUL7 or IRS1 is critical to SV40 oncogenic transformation (7-9). However, the functional effect of LT interaction with CUL7/IRS1 and their pathophysiological interrelation remains mostly unknown.CUL7 is a scaffold protein responsible for assembling the multisubunit Cullin-RING E3 ubiquitin ligase 7 (CRL7) that consists of the RING-finger protein ROC1 and the Skp1-Fbw8 substrate-targeting subunit (10, 11). Genetic studies documented a pivotal growth-regulatory role of CRL7. Both cul7 (12) and fbw8 (13) null mice exhibit intrauterine growth retardation. In addition, CUL7 germ-line mutations were linked to 3-M syndrome, a hereditary disorder characterized by pre-and postnatal growth retardation in hum...

Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion

“…In this regard, cactin was not identified in a mass-spectrometrybased study where CUL7, OBSL1 and CCDC8 were over-expressed in HEK 293 cells and used as bait (Hanson et al, 2014). Because we performed our experiments in HEK 293T cells (HEK 293 cells stably expressing the SV40 large T antigen), it is possible that the SV40 large T antigen, which is known to associate with CUL7 and inhibit its ubiquitin ligase activity (Hartmann et al, 2014), stimulates cactin interaction with the 3M complex. Alternatively, cactin might be associated with the other elements of the 3M complex only transiently or at very low levels and therefore escaped detection in experiments where CUL7, OBSL1 and CCDC8 were used as bait (Hanson et al, 2014).…”

Cullin E3 Ligases and Their Rewiring by Viral Factors

“…The polyomavirus SV40 (a simian/human virus) encodes the Large T antigen (LTAg)—a well-studied highly oncogenic viral protein. LTAg has recently been shown to bind to the carboxyl terminus of CRL7 ( Figure 3 h) [ 41 ]. This interaction impairs the ability of CRL7 to degrade its cognate substrate, the insulin receptor substrate 1 (IRS-1), a critical component of the insulin and insulin like growth factor 1 (IGF1) -signaling pathways.…”

Automatic network generation describes differential gene data in user friendly and expeditiously analyzable network views

“…This is due to the used transcription factors, which shown to also mainly interact with AKT and similar networks missing ERK effects were painted [59,83,84] and was previously shown in literature [56,85]. Something similar was also shown for the ERK action on IRS1 [86]. This behavior is difficult to avoid because of the hub-like structures of most protein-protein interaction databases available [11].…”