Prashant Sharma

Loss of SUMO1 in mice affects RanGAP1 localization and formation of PML nuclear bodies, but is not lethal as it can be compensated by SUMO2 or SUMO3

Conjugation of the small ubiquitin-like modifier (SUMO) to target proteins regulates numerous biological processes and has been implicated in tumorigenesis and metastasis. The three SUMO isoforms in vertebrates, SUMO1 and the highly similar SUMO2 and SUMO3, can be conjugated to unique as well as overlapping subsets of target proteins. Yet, it is still not clear whether roles for each family member are distinct or whether redundancy exists. Here we describe a mutant mouse line that completely lacks SUMO1, but surprisingly is viable and lacks any overt phenotype. Our study points to compensatory utilization of SUMO2 and/or SUMO3 for sumoylation of SUMO1 targets. The ability of SUMO isoforms to substitute for one another has important implications for rational targeting of the SUMO pathway.

Mutation of SENP1/SuPr-2 reveals an essential role for desumoylation in mouse development.

ABSTRACT The covalent modification of proteins by the small ubiquitin-like protein SUMO has been implicated in the regulation of numerous biological processes, including nucleocytoplasmic transport, genomic stability, and gene transcription. Sumoylation occurs by a multienzyme process similar to ubiquitination and, in Saccharomyces cerevisiae, is reversed by desumoylating enzymes encoded by the Ulp1 and Smt4/Ulp2 genes. The physiological importance of desumoylation has been revealed by mutations in either gene, which lead to nonoverlapping defects in cell cycle transition and meiosis. Several mammalian Ulp homologues have been identified, but, to date, nothing is known of the phenotypic effects of their loss of function. Here, we describe a random retroviral insertional mutation of one homolog, mouse SENP1/SuPr-2. The mutation causes increased steady-state levels of the sumoylated forms of a number of proteins and results in placental abnormalities incompatible with embryonic development. Our findings provide the first insight into the critical importance of regulating sumoylation in mammals.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin–protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73α, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73α for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73α and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1−/− cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73α and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

N4BP1 is a newly identified nucleolar protein that undergoes SUMO-regulated polyubiquitylation and proteasomal turnover at promyelocytic leukemia nuclear bodies

A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate for monoubiquitylation by the E3 ubiquitin ligase Nedd4. Here, we describe Nedd4-mediated polyubiquitylation and proteasomal degradation of N4BP1. In addition, we show that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation. Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme SENP1, which show increased steady-state sumoylation levels. We have localized endogenous N4BP1 predominantly to the nucleolus in primary cells. However, a small fraction is found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, there is considerable accumulation of N4BP1 at PML NBs. These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by ubiquitin and SUMO in the regulation of nucleolar protein turnover.

Nodal signaling from the visceral endoderm is required to maintain Nodal gene expression in the epiblast and drive DVE/AVE migration.

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE. The Nodal gene is expressed in both the VE and in the pluripotent epiblast, which gives rise to the germ layers. Previous findings have provided conflicting evidence as to the relative importance of Nodal signaling from the epiblast vs. VE for AP patterning. Here we show that conditional mutagenesis of the Nodal gene specifically within the VE leads to reduced Nodal expression levels in the epiblast and incomplete or failed DVE/AVE migration. These results support a required role for VE Nodal to maintain normal levels of expression in the epiblast, and suggest signaling from both VE and epiblast is important for DVE/AVE migration.