Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.

No abstract

Signal regulatory protein (SIRP) ␣1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRP␣1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRP␣1, but not of a mutant SIRP␣1 lacking the tyrosine phosphorylation sites, in SIRP␣1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRP␣1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRP␣1 significantly suppressed activation of NF-B and also sensitized Huh7 cells to TNF␣ or cisplatin-induced cell death. In addition, SIRP␣1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRP␣1/SHP-2 complex formation. In conclusion, a negative regulatory effect of SIRP␣1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF-B pathways. contains 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) with 4 tyrosine residues that are phosphorylated in response to a variety of growth factors and integrin-mediated cell adhesion. [1][2][3] This phosphorylation enables recruitment and activation of Src homology-containing phosphotyrosine phosphatase 2 (SHP-2) that in turn dephosphorylates specific protein substrates involved in mediating various physiological effects.SHP-2, a widely expressed cytoplasmic tyrosine phosphatase with 2 Src homology-2 domains, has been implicated in growth factor-induced cell proliferation probably through activation of the Ras-mitogen-activated protein kinase (MAPK) cascade. 4 SIRP␣1/SHP-2 complex negatively or positively regulates intracellular signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix proteins. For example, SIRP␣1 overexpression in NIH3T3 cells inhibited DNA synthesis and MAPK phosphorylation following endothelial growth factor (EGF) or insulin stimulation. 1 In contrast, overexpression of wildtype SHPS-1 (the murine homolog of SIRP␣1) was reported to increase MAPK activity in response to insulin or integrin stimulation. 5 SHPS-1 overexpression suppressed anchorage-independent cell growth and cancer dissemination; however, phosphorylation states of MAPK and c-jun NH2-terminal kinase (JNK) were almost similar. 6 In fibroblasts expressing an SHPS-1 mutant lacking most of the cytoplasmic region, growth factor-induced JNK

c-src codon 531 mutation in colorectal cancer is not the cause of c-src activation.