Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.     

Transforming growth factor-beta-independent regulation of myogenesis by SnoN sumoylation

Recent progress has been made on the role of oncoproteins c-Ski and related SnoN in the control of cellular transformation. c-Ski/SnoN potently repress transforming growth factor-beta (TGF-beta) antiproliferative signaling through physical interaction with signal transducers called Smads. Overexpression of c-Ski/SnoN also induces skeletal muscle differentiation, but how c-Ski/SnoN function in myogenesis is largely unknown. During our investigation on the role of sumoylation in TGF-beta signaling, we inadvertently found that SnoN is modified by small ubiquitin-like modifier-1 (SUMO-1). Here, we biochemically characterize SnoN sumoylation in detail and report the physiological function of the modification. Sumoylation occurs primarily at lysine 50 (Lys-50). PIAS1 and PIASx proteins physically interact with SnoN to stimulate its sumoylation, thus serving as SUMO-protein isopeptide ligases (E3) for SnoN sumoylation. SnoN sumoylation does not alter its metabolic stability or its ability to repress TGF-beta signaling. Notably, loss of sumoylation in the Lys-50 site (via a Lys-to-Arg point mutation) potently activates muscle-specific gene expression and enhances myotube formation. Our study suggests a novel role for SUMO modification in the regulation of myogenic differentiation.     

Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25

Vertebrates express two distinct families of SUMO proteins (SUMO1 and SUMO2/3) that serve distinct functions as posttranslational modifiers. Many proteins are modified specifically with SUMO1 or SUMO2/3, but the mechanisms for paralog selectivity are poorly understood. In a screen for SUMO2/3 binding proteins, we identified Ubiquitin Specific Protease 25 (USP25). USP25 turned out to also be a target for sumoylation, being more efficient with SUMO2/3. Sumoylation takes place within USP25's two ubiquitin interaction motifs (UIMs) that are required for efficient hydrolysis of ubiquitin chains. USP25 sumoylation impairs binding to and hydrolysis of ubiquitin chains. Both SUMO2/3-specific binding and sumoylation depend on a SUMO interaction motif (SIM/SBM). Seven amino acids in the SIM of USP25 are sufficient for SUMO2/3-specific binding and conjugation, even when taken out of structural context. One mechanism for paralog-specific sumoylation may, thus, involve SIM-dependent recruitment of SUMO1 or SUMO2/3 thioester-charged Ubc9 to targets.     

Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass

Sumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin‐related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild‐type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphate‐starvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress‐induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress‐related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.     

SUMO conjugation attenuates the activity of the gypsy chromatin insulator

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. We present evidence suggesting that two protein components of the gypsy chromatin insulator of Dorsophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation.     

Regulation of bcl-2 expression by Ubc9

Posttranslational modifications mediated by ubiquitin-like proteins have been implicated in regulating a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has caught a great deal of attention recently because of its novel and distinguished functions. Sumoylation is a multiple-step process, involving maturation, activation, conjugation and ligation. Ubc9 is an E2 conjugating enzyme essential for sumoylation. We have previously shown that suppression of sumoylation by a dominant negative Ubc9 mutant (Ubc9-DN) in the estrogen receptor (ER) positive MCF-7 cells is associated with alterations of tumor cell's response to anticancer drugs as well as tumor growth in a xenograft mouse carcinoma model. To dissect the underlying mechanism of Ubc9-associated alterations of drug responsiveness and tumor growth, we profiled gene expression for the cells expressing wild type Ubc9 (Ubc9-WT) and Ubc9-DN. We found that several tumorigenesis-related genes were downregulated in the Ubc9-DN cells. Within this group, we found that over 10 genes are known to be regulated by ER. Experiments using the estrogen response element fused to the luciferase reporter showed that the basal level of luciferase activity was significantly reduced in the Ubc9-DN cells when compared to the vector alone or the Ubc9-WT cells. Furthermore, we found that both the stability and the subcellular localization of steroid hormone receptor coactivator-1 (SRC-1) were altered in the Ubc9-DN cells. Together, these results suggest that Ubc9 might regulate bcl-2 expression through the ER signaling pathway, which ultimately contributes to the alterations of drug responsiveness and tumor growth.