Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation

Constitutive and induced protein SUMOylation is involved in the regulation of a variety of cellular processes, such as regulation of gene expression and protein transport, and proceeds mainly in the nucleus of the cell. So far, several hundred SUMOylation targets have been identified, but presumably they represent only a part of the total of proteins which are regulated by SUMOylation. Here, we used the Ubc9 fusion-dependent SUMOylation system (UFDS) to screen for constitutive and induced SUMOylation of 46 randomly chosen proteins with proven or potential nuclear localization. Fourteen new UFDS-substrate proteins were identified of which eight could be demonstrated to be SUMOylated in a UFDS-independent manner in vivo. Of these, three were constitutively SUMOylated (FOS, CRSP9 and CDC37) while the remaining five substrates (CSNK2B, TAF10, HSF2BP, PSMC3 and DRG1) showed a stimulation-dependent SUMOylation induced by the MAP3 kinase MEKK1. Hence, UFDS is appropriate for the identification and characterization of constitutive and, more importantly, induced protein SUMOylation in vivo

CD2AP Regulates SUMOylation of CIN85 in Podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85

Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation

Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion–directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity

PRMT5-mediated regulatory arginine methylation of RIPK3

Abstract The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context-dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found to be a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-κB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-β-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST–YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6–p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1

The spinal muscular atrophy disease protein SMN is linked to the rho-kinase pathway via profilin

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis

Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation

Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion–directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity