Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

• MeSH
• draslíkové kanály řízené napětím chemie   metabolismus   MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• lidé MeSH
• mikrofilamentové proteiny chemie   metabolismus   MeSH
• multimerizace proteinu * MeSH
• proteasomový endopeptidasový komplex chemie   metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 1 chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• ubikvitinace * MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• glykoproteiny metabolismus   MeSH
• HEK293 buňky MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza * MeSH
• homeostáza MeSH
• lidé MeSH
• lipoylace MeSH
• malá interferující RNA metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• receptory CXCR4 metabolismus   MeSH
• signální transdukce * MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• zárodečné buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.

• MeSH
• fosforylace MeSH
• lidé MeSH
• proteiny 14-3-3 * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.

• MeSH
• arrestin genetika   metabolismus   MeSH
• časové faktory MeSH
• endocytóza * MeSH
• genetická transkripce MeSH
• glukosa nedostatek   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• mutace MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinfosfatasa 1 metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• represorové proteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• ubikvitinace MeSH
• Publikační typ
• časopisecké články MeSH

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

• MeSH
• aktivace enzymů MeSH
• biotest metody   MeSH
• chloramfenikol-O-acetyltransferasa genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu * MeSH
• posttranslační úpravy proteinů MeSH
• proteolýza MeSH
• reportérové geny * MeSH
• ubikvitinace * MeSH
• ubikvitinligasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH