ABSTRACT
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ABSTRACT
The negatively charged amino acid-dependent sumoylation motif (NDSM) carries an additional stretch of acidic residues downstream of the consensus Ψ-K-x-E/D sumoylation motif. We have previously shown that acetylation of the SUMO E2 conjugase enzyme, Ubc9, at K65 downregulates its binding to the NDSM and renders a selective decrease in sumoylation of substrates with the NDSM motif. Here, we provide detailed structural, thermodynamic, and kinetics results of the interactions between Ubc9 and its K65 acetylated variant (Ac-Ubc9K65) with three NDSMs derived from Elk1, CBP, and Calpain2 to rationalize the mechanism beneath this reduced binding. Our nuclear magnetic resonance (NMR) data rule out a direct interaction between the NDSM and the K65 residue of Ubc9. Similarly, we found that NDSM binding was entropy-driven and unlikely to be affected by the negative charge by K65 acetylation. Moreover our NMR, mutagenesis and molecular dynamics simulation studies defined the sequence of the NDSM as Ψ-K-x-E/D-x1-x2-(x3/E/D)-(x4/E/D)-xn and determined that K74 and K76 were critical Ubc9 residues interacting with the negatively charged residues of the NDSM.
Subject(s)
Calpain/metabolism , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Sialoglycoproteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , ets-Domain Protein Elk-1/metabolism , Acetylation , Calpain/chemistry , Humans , Kinetics , Molecular Dynamics Simulation , Peptide Fragments/chemistry , Protein Binding , Sialoglycoproteins/chemistry , Thermodynamics , Ubiquitin-Conjugating Enzymes/chemistry , ets-Domain Protein Elk-1/chemistryABSTRACT
The Pin1 prolyl isomerase regulates phosphorylation signaling by controlling protein conformation after phosphorylation, and its upregulation promotes oncogenesis via acting on numerous oncogenic molecules. SUMOylation and deSUMOylation are dynamic mechanisms regulating a spectrum of protein activities. The SUMO proteases (SENP) remove SUMO conjugate from proteins, and their expression is deregulated in cancers. However, nothing is known about the role of SUMOylation in regulating Pin1 function. Here, we show that Pin1 is SUMOylated on Lys6 in the WW domain and on Lys63 in the PPIase domain. Pin1 SUMOylation inhibits its protein activity and oncogenic function. We further identify that SENP1 binds to and deSUMOylates Pin1. Importantly, either overexpression of SENP1 or disruption of Pin1 SUMOylation promotes the ability of Pin1 to induce centrosome amplification and cell transformation. Moreover, SENP1 also increases Pin1 protein stability in cell cultures, and Pin1 levels are positively correlated with SENP1 levels in human breast cancer specimens. These results not only uncover Pin1 SUMOylation on Lys6/63 as a novel mechanism to inhibit its activity and function but also identify a critical role for SENP1-mediated deSUMOylation in promoting Pin1 function during tumorigenesis.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Endopeptidases/metabolism , Peptidylprolyl Isomerase/metabolism , Sumoylation , Amino Acid Sequence , Amino Acid Substitution , Animals , Breast Neoplasms/enzymology , Cell Line , Centrosome/metabolism , Chromosomal Instability , Cyclin D1 , Cysteine Endopeptidases , Female , Gene Knockdown Techniques , Humans , Mice , NIMA-Interacting Peptidylprolyl Isomerase , Oxidative Stress , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/genetics , Protein Binding , Protein Interaction Domains and Motifs , Signal TransductionABSTRACT
While numerous small ubiquitin-like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid-dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ-K-X-E/D consensus motif, such as CBP and Elk-1, but not substrates with core ψ-K-X-E/D motif alone or SUMO-interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia-elicited modulation of sumoylation and target gene expression of CBP and Elk-1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.
Subject(s)
Acetyltransferases/metabolism , Protein Processing, Post-Translational , SUMO-1 Protein/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/physiology , Acetylation , Acetyltransferases/physiology , Cell Hypoxia/genetics , Cell Hypoxia/physiology , Cells, Cultured , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/physiology , RNA, Small Interfering/pharmacology , Sirtuin 1/metabolism , Sirtuin 1/physiology , Sumoylation/drug effects , Sumoylation/genetics , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/genetics , ets-Domain Protein Elk-1/metabolismABSTRACT
Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.
