ABSTRACT
Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.
Subject(s)
Cell Cycle Proteins , Proteomics , Cell Cycle/physiology , Anaphase-Promoting Complex-Cyclosome/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Phosphorylation , Protein Stability , NIMA-Interacting Peptidylprolyl Isomerase/genetics , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , MitosisABSTRACT
Cyclin-dependent kinases (CDKs) mediated phosphorylation inactivates the anaphase-promoting complex (APC/CCDH1), an E3 ubiquitin ligase that contains the co-activator CDH1, to promote G1/S transition. PIN1 is a phosphorylation-directed proline isomerase and a master cancer signaling regulator. However, little are known about APC/CCDH1 regulation after phosphorylation and about PIN1 ubiquitin ligases. Here we uncover a domain-oriented reciprocal inhibition that controls the timely G1/S transition: The non-phosphorylated APC/CCDH1 E3 ligase targets PIN1 for degradation in G1 phase, restraining G1/S transition; APC/CCDH1 itself, after phosphorylation by CDKs, is inactivated by PIN1-catalyzed isomerization, promoting G1/S transition. In cancer, PIN1 overexpression and APC/CCDH1 inactivation reinforce each other to promote uncontrolled proliferation and tumorigenesis. Importantly, combined PIN1- and CDK4/6-inhibition reactivates APC/CCDH1 resulting in PIN1 degradation and an insurmountable G1 arrest that translates into synergistic anti-tumor activity against triple-negative breast cancer in vivo. Reciprocal inhibition of PIN1 and APC/CCDH1 is a novel mechanism to control timely G1/S transition that can be harnessed for synergistic anti-cancer therapy.
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
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.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/physiology , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Apoptosis/genetics , Carrier Proteins/genetics , Casein Kinase II/metabolism , Cell Line , Co-Repressor Proteins , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Models, Molecular , Molecular Chaperones , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , SUMO-1 Protein/metabolism , Stress, PhysiologicalABSTRACT
Small Ubiquitin-like MOdifiers (SUMOs) are ubiquitin-like proteins known to covalently modify large number of cellular proteins. The mammalian SUMO family includes four paralogues, SUMO-1 through SUMO-4. Death-associated protein-6, Daxx, is a 740 residue important transcription corepressor known to represses transcriptional potential of several sumolyted transcription factors. Daxx also plays important role in apoptosis. Both terminals of Daxx harbor separate SUMO Interaction Motifs (SIM), which mediate its interaction with SUMO and hence the sumolyted transcription factors. The C-terminal SIM of Daxx preferentially binds SUMO-1. Practically complete (1)H, (13)C and (15)N resonance assignments for the complex between SUMO-1 and 20 residue Daxx C-terminal SIM peptide are reported here.
