scholarly journals Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2

2018 ◽  
Vol 11 (556) ◽  
pp. eaau0715 ◽  
Author(s):  
JingJing Liu ◽  
Xianlin Zou ◽  
Tetsuya Gotoh ◽  
Anne M. Brown ◽  
Liang Jiang ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Ubiquitin Ligase ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Tumor Development ◽  
Mouse Double Minute ◽  
Period 2 ◽  
Clock Component ◽  
Ubiquitin Ligase Complex ◽  
Double Minute

The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat–containing proteins (β-TrCPs), which are F-box–containing proteins that function as substrate recognition subunits of the SCFβ-TRCPubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

scholarly journals Nuclear stabilisation of p53 requires a functional nucleolar surveillance pathway

2021 ◽  
Author(s):  
Katherine M. Hannan ◽  
Priscilla Soo ◽  
Mei S. Wong ◽  
Justine K. Lee ◽  
Nadine Hein ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Loss Of Function ◽  
Ribonucleoprotein Particle ◽  
Mouse Double Minute ◽  
Genome Wide ◽  
Double Minute ◽  
Precise Molecular Mechanism ◽  
Mammalian Protein

AbstractThe nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed “ribosomopathies”, while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.

scholarly journals Downregulation of urea transporter UT-A1 activity by 14-3-3 protein

2015 ◽  
Vol 309 (1) ◽  
pp. F71-F78 ◽  
Author(s):  
Xiuyan Feng ◽  
Zenggang Li ◽  
Yuhong Du ◽  
Haian Fu ◽  
Janet D. Klein ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Regulatory Mechanism ◽  
Critical Role ◽  
Regulatory Proteins ◽  
Transport Activity ◽  
Urea Transporter ◽  
Glutathione S Transferase ◽  
Mouse Double Minute ◽  
Pulldown Assay ◽  
Double Minute

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

scholarly journals p53 Phosphomimetics Preserve Transient Secondary Structure but Reduce Binding to Mdm2 and MdmX

Biomolecules ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 83 ◽  
Author(s):  
Robin Levy ◽  
Emily Gregory ◽  
Wade Borcherds ◽  
Gary Daughdrill
Keyword(s):  
Secondary Structure ◽  
Posttranslational Modifications ◽  
Negative Charge ◽  
Transactivation Domain ◽  
Negative Regulators ◽  
Mouse Double Minute ◽  
Double Minute ◽  
Kix Domain ◽  
Structural Methods ◽  
Transient Secondary Structure

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.

scholarly journals A Subunit of the Anaphase-Promoting Complex Is a Centromere-Associated Protein in Mammalian Cells

1998 ◽  
Vol 18 (1) ◽  
pp. 468-476 ◽  
Author(s):  
Pia-Marie Jörgensen ◽  
Eva Brundell ◽  
Maria Starborg ◽  
Christer Höög
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Separation Process ◽  
Soluble Form ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Ubiquitin Ligase Complex ◽  
A Subunit

ABSTRACT Sister chromatids in early mitotic cells are held together mainly by interactions between centromeres. The separation of sister chromatids at the transition between the metaphase and the anaphase stages of mitosis depends on the anaphase-promoting complex (APC), a 20S ubiquitin-ligase complex that targets proteins for destruction. A subunit of the APC, called APC-α in Xenopus (and whose homologs are APC-1, Cut4, BIME, and Tsg24), has recently been identified and shown to be required for entry into anaphase. We now show that the mammalian APC-α homolog, Tsg24, is a centromere-associated protein. While this protein is detected only during the prophase to the anaphase stages of mitosis in Chinese hamster cells, it is constitutively associated with the centromeres in murine cells. We show that there are two forms of this protein in mammalian cells, a soluble form associated with other components of the APC and a centromere-bound form. We also show that both the Tsg24 protein and the Cdc27 protein, another APC component, are bound to isolated mitotic chromosomes. These results therefore support a model in which the APC by ubiquitination of a centromere protein regulates the sister chromatid separation process.

scholarly journals A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Michaela Gschweitl ◽  
Anna Ulbricht ◽  
Christopher A Barnes ◽  
Radoslav I Enchev ◽  
Ingrid Stoffel-Studer ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Influenza A ◽  
Endocytic Trafficking ◽  
Dependent Manner ◽  
Ubiquitin Ligase Complex ◽  
Cullin 3 ◽  
Host Cell Entry ◽  
Endosome Maturation

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.

scholarly journals SCF-Fbxo42 promotes synaptonemal complex assembly by downregulating PP2A-B56

2020 ◽  
Vol 220 (2) ◽  
Author(s):  
Pedro Barbosa ◽  
Liudmila Zhaunova ◽  
Simona Debilio ◽  
Verdiana Steccanella ◽  
Van Kelly ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Synaptonemal Complex ◽  
Ubiquitin Ligase ◽  
Posttranslational Modifications ◽  
Meiotic Prophase ◽  
Female Meiosis ◽  
Complex Assembly ◽  
Homologous Chromosomes ◽  
Ubiquitin Ligase Complex ◽  
Segregation Of Chromosomes

Meiosis creates genetic diversity by recombination and segregation of chromosomes. The synaptonemal complex assembles during meiotic prophase I and assists faithful exchanges between homologous chromosomes, but how its assembly/disassembly is regulated remains to be understood. Here, we report how two major posttranslational modifications, phosphorylation and ubiquitination, cooperate to promote synaptonemal complex assembly. We found that the ubiquitin ligase complex SCF is important for assembly and maintenance of the synaptonemal complex in Drosophila female meiosis. This function of SCF is mediated by two substrate-recognizing F-box proteins, Slmb/βTrcp and Fbxo42. SCF-Fbxo42 down-regulates the phosphatase subunit PP2A-B56, which is important for synaptonemal complex assembly and maintenance.

scholarly journals Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Brenna ◽  
Iwona Olejniczak ◽  
Rohit Chavan ◽  
Jürgen A Ripperger ◽  
Sonja Langmesser ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Cyclin Dependent Kinase ◽  
Suprachiasmatic Nuclei ◽  
Nuclear Entry ◽  
Period 2 ◽  
Clock Component ◽  
Free Running ◽  
Free Running Period ◽  
Cryptochrome 1 ◽  
Cyclin Dependent Kinase 5

Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.

scholarly journals A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers

2021 ◽  
Author(s):  
Floris Foijer ◽  
Lin Zhou ◽  
Fernando R Rosas Bringas ◽  
Bjorn Bakker ◽  
Judith E Simon ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Chemotherapy Resistance ◽  
Synthetic Lethal ◽  
Ubiquitin Ligase Complex ◽  
Synthetic Lethal Interactions ◽  
A Subunit ◽  
Scf Ubiquitin Ligase

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the SCF ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work might provide a new angle of how to exploit MELK overexpression in cancers and might thus lead to novel intervention strategies.

scholarly journals Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination

Science ◽  
2018 ◽  
Vol 362 (6419) ◽  
pp. 1177-1182 ◽  
Author(s):  
M. Steklov ◽  
S. Pandolfi ◽  
M. F. Baietti ◽  
A. Batiuk ◽  
P. Carai ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Human Disease ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Ras Signaling ◽  
Ras Proteins ◽  
Ubiquitin Ligase Complex ◽  
Cullin 3 ◽  
Guanosine Triphosphatase

The leucine zipper–like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

scholarly journals CK1δ/ε protein kinase primes the PER2 circadian phosphoswitch

2018 ◽  
Vol 115 (23) ◽  
pp. 5986-5991 ◽  
Author(s):  
Rajesh Narasimamurthy ◽  
Sabrina R. Hunt ◽  
Yining Lu ◽  
Jean-Michel Fustin ◽  
Hitoshi Okamura ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Splice Variant ◽  
Carboxyl Terminus ◽  
Casein Kinase 1 ◽  
Multisite Phosphorylation ◽  
Period 2 ◽  
Cellular Environment ◽  
Clock Component ◽  
Biophysical Analysis ◽  
Carboxyl Terminal

Multisite phosphorylation of the PERIOD 2 (PER2) protein is the key step that determines the period of the mammalian circadian clock. Previous studies concluded that an unidentified kinase is required to prime PER2 for subsequent phosphorylation by casein kinase 1 (CK1), an essential clock component that is conserved from algae to humans. These subsequent phosphorylations stabilize PER2, delay its degradation, and lengthen the period of the circadian clock. Here, we perform a comprehensive biochemical and biophysical analysis of mouse PER2 (mPER2) priming phosphorylation and demonstrate, surprisingly, that CK1δ/ε is indeed the priming kinase. We find that both CK1ε and a recently characterized CK1δ2 splice variant more efficiently prime mPER2 for downstream phosphorylation in cells than the well-studied splice variant CK1δ1. While CK1 phosphorylation of PER2 was previously shown to be robust to changes in the cellular environment, our phosphoswitch mathematical model of circadian rhythms shows that the CK1 carboxyl-terminal tail can allow the period of the clock to be sensitive to cellular signaling. These studies implicate the extreme carboxyl terminus of CK1 as a key regulator of circadian timing.

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