scholarly journals Regulation of Mdm2-Directed Degradation by the C Terminus of p53

1998 ◽  
Vol 18 (10) ◽  
pp. 5690-5698 ◽  
Author(s):  
Michael H. G. Kubbutat ◽  
Robert L. Ludwig ◽  
Margaret Ashcroft ◽  
Karen H. Vousden
Keyword(s):  
P53 Protein ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Tumor Suppressor Protein ◽  
C Terminus ◽  
The Stability ◽  
P53 Tumor Suppressor Protein ◽  
Inducible Gene ◽  
Oligomerization Domain

ABSTRACT The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.

scholarly journals Amyloid-ß promotes neurotoxicity by Cdk5-induced p53 stabilization

2018 ◽  
Author(s):  
Rebeca Lapresa ◽  
Jesús Agulla ◽  
Irene Sánchez-Morán ◽  
Juan P. Bolaños ◽  
Angeles Almeida
Keyword(s):  
Neuronal Apoptosis ◽  
Therapeutic Strategy ◽  
Neuronal Damage ◽  
P53 Protein ◽  
Protein A ◽  
Tumor Suppressor Protein ◽  
Protein Accumulation ◽  
Brain Areas ◽  
P53 Tumor Suppressor Protein

ABSTRACTThe p53 tumor suppressor protein, a key regulator of cell apoptosis, has been described to accumulate in affected brain areas from Alzheimer’s disease (AD) patients. However, whether p53 plays any role in AD pathogenesis remains unknown. Here, we found that exposure of neurons to oligomers of the amyloidogenic fragment 25-35 of the Aß peptide (Aβ25-35) activated Cdk5, which promoted p53 protein phosphorylation and stabilization. Moreover, Aβ25-35-mediated mitochondrial dysfunction and neuronal apoptosis were prevented by both genetic and pharmacological inhibition of either p53 or Cdk5 activities. To confirm this mechanism in vivo, Aβ25-35 was stereotaxically injected in the cerebral right ventricle of mice, a treatment that caused p53 protein accumulation, dendrite disruption and neuronal death. Furthermore, these effects were prevented in p53 knockout mice or by pharmacologically inhibiting p53. Thus, Aβ25-35 triggers Cdk5 activation to induce p53 phosphorylation and stabilization, which leads to neuronal damage. Inhibition of the Cdk5-p53 pathway may therefore represent a novel therapeutic strategy against Aβ-induced neurodegeneration.

scholarly journals TIP30 directly binds p53 tumor suppressor protein in vitro

2012 ◽  
Vol 34 (5) ◽  
pp. 495-500 ◽  
Author(s):  
Si-Hyung Lee ◽  
Sung-Kyu Ju ◽  
Tae-Young Lee ◽  
Sung-Ho Huh ◽  
Kyou-Hoon Han
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Protein ◽  
P53 Tumor Suppressor ◽  
P53 Tumor Suppressor Protein

scholarly journals Functional Dissection of Human and Mouse POT1 Proteins

2008 ◽  
Vol 29 (2) ◽  
pp. 471-482 ◽  
Author(s):  
Wilhelm Palm ◽  
Dirk Hockemeyer ◽  
Tatsuya Kibe ◽  
Titia de Lange
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Dna Damage Response ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Telomeric Dna ◽  
C Terminus ◽  
Damage Response ◽  
End Resection

ABSTRACT The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5′-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5′-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5′-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

scholarly journals Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Stephan Tetenborg ◽  
Helen Y. Wang ◽  
Lena Nemitz ◽  
Anne Depping ◽  
Alexsandra B. Espejo ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Binding Affinity ◽  
Protein Microarray ◽  
Pdz Domain ◽  
Phosphorylation Site ◽  
Binding Domain ◽  
Dependent Manner ◽  
C Terminus ◽  
Activity Dependent

Abstract Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

scholarly journals A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

2020 ◽  
Vol 168 (5) ◽  
pp. 515-533
Author(s):  
Jose M González de Cózar ◽  
Maria Carretero-Junquera ◽  
Grzegorz L Ciesielski ◽  
Sini M Miettinen ◽  
Markku Varjosalo ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Shotgun Proteomics ◽  
Binding Domain ◽  
The Novel ◽  
Binding Model ◽  
Interaction Partners ◽  
The Stability ◽  
Deletion Series

Abstract In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

scholarly journals The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins

2009 ◽  
Vol 419 (3) ◽  
pp. 595-602 ◽  
Author(s):  
Tawnya Webber ◽  
Sarsati Gurung ◽  
Justin Saul ◽  
Trenton Baker ◽  
Michelle Spatara ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Critical Role ◽  
Oligomeric Proteins ◽  
C Terminus ◽  
Monomeric Proteins ◽  
P22 Tailspike ◽  
Tailspike Protein ◽  
Oligomerization Domain

TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimaera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimaera appears to involve hydrophobic interactions and a refolding sequence, both of which are analogous to the native TSP. These results underscore the importance of the TSP C-terminus in the assembly of the mature trimer and demonstrate its potential utility as a model to study the folding and assembly of the TSP C-terminus in isolation.

scholarly journals Regulation of p53 Function and Stability by Phosphorylation

1999 ◽  
Vol 19 (3) ◽  
pp. 1751-1758 ◽  
Author(s):  
Margaret Ashcroft ◽  
Michael H. G. Kubbutat ◽  
Karen H. Vousden
Keyword(s):  
Dna Damage ◽  
Protein Kinases ◽  
Phosphorylation Site ◽  
Tumor Suppressor Protein ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Mutant Proteins ◽  
Encoding Gene ◽  
P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

scholarly journals Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction

2001 ◽  
Vol 82 (10) ◽  
pp. 2341-2351 ◽  
Author(s):  
Kelly J. Woytek ◽  
Dhandapani Rangasamy ◽  
Cynthia Bazaldua-Hernandez ◽  
Mike West ◽  
Van G. Wilson
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Genome Replication ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Recognition Element ◽  
C Terminus

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

scholarly journals Salicylic acid-driven association of LENRV and NIMIN1/NIMIN2 binding domain regions in the C-terminus of tobacco NPR1 transduces SAR signal

2019 ◽  
Author(s):  
David Neeley ◽  
Evelyn Konopka ◽  
Anna Straub ◽  
Felix Maier ◽  
Artur J. P. Pfitzner ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Systemic Acquired Resistance ◽  
Binding Domain ◽  
List Type ◽  
Animal Cells ◽  
Conserved Sequence ◽  
Physical Association ◽  
C Terminus ◽  
Sa Signaling

SummaryNONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES1 (NPR1) is the central regulator of salicylic acid (SA)-induced PR-1 gene expression and systemic acquired resistance (SAR). The mechanism how SA is transduced through NPR1 is discussed controversially. Previously, we showed that Arabidopsis and tobacco (Nt) NPR1 contain two domains in their C-terminal thirds with relevance to SA signaling. SA sensitivity of NPR1 relies on the arginine residue in the LENRV motif, and SA-induced NIM1-INTERACTING (NIMIN, N) proteins bind to a highly conserved sequence termed N1/N2 binding domain (BD).We demonstrate that LENRV and N1/N2BD regions of tobacco NPR1, separated from each other, interact in yeast, in vitro, in plant and in animal cells. Physical association of LENRV and N1/N2BD parts is enhanced considerably by SA and functional analogs, but not by a non-functional analog. Furthermore, physical association requires R431 and is most effective with intact LENRV and N1/N2BD interfaces.Association of separated LENRV and N1/N2BD parts by SA reconstitutes a functional NtNPR1 C-terminus, displaying transcription activity and able to interact with TGA transcription factors at two distinct sites.Tobacco NIMIN proteins can assemble LENRV and N1/N2BD parts into ternary complexes suggesting that NIMINs shape the NPR1 C-terminus to modulate SA signaling.

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