The N-terminal domain of human holocarboxylase synthetase facilitates biotinylation via direct interaction with the substrate protein

Human holocarboxylase synthetase shows a high degree of sequence homology in the catalytic domain with bacterial biotin ligases such as Escherichia coli BirA, but differs in the length and sequence of the N-terminus. Despite several studies having been undertaken on the N-terminal region of hHCS, the role of this region remains unclear. We determined the structure of the N-terminal domain of hHCS by limited proteolysis and showed that this domain has a crucial effect on the enzymatic activity. The domain interacts not only with biotin acceptor protein, but also with the catalytic domain of hHCS, as shown by nuclear magnetic resonance (NMR) experiments. We propose that the N-terminal domain of hHCS recognizes the charged region of biotin acceptor protein, distinctly from the recognition by the catalytic domain.

Structured summary

MINT-7543113: hHCS (uniprotkb:P50747) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543096, MINT-7543129: ACC75 (uniprotkb:O00763) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543053: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by nuclear magnetic resonance (MI:0077)MINT-7543070: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by enzymatic study (MI:0415)     

Structure of the p53 C-terminus bound to 14-3-3: Implications for stabilization of the p53 tetramer

The adaptor protein 14-3-3 binds to and stabilizes the tumor suppressor p53 and enhances its anti-tumour activity. In the regulatory C-terminal domain of p53 several 14-3-3 binding motifs have been identified. Here, we report the crystal structure of the extreme C-terminus (residues 385-393, p53pT387) of p53 in complex with 14-3-3σ at a resolution of 1.28 Å. p53pT387 is accommodated by 14-3-3 in a yet unrecognized fashion implying a rationale for 14-3-3 binding to the active p53 tetramer. The structure exhibits a potential binding site for small molecules that could stabilize the p53/14-3-3 protein complex suggesting the possibility for therapeutic intervention.

Structured summary

MINT-7711943: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7711931: 14-3-3 sigma (uniprotkb:P31947) and p53 (uniprotkb:P04637) bind (MI:0407) by isothermal titration calorimetry (MI:0065)     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Characterisation of the interaction of colicin A with its co-receptor TolA

Colicin A enters Escherichia coli cells through interaction with endogenous TolA and TolB proteins. In vitro, binding of the colicin A translocation domain to TolA leads to unfolding of TolA. Through NMR studies of the colicin A translocation domain and polypeptides representing the individual TolA and TolB binding epitopes of colicin A we question if the unfolding of TolA induced by colicin A is likely to be physiologically relevant. The NMR data further reveals that the colicin A binding site on TolA is different from that for colicin N which explains why there is a difference in colicin toxicity for E. coli carrying a TolA-III homologue from Yersina enterocolitica in place of its own TolA-III.

Structured summary

MINT-7888512: TolA (uniprotkb:P19934) and Col-A (uniprotkb:P04480) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888526: TolA (uniprotkb:P19934) and TolB (uniprotkb:P0A857) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888999: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by molecular sieving (MI:0071)MINT-7888982: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Tropomyosin-binding properties of the CHASM protein are dependent upon its calponin homology domain

The calponin homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the calponin homology (CH) domain or expression of the CH domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0 × 10⁻⁶ M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)-CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH domain.

Structured summary

MINT-7966126: CHASM (uniprotkb:Q99LM3), Tropomyosin alpha (uniprotkb:P04268) and Tropomyosin beta (uniprotkb:P19352) physically interact (MI:0915) by isothermal titration calorimetry (MI:0065)MINT-7966073: CHASM (uniprotkb:Q99LM3) physically interacts (MI:0914) with Tropomyosin beta (uniprotkb:P58776) and Tropomyosin alpha (uniprotkb:P58772) by pull down (MI:0096)MINT-7966187: Tropomyosin alpha (uniprotkb:P04268) and Tropomyosin beta (uniprotkb:P19352) physically interact (MI:0915) with CHASM (uniprotkb:Q99LM3) by pull down (MI:0096)MINT-7966090: CHASM (uniprotkb:Q99LM3) binds (MI:0407) to Tropomyosin alpha (uniprotkb:P04268) by pull down (MI:0096)     

Reconstitution of the mitochondrial Hsp70 (mortalin)-p53 interaction using purified proteins - Identification of additional interacting regions

Previous studies have shown that the mammalian mitochondrial 70 kDa heat-shock protein (mortalin) can also be detected in the cytosol. Cytosolic mortalin binds p53 and by doing so, prevents translocation of the tumor suppressor into the nucleus. In this study, we developed a novel binding assay, using purified proteins, for tracking the interaction between p53 and mortalin. Our results reveal that: (i) P53 binds to the peptide-binding site of mortalin which enhances the ability of the former to bind DNA. (ii) An additional previously unknown binding site for mortalin exists within the C-terminal domain of p53.

Structured summary

MINT-7557591: p53 (uniprotkb:P04637) binds (MI:0407) to DnaK (uniprotkb:P0A6Y8) by affinity chromatography technology (MI:0004)MINT-7557644: mortalin (uniprotkb:P38646) binds (MI:0407) to p53 (uniprotkb:P04637) by pull down (MI:0096)MINT-7557580, MINT-7557611: p53 (uniprotkb:P04637) binds (MI:0407) to mortalin (uniprotkb:P38646) by affinity chromatography technology (MI:0004)     

An S-locus receptor-like kinase in plasma membrane interacts with calmodulin in Arabidopsis

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca²⁺-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.

Structured summary

MINT-6800947:CBRLK1 (uniprotkb:Q9ZT06) and AtCaM2 (uniprotkb:P25069) bind (MI:0407) by electrophoretic mobility shift assay (MI:0413)MINT-6800966:AtCaM2 (uniprotkb:P25069) and CBRLK1 (uniprotkb:Q9ZT06) bind (MI:0407) by competition binding (MI:0405)MINT-6800930:CBRLK1 (uniprotkb:Q9ZT06) binds (MI:0407) to AtCaM2 (uniprotkb:P25069) by far Western blotting (MI:0047)MINT-6800978:AtCaM2 (uniprotkb:P25069) physically interacts (MI:0218) with CBRLK1 (uniprotkb:Q9ZT06) by cytoplasmic complementation assay (MI:0228)     

A scanning peptide array approach uncovers association sites within the JNK/βarrestin signalling complex

βarrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/βarrestin signalling complex. We show that βarrestin 1 and βarrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known ‘common docking’ motif. In the N-domain and C-terminus of βarrestin 1 and βarrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the βarrestin 1 and βarrestin 2 we identify a novel MKK4 docking site.

Structured summary

MINT-7263196, MINT-7263175: Arrestin beta-2 (uniprotkb:P32121) binds (MI:0407) to ASK1 (uniprotkb:Q99683) by peptide array (MI:0081)MINT-7263136: JNK3 (uniprotkb:P53779) binds (MI:0407) to Arrestin beta-1 (uniprotkb:P49407) by peptide array (MI:0081)MINT-7263161: JNK3 (uniprotkb:P53779) binds (MI:0407) to Arrestin beta-2 (uniprotkb:P32121) by peptide array (MI:0081)MINT-7263304: Arrestin beta-1 (uniprotkb:P49407) physically interacts (MI:0915) with ASK1 (uniprotkb:Q99683) by anti tag coimmunoprecipitation (MI:0007)MINT-7263286: Arrestin beta-2 (uniprotkb:P32121) binds (MI:0407) to MKK4 (uniprotkb:P45985) by peptide array (MI:0081)MINT-7263231, MINT-7263254: Arrestin beta-1 (uniprotkb:P49407) binds (MI:0407) to ASK1 (uniprotkb:Q99683) by peptide array (MI:0081)MINT-7263269: Arrestin beta-1 (uniprotkb:P49407) binds (MI:0407) to MKK4 (uniprotkb:P45985) by peptide array (MI:0081)     

POSH2 is a RING finger E3 ligase with Rac1 binding activity through a partial CRIB domain

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.

Structured summary

MINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)     

BS69 negatively regulates the canonical NF-κB activation induced by Epstein-Barr virus-derived LMP1

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-κB signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-κB activation and up-regulates IL-6 mRNA expression and IκB degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).

Structured summary

MINT-7032462: LMP1 (uniprotkb:P03230) physically interacts (MI:0218) with TRADD (uniprotkb:Q15628) by anti bait coimmunoprecipitation (MI:0006)MINT-7032451: BS69 (uniprotkb:Q15326) and LMP1 (uniprotkb:P03230) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7032478: LMP1 (uniprotkb:P03230) physically interacts (MI:0218) with BRAM1 (uniprotkb:Q15326) by anti bait coimmunoprecipitation (MI:0006)     

A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase

We show that the monomeric form of Shigella IpaH9.8 E3 ligase catalyses the ubiquitination of human U2AF35 in vitro, providing a molecular mechanism for the observed in vivo effect. We further discover that under non-reducing conditions IpaH9.8 undergoes a domain swap driven by the formation of a disulfide bridge involving the catalytic cysteine and that this dimer is unable to catalyse the ubiquitination of U2AF35. The crystal structure of the domain-swapped dimer is presented. The redox inactivation of IpaH9.8 could be a mechanism of regulating the activity of the IpaH9.8 E3 ligase in response to cell damage so that the host cell in which the bacteria resides is maintained in a benign state suitable for bacterial survival.

Structured summary

MINT-7993779: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0408) by X-ray crystallography (MI:0114) MINT-7993812: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by affinity chromatography technology (MI:0004) MINT-7993790: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by blue native page (MI:0276)     

Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover

Nbr1, a ubiquitous kinase scaffold protein, contains a PB1, and a ubiquitin-associated (UBA) domain. We show here that the nbr1 UBA domain binds to lysine-48 and -63 linked polyubiquitin-B chains. Nbr1 also binds to the autophagic effector protein LC3-A via a novel binding site. Ubiquitin-binding, but not PB1-mediated p62/SQSTM1 interaction, is required to target nbr1 to LC3 and polyubiquitin-positive bodies. Nbr1 binds additionally to proteins implicated in ubiquitin-mediated protein turnover and vesicle trafficking: ubiquitin-specific peptidases USP8, and the endosomal transport regulator p14/Robld3. Nbr1 thus contributes to specific steps in protein turnover regulation disrupted in several hereditary human diseases.

Structured summary

MINT-7034452: USP8 (uniprotkb:P40818) physically interacts (MI:0218) with NBR1 (uniprotkb:Q14596) by pull down (MI:0096)MINT-7034438: SQSTM1 (uniprotkb:Q13501) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034309: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034323: NBR1 (uniprotkb:P97432) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034233: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with USP8 (uniprotkb:P40818) by two hybrid (MI:0018)MINT-7034207: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Robld3 (uniprotkb:Q9JHS3) by two hybrid (MI:0018)MINT-7034400, MINT-7034418: NBR1 (uniprotkb:Q14596) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034167: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin B (uniprotkb:Q78XY9) by two hybrid (MI:0018)MINT-7034470: NBR1 (uniprotkb:Q14596) and USP8 (uniprotkb:P40818) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034194: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3-A (uniprotkb:Q91VR7) by two hybrid (MI:0018)MINT-7034336: SQSTM1 (uniprotkb:Q13501) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034375: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3 (uniprotkb:Q9H492) by pull down (MI:0096)MINT-7034350: NBR1 (uniprotkb:Q14596) and Ubiquitin (uniprotkb:P62988) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034181: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Tmed10 (uniprotkb:Q9D1D4) by two hybrid (MI:0018)MINT-7034220: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with ube2o (uniprotkb:Q6ZPJ3) by two hybrid (MI:0018)     

Characterization of GmCaMK1, a member of a soybean calmodulin-binding receptor-like kinase family

Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca²⁺ signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca²⁺-dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa.

Structured summary

MINT-8051564: AtCRLK2 (uniprotkb:Q9LFV3) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051416: GmCaMK3 (uniprotkb:C6ZRS6) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051258: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-8051400: GmCaMK2 (uniprotkb: C6ZRY5) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051242, MINT-8051295, MINT-8051313, MINT-8051327, MINT-8051341, MINT-8051355: GmCaMK1 (genbank_protein_gi:223452504) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051467: GmCaMK4 (uniprotkb: C6TIQ0) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051276: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by comigration in non denaturing gel electrophoresis (MI:0404)MINT-8051374: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by mass spectrometry studies of complexes (MI:0069)     

Coilin interacts with Ku proteins and inhibits in vitro non-homologous DNA end joining

Coilin is a nuclear protein that plays a role in Cajal body formation. The function of nucleoplasmic coilin is unknown. Here we report that coilin interacts with Ku70 and Ku80, which are major players in the DNA repair process. Ku proteins compete with SMN and SmB′ proteins for coilin interaction sites. The binding domain on coilin for Ku proteins cannot be localized to one discrete region, and only full-length coilin is capable of inhibiting in vitro non-homologous DNA end joining (NHEJ). Since Ku proteins do not accumulate in CBs, these findings suggest that nucleoplasmic coilin participates in the regulation of DNA repair.

Structured summary

MINT-8052983:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SmB′ (uniprotkb:P14678) by pull down (MI:0096)MINT-8052941:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by competition binding (MI:0405)MINT-8052765:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052971:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SMN (uniprotkb:Q16637) by pull down (MI:0096)MINT-8052957:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by competition binding (MI:0405)MINT-8052894, MINT-8052908:coilin (uniprotkb:P38432) binds (MI:0407) to Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052804:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by anti bait coimmunoprecipitation (MI:0006)MINT-8052925:coilin (uniprotkb:P38432) binds (MI:0407) to Ku70 (uniprotkb:P12956) by pull down (MI:0096)MINT-8052786:Ku80 (uniprotkb:P13010) physically interacts (MI:0914) with coilin (uniprotkb:P38432) and Ku70 (uniprotkb:P12956) by anti bait coimmunoprecipitation (MI:0006)MINT-8052776:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by pull down (MI:0096)     

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

Identification of ptpro as a novel target gene of Wnt signaling and its potential role as a receptor for Wnt

Wnt/β-catenin signaling plays critical roles in embryonic development and tissue homeostasis in adults by controlling the expression of target genes. We found that expression of ptpro, which encodes a protein tyrosine phosphatase receptor type O (PTPRO), was induced by Wnt/β-catenin signaling in a T cell factor/lymphoid enhancer factor dependent manner. Biochemical assays found that PTPRO interacted with Wnt via its extracellular domain. In addition, ectopic expression of this extracellular domain inhibited Wnt-mediated reporter activity. These results suggest that ptpro is a target gene of Wnt/β-catenin signaling and that PTPRO may function as a novel receptor for Wnt.

Structured summary

MINT-7992076: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt3a (uniprotkb:P27467) by anti tag coimmunoprecipitation (MI:0007)MINT-7992094: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt-3a (uniprotkb:P27467) by cross-linking study (MI:0030)     

Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover

The E3 ubiqutin ligase, murne double-minute clone 2 (MDM2), promotes the degradation of p53 under normal homeostatic conditions. Several serine residues within the acidic domain of MDM2 are phosphorylated to maintain its activity but become hypo-phosphorylated following DNA damage, leading to inactivation of MDM2 and induction of p53. However, the signalling pathways that mediate these phosphorylation events are not fully understood. Here we show that the oncogenic and cell cycle-regulatory protein kinase, polo-like kinase-1 (PLK1), phosphorylates MDM2 at one of these residues, Ser260, and stimulates MDM2-mediated turnover of p53. These data are consistent with the idea that deregulation of PLK1 during tumourigenesis may help suppress p53 function.

Structured summary

MINT-7266353: MDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)MINT-7266344, MINT-7266329: MDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by anti bait coimmunoprecipitation (MI:0006)MINT-7266250: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) p53 (uniprotkb:P04637) by protein kinase assay (MI:0424)MINT-7266241, MINT-7266318: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) MDM2 (uniprotkb:P23804) by protein kinase assay (MI:0424)MINT-7266231, MINT-7266805, MINT-7266264, MINT-7266299: PLK1 (uniprotkb:P53350) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)     

Nuclear import of Pin1 is mediated by a novel sequence in the PPIase domain

Pin1 actively regulates diverse biological/pathological processes, but little is known about the regulatory mechanisms of its cellular localization. In this study, we report that the endogenous Pin1 is distributed in both nucleus and cytoplasm. We found that point mutations of several basic amino acids in the PPIase domain of Pin1 significantly compromise its nuclear localization. Such inhibition is independent of Pin1 enzymatic activity, and is mainly due to the defects in the nuclear import. A novel sequence harboring these residues was identified as a putative nuclear localization signal (NLS) of Pin1. Importin α5 of the nuclear import machinery was found to interact with Pin1.

Structured summary:

MINT-6803320: PIN1 (uniprotkb:Q13255) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803333: importin alpha 3 (uniprotkb:O00505) and PIN1 (uniprotkb:Q13255) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)MINT-6803357: PIN1 (uniprotkb:Q13255) physically interacts (MI:0218) with importin alpha 5 (uniprotkb:P52294) by anti bait coimmunoprecipitation (MI:0006)MINT-6803345: St3 (uniprotkb:P40763) and importin alpha 5 (uniprotkb:P52294) physically interact (MI:0218) by anti tag coimmunoprecipitation (MI:0007)