NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins

N-methyl-d-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the brain. Here we demonstrate interactions between the NR2A and NR2B subunits of NMDARs with flotillin-1 (flot-1), a lipid raft-associated protein. When mapped, analogous regions in the far distal C-termini of NR2A and NR2B mediate binding to flot-1, and the prohibitin homology domain of flot-1 contains binding sites for NR2A and NR2B. Although NR2B can also directly bind to flot-2 via a separate region of its distal C-terminus, NMDARs were significantly more colocalized with flot-1 than flot-2 in cultured hippocampal neurons. Overall, this study defines a novel interaction between NMDARs and flotillins.

Structured summary

MINT-7013094: NR2A (uniprotkb:Q00959), NR2B (uniprotkb:Q00960) and Flot2 (uniprotkb:Q9Z2S9) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013147: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)MINT-7013189: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013033: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013178: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013197, MINT-7013210: NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) physically interact (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013002: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013117: Flot1 (uniprotkb:Q9Z1E1), NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013171: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by anti bait coimmunoprecipitation (MI:0006)MINT-7013017: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013054: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013129: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013155: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013074: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by two hybrid (MI:0018)MINT-7013162: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)     

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8

Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.

Structured summary

MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007)MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007)MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)     

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex

The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB^R321A mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.

Structured summary:

MINT-7385426:Dlp1 (uniprotkb:Q86YH6) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385083, MINT-7385058:IspB (uniprotkb:P0AD57) and IspB (uniprotkb:P0AD57) bind (MI:0407) by blue native page (MI:0276)MINT-7385413:Dlp1 (uniprotkb:O13851) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385024:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dps1 (uniprotkb:O43091) by pull down (MI:0096)MINT-7385041:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dlp1 (uniprotkb:O13851) by pull down (MI:0096)MINT-7385388:IspB (uniprotkb:P0AD57) and Dps1 (uniprotkb:O43091) physically interact (MI:0915) by blue native page (MI:0276)     

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)     

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.

Structured summary

MINT-7894271: ING2 (uniprotkb:Q9H160) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull-down (MI:0096)MINT-7894319, MINT-7894339: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894301: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with ING2 (uniprotkb:Q9H160) by anti bait co-immunoprecipitation (MI:0006)MINT-7894358: ING1b (uniprotkb:Q9UK53-2) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894249: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag co-immunoprecipitation (MI:0007)     

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)     

Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity

Clostridium thermocellum cellulase 9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60-70% of the intact Cel9I endoglucanase activity.

Structured summary:

MINT-6946626:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-6946649:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by molecular sieving (MI:0071)MINT-6946687:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-6946706:Cel9I (uniprotkb:Q02934) binds (MI:0407) to Cel9I (uniprotkb:Q02934) by pull down (MI:0096)     

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product

Recently, it was reported that the product of Birt-Hogg-Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5′-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN-AMPK complex. These results suggest that function(s) of FLCN-AMPK-FNIP complex is regulated by Ser62 phosphorylation.

Structured summary

MINT-7298145, MINT-7298166: Flcn (uniprotkb:Q76JQ2) physically interacts (MI:0915) with AMPK alpha 1 (uniprotkb:P54645) by anti tag coimmunoprecipitation (MI:0007)MINT-7298267: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) tsc2 (uniprotkb:P49816) by protein kinase assay (MI:0424)MINT-7298182: FNIP1 (uniprotkb:Q8TF40) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)MINT-7298132: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) Flcn (uniprotkb:Q76JQ2) by protein kinase assay (MI:0424)MINT-7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)     

Dynamin-1 co-associates with native mouse brain BKCa channels: Proteomics analysis of synaptic protein complexes

In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BK_Ca) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling.

Structured summary

MINT-7543319: Snap-25 (uniprotkb:P60879) physically interacts (MI:0914) with Tubulin beta-5 chain (uniprotkb:P99024), V-type proton ATPase subunit d 1 (uniprotkb:P51863), Zinc finger homeobox protein 3 (uniprotkb:Q61329), Tubulin beta-2A chain (uniprotkb:Q7TMM9), Synaptophysin (uniprotkb:Q62277), Gapdh (uniprotkb:P16858), Basement membrane-specific heparan sulfate proteoglycan core protein (uniprotkb:Q05793), Tubulin alpha-4A chain (uniprotkb:P68368), Tubulin alpha-1A chain (uniprotkb:P68369), Microtubule-associated protein 6 (uniprotkb:Q7TSJ2), AP-2 complex subunit beta (uniprotkb:Q9DBG3), Phosphofurin acidic cluster sorting protein 1 (uniprotkb:Q8K212), AP-2 complex subunit alpha-1 (uniprotkb:P17426), Kinesin-1 heavy chain (uniprotkb:Q617r68), Kinesin heavy chain isoform 5C (uniprotkb:P28738), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2) and Nck-associated protein 1 (uniprotkb:P28660) by anti bait co-immunoprecipitation (MI:0006)MINT-7543636: Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) physically interacts (MI:0914) with AMP deaminase 2 (uniprotkb:Q9DBT5), Gamma-tubulin complex component 4 (uniprotkb:Q9D4F8), Gamma-tubulin complex component 2 (uniprotkb:Q921G8), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2), Phosphoinositide 3-kinase regulatory subunit 4 (uniprotkb:Q8VD65), Beta-centractin (uniprotkb:Q8R5C5), KIAA1107 (uniprotkb:Q80TK0), Sodium/potassium-transporting ATPase subunit alpha-2 (uniprotkb:Q6PIE5), Sodium/potassium-transporting ATPase subunit alpha-3 (uniprotkb:Q6PIC6), Phosphatidylinositol 3-kinase catalytic subunit type 3 (uniprotkb:Q6PF93), KH domain-containing, RNA-binding, signal transduction-associated protein 1 (uniprotkb:Q60749), Tubulin gamma-1 chain (uniprotkb:P83887), Heat shock cognate 71 kDa protein (uniprotkb:P63017), Alpha-centractin (uniprotkb:P61164), Gamma-tubulin complex component 3 (uniprotkb:P58854), Dynamin-1 (uniprotkb:P39053), Kinesin heavy chain isoform 5C (uniprotkb:P28738), Elongation factor 1-alpha 1 (uniprotkb:P10126), Kinesin light chain 2 (uniprotkb:O88448), Activated CDC42 kinase 1 (uniprotkb:O54967) and Syntaxin-binding protein 1 (uniprotkb:O08599) by anti bait co-immunoprecipitation (MI:0006)MINT-7544031: Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) physically interacts (MI:0914) with Syntaxin-binding protein 1 (uniprotkb:O08599), Syntaxin-1A (uniprotkb:O35526) and Dynamin-1 (uniprotkb:P39053) by anti bait co-immunoprecipitation (MI:0006)MINT-7543287: Syntaxin-1A (uniprotkb:O35526) physically interacts (MI:0914) with Vamp2 (uniprotkb:P63044), Snap-25 (uniprotkb:P60879), munc-18 (uniprotkb:O08599) and BK_Ca alpha subunit (uniprotkb:Q08460) by anti bait co-immunoprecipitation (MI:0006)MINT-7543972: Vamp-2 (uniprotkb:P63044) physically interacts (MI:0914) with Dynamin-1 (uniprotkb:P39053), Snap-25 (uniprotkb:P60879), Syntaxin-1A (uniprotkb:O35526) and Synaptophysin (uniprotkb:Q62277) by anti bait co-immunoprecipitation (MI:0006)MINT-7543728: Dynamin-1 (uniprotkb:P39053) physically interacts (MI:0914) with Clathrin heavy chain 1 (uniprotkb:Q68FD5) and Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) by anti bait co-immunoprecipitation (MI:0006)MINT-7543905: Snap-25 (uniprotkb:P60879) physically interacts (MI:0914) with Syntaxin-1A (uniprotkb:O35526) and Vamp-2 (uniprotkb:P63044) by anti bait co-immunoprecipitation (MI:0006)MINT-7543476: Vamp-2 (uniprotkb:P63044) physically interacts (MI:0914) with Syntaxin-7 (uniprotkb:O70439), Neuronal membrane glycoprotein M6-a (uniprotkb:P35802), Syntaxin-1B (uniprotkb:P61264), Beta-soluble NSF attachment protein (uniprotkb:P28663), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-3 (uniprotkb:Q61011), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (uniprotkb:P62874), Guanine nucleotide-binding protein G(o) subunit alpha (uniprotkb:P18872), V-type proton ATPase subunit d 1 (uniprotkb:P51863), Zinc transporter 3 (uniprotkb:P97441), Sodium/potassium-transporting ATPase subunit alpha-2 (uniprotkb:Q6PIE5), Sodium/potassium-transporting ATPase subunit alpha-3 (uniprotkb:Q6PIC6), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2), Potassium-transporting ATPase alpha chain 1 (uniprotkb:Q64436), Synaptophysin (uniprotkb:Q62277), Syntaxin-1A (uniprotkb:O35526) and Dynamin-1 (uniprotkb:P39053) by anti bait co-immunoprecipitation (MI:0006)     

Interaction of the human somatostatin receptor 3 with the multiple PDZ domain protein MUPP1 enables somatostatin to control permeability of epithelial tight junctions

The presence of heterotrimeric G-proteins at epithelial tight junctions suggests that these cellular junctions are regulated by so far unknown G-protein coupled receptors. We identify here an interaction between the human somatostatin receptor 3 (hSSTR3) and the multiple PDZ protein MUPP1. MUPP1 is a tight junction scaffold protein in epithelial cells, and as a result of the interaction with MUPP1 the hSSTR3 is targeted to tight junctions. Interaction with MUPP1 enables the receptor to regulate transepithelial permeability in a pertussis toxin sensitive manner, suggesting that hSSTR3 can activate G-proteins locally at tight junctions.

Structured summary:

MINT-6800756, MINT-6800770: MUPP1 (uniprotkb:O75970) and hSSTR3 (uniprotkb:P32745) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800587:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800562:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by two hybrid (MI:0018)MINT-6800622:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with PIST (uniprotkb: Q9HD26), Hsp70 (uniprotkb:P08107), Maguk p55 (uniprotkb: Q8N3R9), MAGI3 (uniprotkb:Q5TCQ9), ZO-2 (uniprotkb:Q9UDY2), ZO-1 (uniprotkb:Q07157) and MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800607, MINT-6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)     

Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the findings were confirmed in planta by bimolecular fluorescence complementation (BiFC) assay. Results indicated that although all CESA proteins can interact with each other, only CESA4 is able to form homodimers. A model is proposed for the secondary rosette structure. The RING-motif proved not to be essential for the interaction between the CESA proteins.

Structured summary

MINT-6951243: PIP2-1 (uniprotkb:P43286) physically interacts (MI:0218) with PIP2-1 (uniprotkb:P43286) by bimolecular fluorescence complementation (MI:0809)MINT-6950816: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) withCESA4 (uniprotkb:Q84JA6) by membrane bound complementation assay (MI:0230)MINT-6951056, MINT-6951071, MINT-6951088, MINT-6951103: CESA7 (uniprotkb:Q9SWW6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6950949, MINT-6950990: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by membrane bound complementation assay (MI:0230)MINT-6950909, MINT-6951030: CESA4 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951042: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6951004, MINT-6951016: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951217, MINT-6951230: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)MINT-6951120, MINT-6951140, MINT-6951156, MINT-6951170, MINT-6951185: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA7 (uniprotkb:Q9SWW6) by bimolecular fluorescence complementation (MI:0809)MINT-6951199: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)     

14-3-3 Binding to Pim-phosphorylated Ser166 and Ser186 of human Mdm2 - Potential interplay with the PKB/Akt pathway and p14

Here we show that 14-3-3 proteins bind to Pim kinase-phosphorylated Ser166 and Ser186 on the human E3 ubiquitin ligase mouse double minute 2 (Mdm2), but not protein kinase B (PKB)/Akt-phosphorylated Ser166 and Ser188. Pim-mediated phosphorylation of Ser186 blocks phosphorylation of Ser188 by PKB, indicating potential interplay between the Pim and PKB signaling pathways in regulating Mdm2. In cells, expression of Pim kinases promoted phosphorylation of Ser166 and Ser186, interaction of Mdm2 with endogenous 14-3-3s and p14^ARF, and also increased the amount of Mdm2 protein by a mechanism that does not require Pim kinase activities. The implications of these findings for regulation of the p53 pathway, oncogenesis and drug discovery are discussed.

Structured summary

MINT-6823587:PIM3 (uniprotkb:Q86V86) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823623:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with p14ARF (uniprotkb:Q8N7268N726) by coimmunoprecipitation (MI:0019)MINT-6823537:PKB (uniprotkb:P31749) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823574:PIM2 (uniprotkb:QP1W9) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823555:PIM1 (uniprotkb:P11309)P phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)     

FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53

The p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25 kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.

Structured summary

MINT-6823686:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by anti bait coimmunoprecipitation (MI:0006)MINT-6823707, MINT-6823722:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q62446) by pull down (MI:0096)MINT-6823775:P53 (uniprotkb:Q04637) physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)MINT-6823735, MINT-6823749:FKBP25 (uniprotkb:Q62446) binds (MI:0407) to MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823761:Ubiquitin (UNIPROTKB:62988)P physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823669:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by two hybrid (MI:0018)     

A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity

Metabotropic glutamate receptor subtype 1a (mGluR1a) associates with the proteins mediating its receptor activity, suggesting a complex-controlled function of mGluR1a. Here, using glutathione-S-transferase pull-down, co-immnoprecipitation and immnoflurescence assays in vitro and in vivo, we have found CFTR-associated ligand (CAL) to be a novel binding partner of mGluR1a, through its PSD95/discslarge/ZO1homology domain. Deletion of mGluR1a-carboxyl terminus (CT) or mutation of Leu to Ala in the CT of mGluR1a reduces the association, indicating the essential binding region of mGluR1a for CAL. Functionally, the interaction of mGluR1a with CAL was shown to inhibit mGluR1a-mediated ERK1/2 activation, without an apparent effect, via the C-terminal-truncated receptor. These findings might provide a novel mechanism for the regulation of mGluR1a-mediated signaling through the interaction with CAL.

Structured summary

MINT-6797987, MINT-6798009:

NHERF-2 (uniprotkb:Q15599) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by proteinarray (MI:0089)

MINT-6798026, MINT-6798048, MINT-6798066:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by pull down (MI:0096)

MINT-6797953, MINT-6797970:

NHERF-1 (uniprotkb:O14745) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6797935:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6798084:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by filter binding (MI:0049)

MINT-6798134:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by anti tag coimmunoprecipitation (MI:0007)

MINT-6798158:

CAL (uniprotkb:B4F775) physically interacts (MI:0218) with mGluR1a (uniprotkb:Q9R0W0) by anti bait coimmunoprecipitation (MI:0006)

MINT-6798233:

CAL (uniprotkb:Q9HD26) colocalizes (MI:0403) with mGluR1a (uniprotkb:Q9R0W0) by fluorescence microscopy (MI:0416)

     

Transcriptional activation of hypoxia-inducible factor-1α by HDAC4 and HDAC5 involves differential recruitment of p300 and FIH-1

The interplay between hypoxia-inducible factor-1α (HIF-1α) and histone deacetylase (HDACs) have been well studied; however, the mechanism of cross-talk is unclear. Here, we investigated the roles of HDAC4 and HDAC5 in the regulation of HIF-1α function and its associated mechanisms. HDAC4 and HDAC5 enhanced transactivation by HIF-1α without stabilizing HIF-1α. HDAC4 and HDAC5 physically associated with HIF-1α through the inhibitory domain (ID) that is the binding site for factor inhibiting HIF-1 (FIH-1). In the presence of these HDACs, binding of HIF-1α to FIH-1 decreased, whereas binding to p300 increased. These results indicate that HDAC4 and HDAC5 increase the transactivation function of HIF-1α by promoting dissociation of HIF-1α from FIH-1 and association with p300.

Structured summary:

MINT-6802187:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with FIH1 (uniprotkb:Q9NWT6) by anti bait coimmunoprecipitation (MI:0006)MINT-6802058:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with HDAC4 (uniprotkb:P56524) by pull down (MI:0096)MINT-6802021:HIF1 alpha (uniprotkb:Q61221) physically interacts (MI:0218) with HDAC4 (uniprotkb:P56524) by anti bait coimmunoprecipitation (MI:0006)MINT-6802036:HIF1 alpha (uniprotkb:Q61221) physically interacts (MI:0218) with HDAC5 (uniprotkb:Q9UQL6) by anti bait coimmunoprecipitation (MI:0006)MINT-6802102:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with HDAC5 (uniprotkb:Q9UQL6) by pull down (MI:0096)MINT-6802121, MINT-6802156:P300 (uniprotkb:Q09472) physically interacts (MI:0218) with HIF1 alpha (uniprotkb:Q16665) by anti bait coimmunoprecipitation (MI:0006)     

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)     

The fast-mobility isoform of mouse Mcl-1 is a mitochondrial matrix-localized protein with attenuated anti-apoptotic activity

The full-length pro-survival protein Mcl-1 predominantly resides on the outer membrane of mitochondria. Here, we identified a mitochondrial matrix-localized isoform of Mcl-1 that lacks 33 amino acid residues at the N-terminus which serve both as a mitochondrial targeting and processing signal. Ectopically-expressed Mcl-1 without the N-terminal 33 residues failed to enter the mitochondrial matrix but retained wt-like activities both for interaction with BH3-only proteins and anti-apoptosis. In contrast, the mitochondrial matrix-localized isoform failed to interact with BH3-only proteins and manifested an attenuated anti-apoptotic activity. This study reveals that import of Mcl-1 into the mitochondrial matrix results in the attenuation of Mcl-1’s anti-apoptotic function.

Structured summary

MINT-7965637: NOXA (uniprotkb:Q9JM54) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965699: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with Bim (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7965655: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with NOXA (uniprotkb:Q9JM54) by anti bait coimmunoprecipitation (MI:0006)MINT-7965711: Bim (uniprotkb:O43521) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965673: PUMA (uniprotkb:Q9BXH1) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965685: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with PUMA (uniprotkb:Q9BXH1) by anti bait coimmunoprecipitation (MI:0006)     

Beta-2 adrenergic receptor mediated ERK activation is regulated by interaction with MAGI-3

The beta-2 adrenergic receptor (β2AR) has a carboxyl terminus motif that can interact with PSD-95/discs-large/ZO1 homology (PDZ) domain-containing proteins. In this paper, we identified membrane-associated guanylate kinase inverted-3 (MAGI-3) as a novel binding partner of β2AR. The carboxyl terminus of β2AR binds with high affinity to the fifth PDZ domain of MAGI-3, with the last four amino acids (D-S-L-L) of the receptor being the key determinants of the interaction. In cells, the association of full-length β2AR with MAGI-3 occurs constitutively and is enhanced by agonist stimulation of the receptor. Our data also demonstrated that β2AR-stimulated extracellular signal-regulated kinase-1/2 (ERK1/2) activation was substantially retarded by MAGI-3 expression. These data suggest that MAGI-3 regulates β2AR-mediated ERK activation through the physical interaction between β2AR and MAGI-3.

Structured summary

MINT-7716556: beta2AR (uniprotkb:P07550) physically interacts (MI:0915) with MAGI-3 (uniprotkb:Q5TCQ9) by anti tag coimmunoprecipitation (MI:0007)MINT-7716593: beta2AR (uniprotkb:P18762) physically interacts (MI:0915) with MAGI-3 (uniprotkb:Q9EQJ9) by anti bait coimmunoprecipitation (MI:0006)MINT-7716630: MAGI-3 (uniprotkb:Q5TCQ9) and beta2AR (uniprotkb:P07550) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7716382, MINT-7716335: MAGI-3 (uniprotkb:Q5TCQ9) physically interacts (MI:0915) with beta2AR (uniprotkb:P07550) by pull down (MI:0096)MINT-7716320, MINT-7716422, MINT-7716502, MINT-7716450, MINT-7716470: beta2AR (uniprotkb:P07550) binds (MI:0407) to MAGI-3 (uniprotkb:Q5TCQ9) by pull down (MI:0096)