Physical and functional interaction of KV10.1 with Rabaptin-5 impacts ion channel trafficking

K_V10.1 is a potassium channel expressed in brain and implicated in tumor progression. We have searched for proteins interacting with K_V10.1 and identified Rabaptin-5, an effector of the Rab5 GTPase. Both proteins co-localize on large early endosomes induced by Rab5 hyperactivity. Silencing of Rabaptin-5 induces down-regulation of recycling of K_V10.1 channel in transfected cells and reduction of K_V10.1 current density in cells natively expressing K_V10.1, indicating a role of Rabaptin-5 in channel trafficking. K_V10.1 co-localizes, but does not physically interact, with Rab7 and Rab11. Our data highlights the complex control of the amount of K_V10.1 channels on the cell surface.Structured summary of protein interactionsRabaptin-5 physically interacts with Kv10.1 by anti bait coimmunoprecipitation (View interaction)Rabaptin-5 physically interacts with Rabaptin-5 by two hybrid (View interaction)Kv10.1 physically interacts with Kv10.1 by two hybrid (View interaction)Kv10.1 physically interacts with Rabaptin-5 by anti bait coimmunoprecipitation (View Interaction: 1, 2)RAB11 and Kv10.1 colocalize by fluorescence microscopy (View interaction)Kv10.1 and Rabaptin-5 colocalize by fluorescence microscopy (View interaction)Kv10.1 physically interacts with Rabaptin-5 by two hybrid (View Interaction: 1, 2)Kv10.1 and RAB7 colocalize by fluorescence microscopy (View interaction)     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

The tumor suppressor RECK interferes with HER-2/Neu dimerization and attenuates its oncogenic signaling

Our previous study demonstrates that HER-2/Neu oncogene inhibits a matrix metalloproteinase inhibitor and tumor metastasis suppressor RECK to promote metastasis. Conversely, the effect of RECK on the oncogenic function of HER-2/Neu is unknown. Ectopic expression of RECK in 293T cells and HER-2/Neu-overexpressing breast cancer cells shows that RECK and HER-2/Neu are co-localized and these two proteins can be co-immunoprecipitated. RECK inhibits HER-2/Neu receptor dimerization and autophosphorylation, which causes reduction of ERK and AKT kinase activity and down-regulation of HER-2/Neu target genes. RECK expression is reduced in 58.8% of breast cancer tissues and is associated with lymph node invasion supporting its anti-metastatic role. Collectively, we provide the first evidence that RECK can negatively regulate oncogenic activity of HER-2/Neu by inhibiting receptor dimerization.

Structured summary

HER-2/Neuphysically interacts with HER-2/Neu by blue native page (View interaction)HER-2/Neuphysically interacts with RECK by coimmunoprecipitation (View interaction)HER-2/Neu and RECKcolocalize by fluorescence microscopy (View Interaction 1, 2)HER-2/Neuphysically interacts with RECK by anti bait coimmunoprecipitation (View interaction)     

Annexin A9 is a periplakin interacting partner in membrane-targeted cytoskeletal linker protein complexes

Periplakin regulates keratin organisation and participates in the assembly of epidermal cornified envelopes. A proteomic approach identified annexin A9 as a novel interacting partner for periplakin N-terminus. The presence of annexin A9 in complexes with periplakin was confirmed by immunoblotting of proteins immunoprecipitated by anti-HA or anti-annexin A9 antibodies. Both endogenous and GFP-tagged annexin A9 co-localise with endogenous periplakin and transfected periplakin N-terminus at MCF-7 cell borders and aggregate after Okadaic acid treatment. Annexin A9 and periplakin co-localise in the epidermis and annexin A9 is up-regulated in differentiating keratinocytes, but the epidermal annexin A9 expression does not require periplakin.Structured summary of protein interactionsAnnexin-9 physically interacts with Periplakin by anti bait co-immunoprecipitation (View interaction) Periplakin physically interacts with Annexin-9 by anti tag co-immunoprecipitation (View Interaction: 1, 2) Periplakin and Annexin-9 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3) Keratin-8 and Periplakin colocalize by fluorescence microscopy (View interaction)     

Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

Cellular nucleic acid binding protein (CNBP) contains seven zinc finger (ZF) repeats and an arginine and glycine (RG) rich sequence between the first and the second ZF. CNBP interacts with protein arginine methyltransferase PRMT1. Full-length but not RG-deleted or mutated CNBP can be methylated. Treatment with a methylation inhibitor AdOx reduced CNBP methylation, but did not affect the concentrated nuclear localization of CNBP. Nevertheless, arginine methylation of CNBP appeared to interfere with its RNA binding activity. Our findings show that arginine methylation of CNBP in the RG motif did not change the subcellular localization, but regulated its RNA binding activity.Structured summary of protein interactionsPRMT1 binds to CNBP by pull down (View interaction)PRMT1 methylates CNBP by enzymatic study (View interaction)CNBP physically interacts with PRMT1 by anti tag coimmunoprecipitation (View interaction)     

Acyl-CoA binding domain containing 3 (ACBD3) recruits the protein phosphatase PPM1L to ER-Golgi membrane contact sites

The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER-Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER-Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER-Golgi interface.

Structured summary of protein interactions

ACBD3 and PPM1Lcolocalize by fluorescence microscopy (View interaction)FYCO1physically interacts with PPM1L by pull down (View interaction)SEC14L2physically interacts with PPM1L by pull down (View interaction)ACBD3physically interacts with PPM1L by pull down (View interaction)SEC14L1physically interacts with PPM1L by pull down (View interaction)PPM1Lphysically interacts with ACBD3 by two hybrid (View interaction)     

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure

Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.

Structured summary of protein interactions

GM130 and Piltcolocalize by fluorescence microscopy (View interaction)Arf6(Q67L)physically interacts with Pilt by two hybrid (View Interaction: 1, 2)Piltphysically interacts with Arf6(Q67L) by pull down (View interaction)     

FERM domain-containing protein FRMD5 regulates cell motility via binding to integrin β5 subunit and ROCK1

FRMD5 is a novel FERM domain-containing protein depicted in tumor progression. However, the mechanisms underlying FRMD5 inhibition of cell migration is largely unknown. Here, we show that FRMD5 regulates cell migration by interacting with integrin β5 cytoplasmic tail and ROCK1 in human lung cancer cells. FRMD5 promotes cell–matrix adhesion and cell spreading on vitronectin, and thus inhibits cell migration. Furthermore, FRMD5 interacts with ROCK1 and inhibits its activation that leads to the inhibition of myosin light chain phosphorylation and the actin stress fiber formation. Taken together, these findings demonstrate that the putative tumor suppressive protein FRMD5 regulates tumor cell motility via a dual pathway involving FRMD5 binding to integrin β5 tail and to ROCK1.     

Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA.

Structured summary

TrkAphysically interacts with TrkA by protein complementation assay (View interaction)TrkAphysically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkAphysically interacts with TrkA by cross-linking study (View interaction)     

Replacement of charged and polar residues in the coiled-coiled interface of huntingtin-interacting protein 1 (HIP1) causes aggregation and cell death

HIP1 crystal structures solved in our laboratory revealed abnormalities in the coiled-coil region, suggesting intrinsic plasticity. To test this, specific amino acids in the coiled-coil were mutated. The apparent thermal stability of HIP1 was altered when Thr528 and Glu531 were replaced by leucine, and was enhanced when Lys510 was also mutated. In cells, HIP1 mutant expression produced aggregation. MTS and flow cytometry indicate a correlation between aggregated HIP1 and enhanced cell death. These data support the idea that flexibility of the HIP1 coiled-coil domain is important for normal function and may lead to new insights into Huntington’s disease.

Structured summary of protein interactions

HIP1physically interacts with 23QHtt by anti tag coimmunoprecipitation (View interaction.)     

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in?vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury. Structured digital abstract ? Sirp-alpha?physically interacts?with?Nephrin?by?anti bait coimmunoprecipitation?(View interaction) ? Sirp-alpha?physically interacts?with?Nephrin?by?anti tag coimmunoprecipitation?(View interaction).     

A single amino acid change in the transmembrane domain of the VirB8 protein affects dimerization,interaction with VirB10 and Brucella suis virulence

VirB8 is a critical component of the Brucella suis type IV secretion system (T4SS). We previously showed that the transmembrane (TM) domain plays an essential role in interactions of this protein with itself and the other proteins of the T4SS. We report that a point mutation in this TM domain stabilizes homodimers of VirB8 and heterodimers with VirB10. A similar variant of Agrobacterium tumefaciens VirB8 showed the same phenotype. The B. suis VirB8 variant was unable to complement a virB8 mutant and displayed a dominant negative phenotype when expressed in wild type B. suis. We suggest that interaction of VirB8 with VirB10 could play a major role in the correct function of the B. suis VirB T4SS.Structured summary of protein interactionsAtVirB8 physically interacts with AtVirB10 by two hybrid (View interaction)TraJ physically interacts with TraJ by two hybrid (View Interaction 1, 2)AtVirB8 physically interacts with AtVirB8 by two hybrid (View interaction)VirB10 physically interacts with VirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB8 by two hybrid (View Interaction 1, 2)VirB10 physically interacts with VirB8 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB10 by two hybrid (View interaction)VirB8 physically interacts with VirB10 by two hybrid (View interaction)AtVirB10 physically interacts with AtVirB8 by two hybrid (View interaction)     

Piceatannol attenuates cardiac hypertrophy in an animal model through regulation of the expression and binding of the transcription factor GATA binding factor 6

Piceatannol is found in grapes, passion fruit, and Japanese knotweed. Piceatannol pretreatment suppresses cardiac hypertrophy induced by isoproterenol as assessed by heart weight/body weight ratio, cross-sectional area, and expression of hypertrophic markers. The anti-hypertrophic effect of piceatannol in rat neonatal cardiomyocytes is the same as that in vivo. Piceatannol inhibits lentiviral-GATA6-induced cardiomyocyte hypertrophy. Furthermore, piceatannol reduces the interaction between GATA4 and GATA6 as well as the DNA-binding activity of endogenous GATA6 in the ANP promoter. Our results suggest that piceatannol may be a novel therapeutic agent for the prevention of cardiac hypertrophy.Structured summary of protein interactionsGATA6 physically interacts with GATA4 by anti V5 tag coimmunoprecipitation (View interaction)     

p120, a p120-related protein (p100), and the cadherin/catenin complex

Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, beta and gamma, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60src. In this paper, we show that endothelial and epithelial cells express p120 and p100, a 100-kD, p120- related protein. Peptide sequencing of p100 establishes it as highly related to p120. p120 and p100 both appear associated with the cadherin/catenin complex, but independent p120/catenin and p100/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/p100 antibody, and of p120/p100 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and beta-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and beta-catenin is similar in MDCK cells, but only approximately 20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

EDC4 interacts with and regulates the dephospho-CoA kinase activity of CoA synthase

Coenzyme A synthase (CoAsy) is a bifunctional enzyme which facilitates the last two steps of Coenzyme A biogenesis in higher eukaryotes. Here we describe that CoAsy forms a complex with enhancer of mRNA-decapping protein 4 (EDC4), a central scaffold component of processing bodies. CoAsy/EDC4 complex formation is regulated by growth factors and is affected by cellular stresses. EDC4 strongly inhibits the dephospho-CoA kinase activity of CoAsy in vitro. Transient overexpression of EDC4 decreases cell proliferation, and further co-expression of CoAsy diminishes this effect. Here we report that EDC4 might contribute to regulation of CoA biosynthesis in addition to its scaffold function in processing bodies.

Structured summary of protein interactions

CoAsyphysically interacts with EDC4 by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3)     

STAT3 repressed USP7 expression is crucial for colon cancer development

Interleukin-6 (IL-6) induced STAT3 activation is viewed as crucial for multiple tumor growth and metastasis, including colon cancer. However, the molecular mechanisms remain largely unexplored. Here, we show that expression of ubiquitin-specific protease 7 (USP7), a deubiquitylating enzyme, is decreased in STAT3-positive tumors. IL-6 administration or transfection of a constitutively activated STAT3 in SW480 cells also repressed USP mRNA expression. Using luciferase reporter and ChIP assay, we found that STAT3 bound to the promoter region of USP7 and inhibited its activity through recruiting HDAC1. As a result of the decline of USP7 expression, endogenous P53 protein level was decreased. Thus, our results suggest a previously unknown STAT3-USP7-P53 molecular network controlling colon cancer development.

Structured summary of protein interactions:

STAT3physically interacts with HDAC1 by anti bait coimmunoprecipitation (View interaction)