Yeast Two-hybrid Screen For Proteins Research Articles

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Yeast two-hybrid screening for proteins that interact with PFT in wheat

Fusarium head blight (FHB) is a devastating disease of wheat worldwide. Fhb1 is the most consistently reported quantitative trait locus (QTL) for FHB resistance breeding. A pore-forming toxin-like (PFT) gene at Fhb1 was first cloned by map-based cloning and found to confer FHB resistance in wheat. Proteins often interact with each other to execute their functions. Characterization of the proteins interacting with PFT might therefore provide information on the molecular mechanisms of PFT functions. In this study, a high-quality yeast two-hybrid (Y2H) library using RNA extracted from Fusarium graminearum (Fg)-infected wheat spikes of Sumai 3 was constructed. The agglutinin domains of PFT exhibited no self-activation and toxicity to yeast cells and were used as bait to screen the Y2H library. Twenty-three proteins that interact with PFT were obtained, which were mainly involved in the ubiquitination process, clathrin coat assembly, the oxidation-reduction process, and protein phosphorylation. The expression pattern of these interacting genes was analyzed by quantitative real-time PCR. This study clarifies the protein interactions of PFT and raises a regulatory network for PFT regarding FHB resistance in wheat.

A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover

The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196-Leu2197 We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.

Upregulation of MIIP regulates human breast cancer proliferation, invasion and migration by mediated by IGFBP2

AimsThe migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). This study aims to evaluate the biological effects of MIIP in breast cancer by targeting IGFBP2. Materials and methodsReverse transcription quantitative real-time polymerase chain reaction and Western blotting were used to evaluate the abnormal expression of MIIP and IGFBP2 in breast cancer tissue or breast cancer cell lines. Transfection assay was used to overexpress MIIP protein in breast cancer cells. MTT assay and colony formation assay were used to detect cell viability of breast cancer cells after MIIP overexpression. Transwell and wound-healing assays were used to detect cell invasion and migration after MIIP overexpression. ResultsMIIP was significantly decreased and IGFBP2 was significantly increased in breast cancer tissues versus para cancerous. Breast cancer tissues of HER2 overexpression and Basal-like were more significant than Luminal A and Luminal B. MIIP was obviously downregulated and IGFBP2 was upregulated in MDA-MB-231, SKBR3 and MCF-7 versus MCF-10A especially in MDA-MB-231. Cell proliferation, cell migration and cell invasion were significantly inhibited after overexpression of MIIP. IGFBP2 was downregulated after overexpression of MIIP. The effects of MIIP on cell proliferation, cell migration and invasion were significantly reversed by IGFBP2. ConclusionThe abnormal expression of MIIP in breast cancer affects the cell biological effects. IGFBP2 was regulated via MIIP which may be associated with these biological effects. These results reveal that MIIP can be a potential target for breast cancer treatment.

Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein.

Alzheimer's disease (AD) is a neurodegenerative disorder in which amyloid β plaques are a pathological characteristic. Little is known about the physiological functions of amyloid β precursor protein (APP). Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1, members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease (PMD) and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

Calpain Cleaves Most Components in the Multiple Aminoacyl-tRNA Synthetase Complex and Affects Their Functions

Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNA(Gln) and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca(2+) and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC.

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism

Thromboxane A(2) (TXA(2)) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA(2) receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro- and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA(2) synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro- and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues.

MIIP, a Cytoskeleton Regulator that Blocks Cell Migration and Invasion, Delays Mitosis, and Suppresses Tumorogenesis

The migration and invasion inhibitory protein (MIIP) was initially discovered in a yeast two-hybrid screen for proteins that interact and inhibit the migration and invasion-promoting protein insulin-like growth factor binding protein 2 (IGFBP2). Recent studies have shown that MIIP not only modulates IGFBP2 but also regulates microtubule by binding to and inhibiting HDAC6, a class 2 histone deacetylase that deacetylates α-tubulin, heat-shock protein 90 (HSP90), and cortactin. In addition, MIIP also regulates the mitosis checkpoint, another microtubule-associated process. The location of the MIIP gene in chromosomal region 1p36, a commonly deleted region in a broad spectrum of human cancers, and the observation that MIIP attenuates tumorigenesis in a mouse model suggest that it functions as a tumor-suppressor gene. This review summarizes the recent progress in characterizing this novel protein, which regulates cell migration and mitosis, two processes that rely on the highly coordinated dynamics of the microtubule and cytoskeleton systems.

Delivering AMPA Receptors to the Synapse

Incorporation of AMPA-type glutamate receptors containing the GluR1 subunit into the postsynaptic membrane is associated with long-term potentiation (LTP), a form of synaptic plasticity that may underlie some forms of memory. The trafficking of GluR1-containing receptors to the cell surface is regulated by multiple interactions that occur with the C-terminal domain (CTD), including interactions with kinases that phosphorylate the CTD. Phosphorylation of Ser 845 , reportedly mediated by the adenosine 3′,5′-monophosphate (cAMP)-regulated enzyme protein kinase A (PKA), appears to be required for GluR1 synaptic insertion. Serulle et al . provide evidence that Ser 845 can also be phosphorylated by guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase II (cGKII), thereby providing a mechanism for nitric oxide signaling downstream of NMDA-type glutamate receptors to increase synaptic strength and contribute to LTP. cGKII was identified in a modified yeast two-hybrid screen for proteins that interacted with the CTD of GluR1. In vitro binding studies suggested that activation of cGKII (binding to the cGMP analog 8-Br-cGMP) increased its interaction with the GluR1 CTD. In lysates from rat brain, cGKII and GluR1 were present in the same subcellular fraction and coimmunoprecipitated. In cultured hippocampal neurons, endogenous cGKII and GluR1 colocalized and coimmunoprecipitated. The interaction was enhanced when the cells were (i) exposed to 8-Br-cGMP to stabilize cGKII in the active conformation, (ii) exposed to glycine to induce a chemical form of LTP (chemLTP) that involves activation of NMDA receptors and activation of nitric oxide synthase and soluble guanylate cyclase (the enzyme that produces cGMP), or (iii) exposed to a nitric oxide donor. In vitro kinase assays demonstrated that the GluR1 CTD was phosphorylated by cGKII at Ser 845 , the same site phosphorylated by PKA. In cultured neurons, chemLTP also triggered phosphorylation of GluR1 at Ser 845 , and this was blocked by the presence of an NMDA receptor antagonist or cGKII inhibitor. Both cAMP analogs and cGMP analogs increased the abundance of GluR1 at the surface of hippocampal neurons but did not induce LTP. The chemLTP condition stimulated an increase in synaptic GluR1 and induced LTP, which was measured as a sustained increase in miniature excitatory postsynaptic currents. The interaction of GluRI with PKA is indirect, requiring the PDZ scaffolding protein SAP97. The interaction with cGKII appears to be direct and involves Arg 837 and does not require SAP97 as a mediator. With mutants either lacking the SAP97 binding region or containing a point mutation at Arg 837 , the authors demonstrated that the cAMP- and cGMP-mediated GluR1 trafficking and phosphorylation pathways were independent. In hippocampal mouse slices, pharmacological inhibitors of the NO to cGMP pathway or expression of a dominant-negative fragment of the cGKII that disrupted the interaction between cGKII and GluR1 inhibited LTP in response to tetanic stimulation. Thus, it appears that both cAMP and cGMP pathways converge on Ser 845 to regulate GluR1 trafficking. Y. Serulle, S. Zhang, I. Ninan, D. Puzzo, M. McCarthy, L. Khatri, O. Arancio, E. B. Ziff, A GluR1-cGKII interaction regulates AMPA receptor trafficking. Neuron 56 , 670-688 (2007). [PubMed]

New Candidates for the PS Receptor

Prompt removal of cells that die by programmed cell death, or apoptosis, is a key mechanism by which this intentional cell death response does not lead to inflammation. Phosphatidylserine (PS) is a membrane lipid that is exposed on the surface of apoptotic cells, where it is believed to be a key signal for the recognition of such dying cells by phagocytosing cells. Two groups report the identification of proteins that bind PS and appear to be important for recognition and phagocytosis of apoptotic cells. Park et al . identified brain-specific angiogenesis inhibitor 1 (BAI1)--a seven-transmembrane-domain protein with an extended extracellular region containing thrombospondin repeats (TSR) and an RGD domain--in a yeast two-hybrid screen for proteins that interacted with Rac and its guanine nucleotide exchange factor (GNEF), the EMLO and Dock180 complex, which are part of the signaling cascade that triggers the changes in the actin cytoskeleton associated with phagocytosis. Forced expression of BAI1 in nonphagocytic cells or in macrophages enhanced uptake of apoptotic thymocytes or beads mimicking apoptotic cells. (BAI1 was detected in untransfected human monocytes and macrophages, macrophage cell lines, spleen, and bone marrow.) Annexin V, which binds PS, prevented BAI1-stimulated phagocytosis of apoptotic cells, as did exposure of the cells to a BAI1 fragment containing the RGD and TSR domains, but not one lacking the TSR domain. The BAI1 TSR domain fragment also blocked phagocytosis of labeled beads when co-injected into the peritoneum of mice. Depletion of BAI1 in primary mouse astrocytes decreased phagocytosis of the beads or apoptotic thymocytes. Miyanishi et al . screened a panel of antibodies against mouse peritoneal macrophages and found that an antibody that recognized T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4) inhibited PS-dependent engulfment of apoptotic cells. Tim4 mRNA was detected in peritoneal cells, spleen, lymph nodes, and salivary glands, and forced expression of Tim4, which has a single transmembrane domain, in nonphagocytic cells promoted the phagocytosis of apoptotic cells. Injection of the antibody that recognized Tim4 in mice followed by treatment to induce apoptosis in the thymus resulted in impaired phagocytosis of the apoptotic cells and the development of autoantibodies. A fusion protein between the Tim4 extracellular domain and the Fc domain of antibody receptors (Tim4-Fc) bound to apoptotic cells or to PS spots on nitrocellulose filters. Fusion proteins containing the Tim4 or Tim1 IgV domain bound to microtiter plates coated in PS. However, the same domains of the Tim2 and Tim3 family members showed no such binding, nor did the full-length proteins promote apoptotic cell phagocytosis when overexpressed. Exosomes, which are membrane fragments released from cells that may mediate cell communication, also have PS on the surface, and cells expressing either Tim4 or Tim1 bound exosome-like vesicles (based on gold-labeling and electron microscopy). PS appeared to promote heterophilic and homophilic interactions between Tim4 and Tim1. The authors suggest that Tim1 and Tim4 may serve as PS receptors involved in recognition of both apoptotic cells and exosomes. D. Park, A.-C. Tosello-Trampont, M. R. Elliott, M. Lu, L. B. Haney, Z. Ma, A. L. Klibanov, J. W. Mandell, K. S. Ravichandran, BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450 , 430-434 (2007). [PubMed] M. Miyanishi, K. Tada, M. Koike, Y. Uchiyama, T. Kitamura, S. Nagata, Identification of Tim4 as a phosphatidylserine receptor. Nature 450 , 435-439 (2007). [PubMed]

Tyrosine Kinase 2 (Tyk2) Interacts with and Phosphorylates Siva-1, and Auguments the Apoptotic Effect Induced by Siva-1.

Jak-Stat pathway is essential to transduce cytokine signalings. We previously reported that tyrosine kinase 2 (Tyk2), one of the Jak kinases, was essential but Stat1 was dispensable in IFN-alpha-induced B lymphocyte apoptosis. This indicates the existence of other signaling molecules besides Stat1 downstream of activated Tyk2. Therefore, in order to find Tyk2-activated signaling molecules which transduce IFN-alpha signal and induce B lymphocyte apoptosis, we performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Siva-1, which had been originally cloned as a CD27 binding protein. Siva-1 has a death domain (DD) homology region and induces apoptosis in various cells through binding CD27, which belongs to the tumor necrosis factor receptor (TNFR) family. We found that Tyk2 interacts with and phosphorylates Siva-1. Two regions of Siva-1, the N-terminus and the middle portion of the protein containing a death domain homology region, contributed to binding to Tyk2, and two tyrosines of Siva-1, Tyr53 and Tyr162, were phosphorylated by Tyk2. Because Siva-1 is so far thought to be a proapoptotic protein, we assessed whether Tyk2 had any effects on Siva-1-mediated apoptosis through the Tyk2-Siva-1 interaction. First, we established BaF3 cell (IL-3 dependent murine proB cell) lines stably expressing either wild-type Tyk2 (BaF3/WT Tyk2) or constitutively activated Tyk2 which Valine678 was replaced by Phenylalanine (BaF3/V678F Tyk2). In BaF3/V678F Tyk2 cells, Tyk2 and Stats are constitutively activated, and they grow in the absence of IL-3. We transiently transfected either GFP-Siva-1 or control GFP vector into BaF3/parent cells, BaF3/WT Tyk2 cells or BaF3/V678F Tyk2 cells. Thirty-six hours after transfection, we measured annexin V positive cells by flow cytometry on GFP-positive cells. Approximately 24% of both BaF3/parent cells and BaF3/WT Tyk2 cells transfected with GFP-Siva-1 fell into apoptosis, whereas only less than 7% of the cells transfected control vector showed apoptosis. The frequency of this Siva-1-induced apoptosis was increased in BaF3/V678F Tyk2 cells (over 43% vs <10% of expressing control vector), suggesting that activated Tyk2 augmented the apoptotic function of Siva-1. In conclusion, we show that Tyk2 associates with and phosphorylates Siva-1 and that this Tyk2-Siva-1 interaction enhances the apoptotic effect induced by Siva-1. This indicates that Siva-1 might be a probable key molecule downstream of Tyk2 which is activated in response to IFN-alpha.

Structural and Functional Insights into the Regulation of Arabidopsis AGC VIIIa Kinases

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta.

New Substrates for Factor Inhibiting HIF

Factor inhibiting hypoxia-inducible factor (FIH) is an asparaginyl hydroxylase, and hydroxylation of Asn of HIF (hypoxia-inducible factor) by FIH disrupts the interaction of HIF with the transcriptional coactivators CBP/p300. FIH activity is inhibited during hypoxia. Cockman et al . performed a yeast two-hybrid screen for proteins that interacted with FIH and found and confirmed the interaction with three ankryrin repeat domain (ARD)-containing proteins. Other ARD-containing peptides were also found to be substrates for FIH-dependent Asn hydroxylation. The interaction with the p105 precursor of the p50 component of the nuclear factor κB (NF-κB) transcription factor and with IκBα were characterized further. Interactions between the endogenous proteins were demonstrated by coimmunoprecipitation, and the interaction of p105 with FIH required Asn 678 . In vitro assays showed that in the presence of p105 or IκBα, FIH activity was stimulated, and liquid chromatography/mass spectrometry confirmed that p105 and IκBα were hydroxylated in vivo. RNA interference experiments showed that hydroxylation was dependent on FIH and hydroxylation of IκBα was decreased by hypoxic conditions. Despite using several different experimental approaches, the authors failed to show a clear consequence of ARD hydroxylation on NF-κB activity. Thus, it remains to be determined whether Asn hydroxylation is serving a structural role or is a regulatory posttranslational modification that alters the activity or interactions of these newly defined substrates. M. E. Cockman, D. E. Lancaster. I. P. Stolze, K. S. Hewitson, M. A. McDonough, M. L. Coleman, C. H. Coles, X. Yu, R. T. Hay, S. C. Ley, C. W. Pugh, N. J. Oldham, N. Masson, C. J. Schofield, P. J. Ratcliffe, Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc. Natl. Acad. Sci. U.S.A. 103 , 14767-14772 (2006). [Abstract] [Full Text]

MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation

Chromosome condensation is essential for proper segregation of duplicated sister chromatids in mitosis. Mammalian erythroid maturation is also associated with gradual nuclear condensation. However, few proteins that are directly involved in chromosome condensation during erythropoiesis have been identified. In this report, we show that MTB (more than blood), which was initially isolated in a yeast two-hybrid screen for proteins that interact with the basic helix-loop-helix (bHLH) protein stem cell leukemia (SCL), and later identified as the murine homolog of the condensin II subunit CAP-G2, participates in erythroid cell development. MTB interacts with SCL and another hematopoietic bHLH protein, E12, and is recruited to the nucleus by SCL and E12. In addition, MTB can repress SCL/E12-mediated transcriptional activation. Consistent with the model that MTB may function together with SCL/E12 heterodimer during erythroid cell development, MTB is highly expressed in the erythroid lineage and is upregulated upon erythroid differentiation. Moreover, overexpression of MTB promotes the terminal differentiation of the murine erythroleukemia erythroid cell line. Together, these findings demonstrate that the condensin II subunit MTB/mCAP-G2 plays a novel function during erythropoiesis and suggest that key hematopoietic transcription factors such as SCL and E12 may regulate the terminal differentiation of hematopoietic cells through the interaction with condensin complexes.

Interactions of Annexins with the mu Subunits of the Clathrin Assembly Proteins

A number of biochemical and genetic studies have suggested that certain annexins play important roles in the endocytic pathway, possibly involving the generation, localization, or fusion of endocytic compartments. In a yeast two-hybrid screen for proteins that interact with the N-terminal domain of annexin A2 we identified the mu2 subunit of the clathrin assembly protein complex AP-2. The interaction depended upon two copies of a Yxx phi amino acid sequence motif (Y = tyrosine, x = variable residue, phi = bulky, hydrophobic residue) in the annexin that is also characteristic of the binding site for mu2 on the cytoplasmic domains of transmembrane receptors. The interaction between mu2 and full-length annexin A2 was demonstrated in vitro to be direct, to require calcium, and to be functional in the sense that annexin A2 was able to recruit the mu2 to immobilized lipids. Examination of other annexins and mu subunits demonstrated that annexin A2 also binds the mu1 subunit of the AP-1 complex, that annexin A6 binds mu1 and mu2, and that annexin A1 binds only mu1. We propose that annexins can "masquerade" as transmembrane receptors when they are attached to membranes in the presence of calcium and that they might therefore function to initiate calcium-regulated coated pit formation at the cell surface or on intracellular organelles.

NO Deadly Signal

Reminiscent of mild-mannered Clark Kent, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an alter ego with potent and deadly effects. When translocated to the nucleus, GAPDH has been associated with transcriptional regulation, and Hara et al. present results implicating it in control of apoptosis. A yeast two-hybrid screen for proteins interacting with GAPDH turned up Siah1, an E3 ubiquitin ligase. In transfected cells, the authors detected interaction of the proteins and increased localization of GAPDH to the nucleus, an effect that required the nuclear localization signal of Siah1. In human embryonic kidney cells undergoing apoptosis in response to staurosporine, the authors detected modification of GAPDH by mass spectrometry and went on to show in vitro that modification of GAPDH by S -nitrosylation (often a consequence of increased generation of nitric oxide within cells) enhanced association with Siah1. These events appear to be important in regulation of apoptosis. In a macrophage cell line undergoing apoptosis in response to lipopolysaccharide, GAPDH became S -nitrosylated and associated with endogenous GAPDH, and the complex moved to the nucleus. All of these effects were blocked by an inhibitor of the nitric oxide-generating enzyme iNOS (inducible nitric oxide synthase). Depletion of GAPDH with small interfering RNA (siRNA) blocked the appearance of phosphorylated histone H2B, a marker associated with apoptosis. In cerebellar granular neurons undergoing apoptosis in response to activation of glutamate receptors, similar effects were observed. Increased binding of Siah1 to GAPDH was blocked in cells from neuronal NOS (nNOS) knockout mice, and depletion of either GAPDH or Siah1 from wild-type cells with siRNA inhibited apoptosis. Exactly how nuclear Siah1 promotes apoptosis remains to be explored, but its action appears to require its RING finger domain, indicating that Siah1-mediated ubiquitination and consequent degradation of nuclear proteins is one likely mechanism. Nitric oxide-mediated modification of metabolic enzymes has been shown to contribute to cell death through energy depletion. Hara et al. propose that this may be a slower process and one associated more closely with necrosis, whereas the faster nuclear actions of GAPDH, which reflect translocation of only a small portion of cytoplasmic enzyme, appear to be independent of effects on glycolysis. M. R. Hara, N. Agrawal, S. F. Kim, M. B. Cascio, M. Fujimuro, Y. Ozeki, M. Takahashi, J. H. Cheah, S. K. Tankou, L. D. Hester, C. D. Ferris, S. D. Hayward, S. H. Snyder, A. Sawa, S -nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7 , 665-674 (2005). [PubMed]

Abl Tyrosine Kinase and Its Substrate Ena/VASP Have Functional Interactions with Kinesin-1

Relatively little is known about how microtubule motors are controlled or about how the functions of different cytoskeletal systems are integrated. A yeast two-hybrid screen for proteins that bind to Drosophila Enabled (Ena), an actin polymerization factor that is negatively regulated by Abl tyrosine kinase, identified kinesin heavy chain (Khc), a member of the kinesin-1 subfamily of microtubule motors. Coimmunoprecipitation from Drosophila cytosol confirmed a physical interaction between Khc and Ena. Kinesin-1 motors can carry organelles and other macromolecular cargoes from neuronal cell bodies toward terminals in fast-axonal-transport. Ena distribution in larval axons was not affected by mutations in the Khc gene, suggesting that Ena is not itself a fast transport cargo of Drosophila kinesin-1. Genetic interaction tests showed that in a background sensitized by reduced Khc gene dosage, a reduction in Abl gene dosage caused distal paralysis and axonal swellings. A concomitant reduction in ena dosage rescued those defects. These results suggest that Ena/VASP, when not inhibited by the Abl pathway, can bind Khc and reduce its transport activity in axons.

Small GTPase Proteins Rin and Rit Bind to PAR6 GTP-dependently and Regulate Cell Transformation

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.

Janus Kinases Interact with and Phosphorylate Rack-1 (Receptor for Activated C Kinase-1), a WD Motif Containing Protein.

We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.