GST Binding Assays Research Articles

ITSN1 binds the E2-conjugating enzyme UBC9

Aim. Scaffolding protein of the intersectin 1 (ITSN1) associated with malignant cell transformation. A short isoform of ITSN1 (ITSN1-S) can localize to the nucleus and inhibit breast cancer cell proliferation but the exact mechanisms of ITSN1 nuclear export have not been fully elucidated. SUMOylation of ITSN1, or its interaction with components of SUMO modification, may be one of the regulatory mechanisms contributing to the nuclear-cytoplasmic shuffle of ITSN1 in the cell. Methods. Full-length human UBC9 sequence was subcloned in pGEX4T2 vector for in vitro GST-binding assays with overexpressed Omni-ITSN1-S in 293 cell line. Lysates of 293 cells with overexpressed FLAG-UBC9 were used for co-immunoprecipitation with endogenous proteins of ITSN1 and ITSN2. Results. Endogenous ITSN1-S form complexes with full-length overexpressed UBC9 in 293 in vivo. Further analysis revealed that GST-UBC9 binds human full-length short isoform ITSN1-S in vitro. Conclusions. E2-conjugating enzyme of the SUMOylation, UBC9, is confirmed as a novel protein partner for ITSN1 both in vitro and in vivo. Considering the tumor suppressor role of a nuclear ITSN1-S in breast cancer and the unique role UBC9 plays in SUMO-modification of proteins, we suggest a possibility of UBC9 and ITSN1 interaction association with malignant transformation, which can be the ground for the further studies.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions.

Abstract A38: In vitro and in vivo interaction of Cdk6 and Eya2 indicate potential crosstalk

Abstract We have identified a novel association of the developmentally significant protein, Eya2, with cyclin dependent kinase (cdk) 6. Eya2 is part of the conserved retinal determination network that has been shown to function in fly eye development (Rebay, 2005). In mammals, the EYA gene product has been implicated in gonadogenesis, myogenesis, neurognenesis, limb formation, thymus, and kidney development, in part through its ability to regulate cell proliferation (Zhang 2005). Eya2 is overexpressed in several types of cancers including epithelial ovarian (Zhang et al, 2005), cervical (Bierkens et al, 2013), lung adenocarcinoma (Guo et al, 2009), breast and hematopoietic (Patrick, 2013, Wang, 2011). Cdk6 knockout mice display defects in hematopoiesis, including decreased cellularity of the thymus, red blood cells and lymphocytes (Malumbres, 2004). Cdk6 kinase activity is required for thymocyte development (Hu et al, 2011) and this role is partially mediated by modulating Notch target gene expression. The interaction of these two proteins, each important in both development and cancer, and both involved in regulation of cell proliferation and transcription, was first identified in a yeast two-hybrid analysis of a human fetal brain library. Subsequent results from our labs confirm the association of cdk6 and Eya2 in GST binding assays and co-immunoprecipitations. GST binding assays demonstrated that Six4, a cofactor of Eya2, competes with cdk6 for Eya2 binding. Co-immunoprecipitation of transfected cell lysates and native immunoprecipitations also demonstrate binding. Finally, co-expression of cdk6 with Eya2 reduces the half-life of the Eya2 protein in cell lysates. We propose that cdk6 and Eya2 interact to affect the function of cell proliferation and differentiation that is crucial to both the biology of cancer and development. This interaction could provide a mechanism to allow the expansion of the progenitor cell population prior to differentiation.

Abstract 3771: Tank-binding kinase 1 associates with centrosomes and regulates microtubule dynamics and mitosis

Abstract TANK Binding Kinase 1 (TBK1) regulates interferon signaling and NFκB function by acting as a non-canonical IκB kinase. TBK1 also participates in RalB-mediated inflammatory responses and contributes to the survival of non-small cell lung cancers (NSCLC) driven by oncogenic KRAS. In addition, TBK1 could phosphorylate and activate Akt, modulate autophagy and alter the autocrine cytokine network in cancer cells indicating oncogenic roles in addition to its immune regulatory functions. Our studies reveal that TBK1 plays a novel and direct role in promoting mitotic progression of lung cancer cells by phosphorylating multiple substrates during mitosis as well as by modulating microtubule dynamics. Levels of active phospho-TBK1 are elevated in mitotic cells where it localized to centrosomes, mitotic spindles and midbody as seen by confocal microscopy. Inhibition of TBK1 by kinase inhibitors such as BX795, Amlexanox, MRT67037 or depletion of TBK1 by shRNA resulted in mitotic defects and decreased the number of mitotic cells. TBK1 could interact with the centrosomal protein CEP170 and the nuclear mitotic apparatus protein NuMA as seen by GST binding assays, immunoprecipitation-western blot assays and proximity ligation assays. In addition, TBK1 could phosphorylate CEP170 and NuMA in in vitro kinase assays; mass spectrometry analysis revealed that TBK1 phosphorylated 12 serine residues and a threonine residue of CEP170 and five serine residues of NuMA. Inhibition or depletion of TBK1 prevented the centrosomal localization of CEP170 and the association of NuMA with the spindle poles. TBK1 depletion in A549 and H1650 cells resulted in more stable microtubules; further, depletion of TBK1 inhibited the interaction of CEP170 with the microtubule depolymerase Kif2b and the binding of NuMA to Dynein, resulting in mitotic abnormalities or mitotic arrest. In addition, inhibition of specific interaction between TBK1 and CEP170 using a 13 amino acid peptide resulted in inhibition of mitosis, enhanced microtubule stability and mitotic defects. These results are paradigm shifting that suggest TBK1 plays a significant role in regulating microtubule dynamics and mitotic progression of cells and this might have significant relevance to cancer as well as innate immune response. Citation Format: Smitha R. Pillai, Jonathan Nguyen, Joseph Johnson, Eric Haura, Domenico Coppola, Srikumar Chellappan. Tank-binding kinase 1 associates with centrosomes and regulates microtubule dynamics and mitosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2015-3771

Abstract 1516: YAP1 regulates Oct4 activity and Sox2 expression, facilitating self-renewal and vascular mimicry of stem-like cells

Abstract Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that cancer stem-like cells (CSCs) contribute to the genesis and progression of NSCLC. These cells have been shown to form differentiated tumor cells upon receiving appropriate microenvironmental cues and can give rise to multiple components of the tumor, including the vasculature. Earlier work from our laboratory had shown that Hoechst 33342 dye excluding side-population (SP) cells are enriched in cells having stem-like properties. SP cells displayed high self-renewal capacity, were highly drug resistant, and could form metastatic tumors in immunocompromised mice. The present study shows that the transcriptional co-activator YAP1, which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules in matrigel. Inhibition of YAP1 by a small molecule inhibitor Visudyne or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor Sox2. YAP1 could associate with the Sox2 promoter and could transcriptionally induce Sox2 as seen by qRT-PCRs, ChIP assays and promoter luciferase assays; this induction was through a physical interaction of YAP1 with Oct4 protein. The binding of Oct4 to YAP1 could be detected in cell lines as well as NSCLC tumor tissues as seen by immunofluorescence, proximity ligation assays (PLA), immunoprecipitation-western blot experiments and GST binding assays. Interestingly, the interaction was elevated in NSCLC samples compared to normal tissue as seen by PLA. A peptide that could disrupt the interaction between YAP1 and Oct4 abrogated Sox2 expression, self-renewal and vascular mimicry. Depleting YAP1 reduced the expression of EMT genes and prevented the growth and metastasis of tumor xenografts in mice, while overexpression of Sox2 in YAP1 depleted cells could rescue self-renewal of stem-like cells. In conclusion, our results demonstrate that YAP1 can modulate the stem-like functions of cancer stem cells by regulating Sox2, and this regulatory pathway can potentially be targeted for combating NSCLC. Citation Format: Namrata Bora Singhal, Jonathan Nguyen, Courtney Schaal, Deepak Perumal, Sandeep Singh, Domenico Coppola, Srikumar Chellappan. YAP1 regulates Oct4 activity and Sox2 expression, facilitating self-renewal and vascular mimicry of stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1516. doi:10.1158/1538-7445.AM2015-1516

Oncogenic mutations of thyroid hormone receptor β

The C-terminal frame-shift mutant of the thyroid hormone receptor TRβ1, PV, functions as an oncogene. An important question is whether the oncogenic activity of mutated TRβ1 is uniquely dependent on the PV mutated sequence. Using four C-terminal frame-shift mutants-PV, Mkar, Mdbs, and AM-we examined that region in the oncogenic actions of TRβ1 mutants. Remarkably, these C-terminal mutants induced similar growth of tumors in mouse xenograft models. Molecular analyses showed that they physically interacted with the p85α regulatory subunit of PI3K similarly in cells. In vitro GST-binding assay showed that they bound to the C-terminal Src-homology 2 (CSH2) of p85α with markedly higher avidity. The sustained association of mutants with p85α led to activation of the common PI3K-AKT-ERK/STAT3 signaling to promote cell proliferation and invasion and to inhibit apoptosis. Thus, these results argue against the oncogenic activity of PV being uniquely dependent on the PV mutated sequence. Rather, these four mutants could favor a C-terminal conformation that interacted with the CSH2 domain of p85α to initiate activation of PI3K to relay downstream signaling to promote tumorigenesis. Thus, we propose that the mutated C-terminal region of TRβ1 could function as an "onco-domain" and TRβ1 is a potential therapeutic target.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

The GPCR‐associated sorting protein 1 regulates ligand‐induced down‐regulation of GPR55

BACKGROUND AND PURPOSEMany GPCRs, including the CB1 cannabinoid receptor, are down-regulated following prolonged agonist exposure by interacting with the GPCR-associated sorting protein-1 (GASP-1). The CB1 receptor antagonist rimonabant has also recently been described to be an agonist at GPR55, a cannabinoid-related receptor. Here we investigated the post-endocytic properties of GPR55 after agonist exposure and tested whether GASP-1 is involved in this process.EXPERIMENTAL APPROACHWe evaluated the direct protein-protein interaction of GPR55 with GASP-1 using (i) GST-binding assays and (ii) co-immunoprecipitation assays in GPR55-HEK293 cells with endogenous GASP-1 expression. We further tested the internalization, recycling and degradation of GPR55 using confocal fluorescence microscopy and biotinylation assays in the presence and absence of GASP-1 (lentiviral small hairpin RNA knockdown of GASP-1) under prolonged agonist [rimonabant (RIM), lysophosphatidylinositol (LPI)] stimulation.KEY RESULTSWe showed that the prolonged activation of GPR55 with rimonabant or LPI down-regulates GPR55 via GASP-1. GASP-1 binds to GPR55 in vitro, and this interaction was required for targeting GPR55 for degradation. Disrupting the GPR55-GASP-1 interaction prevented post-endocytic receptor degradation, and thereby allowed receptor recycling.CONCLUSION AND IMPLICATIONSThese data implicate GASP-1 as an important regulator of ligand-mediated down-regulation of GPR55. By identifying GASP-1 as a key regulator of the trafficking and, by extension, functional expression of GPR55, we may be one step closer to gaining a better understanding of this receptor in response to cannabinoid drugs.LINKED ARTICLESThis article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

Vascular Endothelial Growth Factor Receptor 2 Direct Interaction with Nephrin Links VEGF-A Signals to Actin in Kidney Podocytes

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

Bimolecular Fluorescence Complementation analysis to reveal protein interactions in herpes virus infected cells

Protein interactions are at the basis of all processes in living organisms. In particular, regulatory proteins do not act alone but participate in multifaceted sets of interactions that are organized into complex networks. In herpes simplex virus (HSV-1) infected cells, viral proteins interact with cellular proteins and with other viral proteins to form the protein complexes required for virus production, including transcription complexes, replication complexes and virion assembly complexes. While a number of methods have been developed to investigate protein–protein interactions such as coimmunoprecipitation, GST-binding assays and yeast 2-hybrid analyses, these approaches require removal of the proteins from the cellular environment and do not provide information on the spatial localization of the protein–protein interaction in living cells. The fluorescence based approach Bimolecular Fluorescence Complementation (BiFC) allows direct visualization of the subcellular localization of the protein complex in living cells. In BiFC, two halves of a fluorescent protein are fused to each of two interacting proteins of interest, resulting in nonfluorescent fusion proteins. Interaction of the protein partners tethers the fused fluorescent fragments in close proximity, which facilitates their association and restoration of fluorescence. Two limitations of BiFC are that there is a delay between the time that the interacting proteins associate and fluorescence complex formation and thus complex formation cannot be measured in real-time, and fluorescence complex formation is irreversible in vivo. Despite these limitations, BiFC is a powerful and sensitive approach that can be performed using standard molecular biology and cell culture protocols and a fluorescence microscope.

Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.

Abstract 4532: Nucleolin inhibits Hdm2-mediated p53 ubiquitination using multiple interactions

Abstract Nucleolin is a multifunctional abundant nucleolar protein whose transcriptional expression is trans-activated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein has defined roles in ribosomal RNA processing and transcriptional regulation of various genes including rRNA. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its sub-nuclear re-localization. Thus, nucleolin has been demonstrated to have both positive and negative effects on cell proliferation. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2 and inhibits Hdm2-mediated degradation of p53 albeit independent of the ARF (Alternate Reading Frame) tumor suppressor protein. Nucleolin also reduces Hdm2 protein levels by an unknown mechanism when over-expressed in cells to levels found in a variety of cancer cells. Here we have delineated that multiple domains of both nucleolin and Hdm2 are involved in distinct nucleolin-Hdm2 interactions in unstressed cells. Our data indicates that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2, as determined by Far-Western as well as GST-binding assays in vitro. While the N-terminus of nucleolin was found associated with the Hdm2 RING domain, the nucleolin RBDs robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2. The C-terminal GAR (Glycine-Arginine Rich) domain of nucleolin, shown previously to be essential for association with p53, did not bind Hdm2 yet was found to stimulate Hdm2-mediated p53-ubiquitination. Conversely, the nucleolin RBD that strongly interacted with Hdm2 was found to inhibit the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. There was no significant effect of over-expression of nucleolin or its various domains on Hdm2-autoubiquitination in cells. Similarly, the phosphorylation status of nucleolin did not significantly influence its binding to Hdm2. As might be expected from a key rRNA processing factor, our findings suggest that the activity of nucleolin towards Hdm2 can be modulated to yield either p53 up- or down-regulation. Our studies can aid in the future design of small molecule-based cancer therapeutics that can stimulate the wild-type p53 checkpoint in variety of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4532.

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr805, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.

Prohibitin physically interacts with MCM proteins and inhibits mammalian DNA replication

Prohibitin, a tumor suppressor protein, has been shown to repress E2F-mediated transcription and arrest cell cycle progression. While prohibitin has been proposed to regulate cell cycle progression by repressing transcriptional targets of E2F1, it is not clear whether other mechanisms are also involved in mediating the growth arrest. Here we demonstrate that prohibitin can function as a potent inhibitor of DNA replication by interacting with members of Minichromosome maintenance complex of proteins (MCM2-7). The data presented here indicates that prohibitin can physically interact with MCM2, MCM5 and MCM7 in in vitro GST binding assays as well as in MCF-7 cells as seen by immunoprecipitation-Western blot experiments. The association was cell cycle dependent, and more pronounced 4-8 hours after serum stimulation of quiescent cells. Prohibitin associated more robustly with MCM2 and MCM5 compared to MCM7, suggesting that prohibitin mainly interacts with the regulatory subunits of the MCM complex. Confirming these results, prohibitin was found to co-localize with MCM2, MCM5 and MCM7 in MCF-7 cells, as seen by double immunofluorescence experiments. Further, Prohibitin strongly inhibited DNA replication in an in vitro replication assay. These results strongly suggest that prohibitin effectively represses replication by interacting with the components of mammalian replication machinery and this might contribute to the growth regulatory properties of prohibitin.

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) in the vast majority of eukaryotes. In human, Dicer has been shown to interact with cellular proteins via its N-terminal domain. Here, we demonstrate the ability of Dicer C-terminus to interact with 5-lipoxygenase (5LO), an enzyme involved in the biosynthesis of inflammatory mediators, in vitro and in cultured human cells. Yeast two-hybrid and GST binding assays delineated the smallest 5-lipoxygenase binding domain (5LObd) of Dicer to its C-terminal 140 amino acids comprising the double-stranded RNA (dsRNA) binding domain (dsRBD). The Dicer 5LObd-5LO association was disrupted upon Ala substitution of Trp residues 13, 75 and 102 in 5LO, suggesting that the Dicer 5LObd may recognize 5LO via its N-terminal C2-like domain. Whereas a catalytically active 5LObd-containing Dicer fragment was found to enhance 5LO enzymatic activity in vitro, human 5LO modified the miRNA precursor processing activity of Dicer. Providing a link between miRNA-mediated regulation of gene expression and inflammation, our results suggest that the formation of miRNAs may be regulated by 5LO in leukocytes and cancer cells expressing this lipoxygenase.

Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Interaction of Hepatitis B Viral Oncoprotein with Cellular Target HBXIP Dysregulates Centrosome Dynamics and Mitotic Spindle Formation

Hepatitis B virus infection is associated with hepatocellular carcinoma, claiming 1 million lives annually worldwide. To understand the carcinogenic mechanism of hepatitis B virus-encoded oncoprotein HBx, we explored the function of HBx interaction with its cellular target HBXIP. Previously, we demonstrated that viral HBx and cellular HBXIP control mitotic spindle formation, regulating centrosome splitting. By using various fragments of HBx, we determined that residues (137)CRHK(140) within HBx are necessary for binding HBXIP. Mutation of the (137)CRHK(140) motif in HBx abolished its ability to bind HBXIP and to dysregulate centrosome dynamics in HeLa and immortal diploid RPE-1 cells. Unlike wild-type HBx, which targets to centrosomes as determined by subcellular fractionation and immunofluorescence microscopy, HBx mutants failed to localize to centrosomes. Overexpression of viral HBx wild-type protein and knockdown of endogenous HBXIP altered centrosome assembly and induced modifications of pericentrin and centrin-2, two essential proteins required for centrosome formation and function, whereas HBXIP nonbinding mutants of HBx did not. Overexpression of HBXIP or fragments of HBXIP that bind HBx neutralized the effects of viral HBx on centrosome dynamics and spindle formation. These results suggest that HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation.

Human Papillomavirus E7 Requires the Protease Calpain to Degrade the Retinoblastoma Protein

Cervical cancers transformed by high risk human papilloma virus (HPV) express the E7 oncoprotein, which accelerates the degradation of the retinoblastoma protein (Rb). Here we show that the E7-mediated degradation of Rb requires the calcium-activated cysteine protease, calpain. E7 bound and activated mu-calpain and promoted cleavage at Rb(810), with mutation of this residue preventing E7-mediated degradation. The calpain cleavage product, Rb(1-810), was unable to mediate cell cycle arrest but retained the ability to repress E6/E7 transcription. E7 also promoted the accelerated proteasomal degradation of Rb(1-810). Calpain inhibitors reduced the viability of HPV-transformed cells and synergized with cisplatin. Calpain, thus, emerges as a central player in E7-mediated degradation of Rb and represents a potential new drug target for the treatment of HPV-associated lesions.

Mammalian SEPT2 Is Required for Scaffolding Nonmuscle Myosin II and Its Kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.

Transforming Growth Factor-β-independent Regulation of Myogenesis by SnoN Sumoylation

Recent progress has been made on the role of oncoproteins c-Ski and related SnoN in the control of cellular transformation. c-Ski/SnoN potently repress transforming growth factor-beta (TGF-beta) antiproliferative signaling through physical interaction with signal transducers called Smads. Overexpression of c-Ski/SnoN also induces skeletal muscle differentiation, but how c-Ski/SnoN function in myogenesis is largely unknown. During our investigation on the role of sumoylation in TGF-beta signaling, we inadvertently found that SnoN is modified by small ubiquitin-like modifier-1 (SUMO-1). Here, we biochemically characterize SnoN sumoylation in detail and report the physiological function of the modification. Sumoylation occurs primarily at lysine 50 (Lys-50). PIAS1 and PIASx proteins physically interact with SnoN to stimulate its sumoylation, thus serving as SUMO-protein isopeptide ligases (E3) for SnoN sumoylation. SnoN sumoylation does not alter its metabolic stability or its ability to repress TGF-beta signaling. Notably, loss of sumoylation in the Lys-50 site (via a Lys-to-Arg point mutation) potently activates muscle-specific gene expression and enhances myotube formation. Our study suggests a novel role for SUMO modification in the regulation of myogenic differentiation.