Biomedical subjects

Xuedong Liu

Publications and source records attributed to Xuedong Liu.

14 recordsLinked to original sources

PubMed2006

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT.

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT http://promoter.colorado.edu, that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species.

Animals↗

PubMed2006

Ubc9 expression is essential for myotube formation in C2C12.

Myogenic differentiation is a fundamental biological process that involves a hierarchical series of events that ultimately leads to muscle-specific gene expression and myofiber formation. Posttranslational modifications of the myogenic regulatory factors have been implicated as important regulatory mechanisms in this process. Here we investigate whether covalent protein modification by a small ubiquitin-like modifier (SUMO) that is known to affect transcription factor activity can impact muscle differentiation. We show that the overall load of sumoylated proteins present in myoblasts diminishes progressively throughout myogenesis. Interestingly, knockdown of the SUMO-conjugating enzyme, Ubc9, severely compromises C2C12 muscle cell terminal differentiation. However, it does not affect the expression, the localization and the activation of MyoD and myogenin. These novel results suggest that protein sumoylation plays a pivotal role in myoblast differentiation and is required to regulate the activity of key targets downstream of MyoD and myogenin.

Animals↗

PubMed2005

Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection.

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1). Timed destruction of p21(Cip1/WAF1) plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCF(Skp2) complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21(Cip1/WAF1). Little is known about how SCF(Skp2) recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCF(Skp2) recognition of p21(Cip1/WAF1) and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21(Cip1/WAF1) requires a functional interaction between p21(Cip1/WAF1) and the cyclin E-Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21(Cip1/WAF1) abolishes its ubiquitination by SCF(Skp2), while mutation of either motif alone has minimal effects, suggesting either contact is sufficient for substrate recruitment. Thus, SCF(Skp2) appears to recognize a trimeric complex consisting of cyclin E-Cdk2-p21(Cip1/WAF1). Furthermore, we show that p21(Cip1/WAF1) can be ubiquitinated at four distinct lysine residues located in the carboxyl-terminal region but not two other lysine residues in the N-terminal region. Any one of these four lysine residues can be targeted for ubiquitination in the absence of the others in vitro, and three of these four lysine residues are also ubiquitinated in vivo, suggesting that there is limited specificity in the selection of ubiquitination sites. Interestingly, mutation of the carboxyl-terminal proline to lysine enables ubiquitin conjugation at the carboxyl terminus of the substrate both in vitro and in vivo. Thus, our results highlight a unique property of the ubiquitination enzymatic reaction in that substrate ubiquitination site selection can be remarkably diverse and occur in distinct spatial areas.

Amino Acid Sequence↗

PubMed2005

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.

Amino Acid Sequence↗

PubMed2005

Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2.

p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF(Skp2), and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF(Skp2). Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF(Skp2). In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF(Skp2).

Amino Acid Motifs↗

PubMed2005

Pathway- and expression level-dependent effects of oncogenic N-Ras: p27(Kip1) mislocalization by the Ral-GEF pathway and Erk-mediated interference with Smad signaling.

Overactivation of Ras pathways contributes to oncogenesis and metastasis of epithelial cells in several ways, including interference with cell cycle regulation via the CDK inhibitor p27(Kip1) (p27) and disruption of transforming growth factor beta (TGF-beta) anti-proliferative activity. Here, we show that at high expression levels, constitutively active N-Ras induces cytoplasmic mislocalization of murine and human p27 via the Ral-GEF pathway and disrupts TGF-beta-mediated Smad nuclear translocation by activation of the Mek/Erk pathway. While human p27 could also be mislocalized via the phosphatidylinositol 3-kinase/Akt pathway, only Ral-GEF activation was effective for murine p27, which lacks the Thr157 Akt phosphorylation site of human p27. This establishes a novel role for the Ral-GEF pathway in regulating p27 localization. Interference with either Smad translocation or p27 nuclear localization was sufficient to disrupt TGF-beta growth inhibition. Moreover, expression of activated N-Ras or specific effector loop mutants at lower levels using retroviral vectors induced p27 mislocalization but did not inhibit Smad2/3 translocation, indicating that the effects on p27 localization occur at lower levels of activated Ras. These findings have important implications for the contribution of activated Ras to oncogenesis and for the conversion of TGF-beta from an inhibitory to a metastatic factor in some epithelial tumors.

Active Transport, Cell Nucleus↗

PubMed2004

Molecular and biochemical characterization of the Skp2-Cks1 binding interface.

SCF(Skp2) is a multisubunit E3 ubiquitin ligase responsible for ubiquitination of cell cycle inhibitor p27. Ubiquitination of p27 requires an adapter protein, Cks1, to be in direct association with Skp2. The exact interface between Skp2 and Cks1 has not been elucidated. Here we have reported the definition of the critical functional interface between Skp2 and Cks1. We have identified eight amino acid residues in two discrete regions of Skp2 that are engaged in Cks1 binding. Mutation of any of these eight residues alone or in combination results in the loss of Cks1 association and negates Skp2-dependent p27 ubiquitination. These eight amino acid residues map on the same side of the Skp2 structure and likely constitute a functional binding surface for Cks1. Four of the eight amino acid residues are located in the largely unstructured carboxyl-terminal tail region of Skp2. These results uncovered the specificity of the Skp2-Cks1 interaction and reveal a critical function for the structurally flexible carboxyl-terminal tail region of Skp2 in Cks1 recognition and substrate ubiquitination.

Amino Acid Motifs↗

PubMed2004

Control of cell cycle-dependent degradation of c-Ski proto-oncoprotein by Cdc34.

It is known that excess amounts of Ski, or any member of its proto-oncoprotein family, causes disruption of the transforming growth factor beta signal transduction pathway, thus causing oncogenic transformation of cells. Previous studies indicate that Ski is a relatively unstable protein whose expression levels can be regulated by ubiquitin-mediated proteolysis. Here, we investigate the mechanism by which the stability of Ski is regulated. We show that the steady-state levels of Ski protein are controlled post-translationally by cell cycle-dependent proteolysis, wherein Ski is degraded during the interphase of the cell cycle but is relatively stable during mitosis. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme Cdc34 mediates cell cycle-dependent Ski degradation both in vitro and in vivo. Overexpression of dominant-negative Cdc34 stabilizes Ski and enhances its ability to antagonize TGF-beta signaling. Our data suggest that regulated proteolysis of Ski is one of the key mechanisms that control the threshold levels of this proto-oncoprotein, and thus prevents epithelial cells from becoming TGF-beta resistant.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Negative regulation of SCFSkp2 ubiquitin ligase by TGF-beta signaling.

TGF-beta is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-beta causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-beta inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-beta treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-beta negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-beta. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-beta-treated cultured cells. In addition, TGF-beta treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF-beta-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-beta occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression.

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.

Animals↗

PubMed2004

Ubiquitination and proteolysis of cancer-derived Smad4 mutants by SCFSkp2.

Smad4/DPC4, a common signal transducer in transforming growth factor beta (TGF-beta) signaling, is frequently inactivated in human cancer. Although the ubiquitin-proteasome pathway has been established as one mechanism of inactivating Smad4 in cancer, the specific ubiquitin E3 ligase for ubiquitination-mediated proteolysis of Smad4 cancer mutants remains unclear. In this report, we identified the SCFSkp2 complex as candidate Smad4-interacting proteins in an antibody array-based screen and further elucidated the functions of SCFSkp2 in mediating the metabolic instability of cancer-derived Smad4 mutants. We found that Skp2, the F-box component of SCFSkp2, physically interacted with Smad4 at the physiological levels. Several cancer-derived unstable mutants exhibited significantly increased binding to Skp2, which led to their increased ubiquitination and accelerated proteolysis. These results suggest an important role for the SCFSkp2 complex in switching cancer mutants of Smad4 to undergo polyubiquitination-dependent degradation.

Amino Acid Motifs↗

PubMed2003

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system.

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.

Amino Acid Sequence↗

PubMed2003

A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1.

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

SUMO-1 modification of MEF2A regulates its transcriptional activity.

Myocyte enhancer factor 2 (MEF2) transcription factors are crucial regulators controlling muscle-specific and growth factor-inducible genes. Numerous studies have reported that the activity of these transcription factors is tightly modulated by posttranslational modifications such as activation by specific phosphorylation as well as repression by class II histone deacetylases (HDACs). We hypothesized that MEF2 could also be regulated by covalent modification by SUMO-1, a reversible posttranslational modification which has been shown to regulate key proteins involved in cell proliferation, differentiation and tumor suppression. In this study, we demonstrate that MEF2A undergoes sumoylation primarily at a single lysine residue (K395) both in vitro and in vivo. We also show that the nuclear E3 ligase, PIAS1, promotes sumoylation of MEF2A. Mutation of lysine 395 to arginine abolishes MEF2A sumoylation and the sumoylation incompetent mutant protein has enhanced transcriptional activity compared to the wild type protein. Our results suggest that protein sumoylation could play a pivotal role in controlling MEF2 transcriptional activity.

Amino Acid Sequence↗