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author:("Liu, xuelong")
1.  High-Throughput Screening AlphaScreen Assay for Identification of Small-Molecule Inhibitors of Ubiquitin E3 Ligase SCFSkp2-Cks1 
Journal of biomolecular screening  2013;18(8):910-920.
Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27Kip1 expression could improve prognosis and prevent excessive cell proliferation. SCFSkp2 is one of the major ubiquitin E3 ligases responsible for degradation of p27Kip1. Ubiquitination of p27Kip1 also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27Kip1. It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27Kip1 degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27Kip1 ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27Kip1 ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.
doi:10.1177/1087057113485789
PMCID: PMC4168015  PMID: 23589337
E3 ligase; inhibitor; Skp2; Cks1; p27kip1; ubiquitin; proteolysis
2.  Measuring the Absolute Abundance of the Smad Transcription Factors Using Quantitative Immunoblotting 
In the age of systems biology, biologists seek to quantify the absolute number of molecules in experimentally treated samples. Immunoblotting remains a technique of choice for assessing the relative differences between the protein levels in different samples. Here we discuss how to exploit immunoblotting for estimating the number of Smad transcription factor molecules per cell. We focus on describing the calculations needed to analyze the data. Our methods are generally applicable to the quantification of other cellular proteins.
doi:10.1007/978-1-60761-738-9_22
PMCID: PMC4168016  PMID: 20694679
Transforming Growth Factor-β; Smad; Cell signaling; Quantitative; Immunoblot; Western blot
3.  Dynamics of TGF-β/Smad Signaling 
FEBS letters  2012;586(14):1921-1928.
The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the noncanonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.
doi:10.1016/j.febslet.2012.03.063
PMCID: PMC4127320  PMID: 22710166
TGF-β; Smad; mathematical model; dose response; switch-like response
4.  Leader cell positioning drives wound-directed collective migration in TGFβ-stimulated epithelial sheets 
Molecular Biology of the Cell  2014;25(10):1586-1593.
Motility analysis of collectively migrating epithelial sheets reveals the interplay between cellular density and leader cell positioning throughout a collective group, as well as the functional role of leader cell constraint in directing a migrating group of cells.
During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFβ-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.
doi:10.1091/mbc.E14-01-0697
PMCID: PMC4019490  PMID: 24623725
5.  Analysis of Ligand Dependent Nuclear Accumulation of Smads in TGF-beta Signaling 
The growth inhibition of dividing cells and most of the transcriptional responses upon TGF-beta treatment depend on the Smad2, Smad3, and Smad4 transcription factors. These proteins shuttle continuously between the cytoplasm and the nucleus, transmitting the ligand status of the TGF-beta receptors to the nuclear transcription machinery. In the absence of TGF-beta ligand, Smads 2/3/4 reside predominantly in the cytoplasm. Following ligand binding to the TGF-beta receptors, the dynamic equilibrium of shuttling Smads 2/3/4 shifts towards a predominantly nuclear state, where a high concentration of these transcription factors drives transcriptional activation and repression of genes required for proper cellular response. Here, we describe live-cell imaging and immunofluorescence microscopy methods for tracking Smads subcellular localization in response to TGF-beta and leptomycin B treatment. In addition, a method of fractionating nuclear and cytoplasmic proteins used to confirm the imaging results was presented. Our results support the notion that the R-Smad shuttling mechanism is distinct from Co-Smad.
doi:10.1007/978-1-60761-738-9_5
PMCID: PMC4083701  PMID: 20694662
TGF-beta; SMAD4; SMAD2; Nuclear Accumulation; Cellular Fractionation; Immunofluorescence; Leptomycin B
6.  Computationally Designed Peptide Inhibitors against the Ubiquitin E3 Ligase SCFFbx4 
doi:10.1002/cbic.201200777
PMCID: PMC4028150  PMID: 23401343
inhibitors; telomere repeat binding factor 1; E3 ligase; protein-protein interactions; ubiquitination
7.  The MPS1 Family of Protein Kinases 
Annual review of biochemistry  2012;81:561-585.
MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs.
doi:10.1146/annurev-biochem-061611-090435
PMCID: PMC4026297  PMID: 22482908
TTK; spindle checkpoint; mitosis; kinetochore; cell cycle
8.  UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores 
Cell Cycle  2013;12(8):1292-1302.
The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.
doi:10.4161/cc.24403
PMCID: PMC3674093  PMID: 23531678
UV-C irradiation; Mps1 kinase; Aurora B; kinetochore association; mitotic delay
9.  The Development of a Novel High Throughput Computational Tool for Studying Individual and Collective Cellular Migration 
PLoS ONE  2013;8(12):e82444.
Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration.
doi:10.1371/journal.pone.0082444
PMCID: PMC3873918  PMID: 24386097
10.  A clinical study of the effects of lead poisoning on the intelligence and neurobehavioral abilities of children 
Background
Lead is a heavy metal and important environmental toxicant and nerve poison that can destruction many functions of the nervous system. Lead poisoning is a medical condition caused by increased levels of lead in the body. Lead interferes with a variety of body processes and is toxic to many organs and issues, including the central nervous system. It interferes with the development of the nervous system, and is therefore particularly toxic to children, causing potentially permanent neural and cognitive impairments. In this study, we investigated the relationship between lead poisoning and the intellectual and neurobehavioral capabilities of children.
Methods
The background characteristics of the research subjects were collected by questionnaire survey. Blood lead levels were detected by differential potentiometric stripping analysis (DPSA). Intelligence was assessed using the Gesell Developmental Scale. The Achenbach Child Behavior Checklist (CBCL) was used to evaluate each child’s behavior.
Results
Blood lead levels were significantly negatively correlated with the developmental quotients of adaptive behavior, gross motor performance, fine motor performance, language development, and individual social behavior (P < 0.01). Compared with healthy children, more children with lead poisoning had abnormal behaviors, especially social withdrawal, depression, and atypical body movements, aggressions and destruction.
Conclusion
Lead poisoning has adverse effects on the behavior and mental development of 2–4-year-old children, prescribing positive and effective precautionary measures.
doi:10.1186/1742-4682-10-13
PMCID: PMC3598508  PMID: 23414525
Child; Lead poisoning; Intelligence development; Neurobehavioral ability
11.  Hydroxyethyl starch 130/0.4 and sodium chloride injection as adjunctive therapy in patients with cerebral hypoperfusion 
BMC Neurology  2012;12:127.
Background
Both severe stenosis and completed occlusion in internal carotid artery or its distal branches have been considered the main reasons of cerebral hypoperfusion, which contributes to the washout disturbances of embolism in low perfusion territories distal to stenosis. An aggravated hypoperfusion state in certain brain region may induce ischemic stroke and further cognitive decline. However, the effective medication for cerebral hypoperfusion is largely unsettled.
Methods/design
By using computed tomography perfusion (CTP) imaging, the trial will evaluate the effectiveness, safety and tolerability of hydroxyethyl starch (HES) 130/0.4 for patients with extra-/intra-cranial artery stenosis and cerebral hypoperfusion. From 5 neurological inpatient wards, 300 patients will be randomly recruited for administered routine medications plus intravascular volume therapies using the equal volume of HES 130/0.4 or 0.9% sodium chloride solution. Cerebral hypoperfusion state after 7-day intervention is the primary outcome measure. The secondary outcome measures includes, impaired renal function, abnormal heart function, hematological changes, neurological dysfunctions and cerebrovascular events in peri-intervention period and/or 3-month follow-up. The sample size will allow the detection of a two-sided 5% significance level between groups in the endpoint with a power of 80%.
Discussion
The trial would provide important efficacy and safety data on the intravascular administration of HES 130/0.4 in patients with unilateral cerebral hypoperfusion. The effects on kidney function, heart function, coagulation, neurological function and cerebralvascular events will be assessed.
Trial registration
ClinicalTrials.gov (Identifier: NCT01192581)
doi:10.1186/1471-2377-12-127
PMCID: PMC3557161  PMID: 23110412
12.  Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses 
Cell Cycle  2011;10(16):2742-2750.
Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.
doi:10.4161/cc.10.16.15927
PMCID: PMC3219542  PMID: 21778823
Mps1; spindle assembly checkpoint; nuclear import; kinase; protein degradation
13.  Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor 
Journal of Biomolecular Screening  2012;17(4):421-434.
Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.
doi:10.1177/1087057111433843
PMCID: PMC3339042  PMID: 22274912
ubiquitin E1; inhibitor; p27kip1; ubiquitin; proteolysis
14.  Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1) 
PLoS ONE  2012;7(1):e29208.
Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells.
doi:10.1371/journal.pone.0029208
PMCID: PMC3261141  PMID: 22279528
15.  Partners in crime: the TGFβ and MAPK pathways in cancer progression 
Cell & Bioscience  2011;1:42.
The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.
doi:10.1186/2045-3701-1-42
PMCID: PMC3275500  PMID: 22204556
TGFβ; Metastasis; ERK; MEK; Ras; Crosstalk; Signal Transduction
16.  Prednisolone and acupuncture in Bell's palsy: study protocol for a randomized, controlled trial 
Trials  2011;12:158.
Background
There are a variety of treatment options for Bell's palsy. Evidence from randomized controlled trials indicates corticosteroids can be used as a proven therapy for Bell's palsy. Acupuncture is one of the most commonly used methods to treat Bell's palsy in China. Recent studies suggest that staging treatment is more suitable for Bell's palsy, according to different path-stages of this disease. The aim of this study is to compare the effects of prednisolone and staging acupuncture in the recovery of the affected facial nerve, and to verify whether prednisolone in combination with staging acupuncture is more effective than prednisolone alone for Bell's palsy in a large number of patients.
Methods/Design
In this article, we report the design and protocol of a large sample multi-center randomized controlled trial to treat Bell's palsy with prednisolone and/or acupuncture. In total, 1200 patients aged 18 to 75 years within 72 h of onset of acute, unilateral, peripheral facial palsy will be assessed. There are six treatment groups, with four treated according to different path-stages and two not. These patients are randomly assigned to be in one of the following six treatment groups, i.e. 1) placebo prednisolone group, 2) prednisolone group, 3) placebo prednisolone plus acute stage acupuncture group, 4) prednisolone plus acute stage acupuncture group, 5) placebo prednisolone plus resting stage acupuncture group, 6) prednisolone plus resting stage acupuncture group. The primary outcome is the time to complete recovery of facial function, assessed by Sunnybrook system and House-Brackmann scale. The secondary outcomes include the incidence of ipsilateral pain in the early stage of palsy (and the duration of this pain), the proportion of patients with severe pain, the occurrence of synkinesis, facial spasm or contracture, and the severity of residual facial symptoms during the study period.
Discussion
The result of this trial will assess the efficacy of using prednisolone and staging acupuncture to treat Bell's palsy, and to determine a best combination therapy with prednisolone and acupuncture for treating Bell's palsy.
Trial Registration
ClinicalTrials.gov: NCT01201642
doi:10.1186/1745-6215-12-158
PMCID: PMC3132722  PMID: 21693007
17.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics 
Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.
Cells respond in real time to the absolute number of TGF-β molecules in their environment.A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.
The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death (Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module (Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.
Members of the TGF-β superfamily are frequently used as morphogens in early embryo development (Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus (Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.
We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses (Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.
We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response (Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 (Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 (Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 (Figure 5E), which is consistent with the graded P-Smad2 response at 45 min (Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive (nHill≈2) (Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 (Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.
In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term (Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.
Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.
doi:10.1038/msb.2011.22
PMCID: PMC3130555  PMID: 21613981
mathematical model; Smad; TGF-β; ultrasensitivity
18.  Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly 
Nucleic Acids Research  2011;39(10):4464-4474.
Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein–protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.
doi:10.1093/nar/gkr030
PMCID: PMC3105382  PMID: 21278418
19.  Structural and Mechanistic Insights into Mps1 Kinase Activation 
Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signaling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7 Å resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the αC helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices αEF and αF that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.
doi:10.1111/j.1582-4934.2008.00605.x
PMCID: PMC2829362  PMID: 19120698
Mps1 structure; kinase activation; phosphorylation
20.  Decoding the quantitative nature of TGF-β/Smad signaling 
Trends in cell biology  2008;18(9):430-442.
How transforming growth factor-β (TGF-β) signaling elicits diverse cell responses remains elusive, despite the major molecular components of the pathway being known. We contend that understanding TGF-β biology requires mathematical models to decipher the quantitative nature of TGF-β/Smad signaling and to account for its complexity. Here, we review mathematical models of TGF-β superfamily signaling that predict how robustness is achieved in bone-morphogenetic-protein signaling in the Drosophila embryo, how changes in receptor-trafficking dynamics can be exploited by cancer cells and how the basic mechanisms of TGF-β/Smad signaling conspire to promote Smad accumulation in the nucleus. These studies demonstrate the power of mathematical modeling for understanding TGF-β biology.
doi:10.1016/j.tcb.2008.06.006
PMCID: PMC2774497  PMID: 18706811
21.  Transforming Growth Factor β Depletion Is the Primary Determinant of Smad Signaling Kinetics▿  
Molecular and Cellular Biology  2009;29(9):2443-2455.
A cell's decision to growth arrest, apoptose, or differentiate in response to transforming growth factor β (TGF-β) superfamily ligands depends on the ligand concentration. How cells sense the concentration of extracellular bioavailable TGF-β remains poorly understood. We therefore undertook a systematic quantitative analysis of how TGF-β ligand concentration is transduced into downstream phospho-Smad2 kinetics, and we found that the rate of TGF-β ligand depletion is the principal determinant of Smad signal duration. TGF-β depletion is caused by two mechanisms: (i) cellular uptake of TGF-β by a TGF-β type II receptor-dependent mechanism and (ii) reversible binding of TGF-β to the cell surface. Our results indicate that cells sense TGF-β dose by depleting TGF-β via constitutive TGF-β type II receptor trafficking processes. Our results also have implications for the role of the TGF-β type II receptor in disease, as tumor cells harboring TGF-β type II receptor mutations exhibit impaired TGF-β depletion, which may contribute to the overproduction of TGF-β and a consequently poor prognosis in cancer.
doi:10.1128/MCB.01443-08
PMCID: PMC2668365  PMID: 19223462
22.  Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †  
Molecular and Cellular Biology  2008;29(5):1306-1320.
Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.
doi:10.1128/MCB.01271-08
PMCID: PMC2643833  PMID: 19103740
23.  Association of v-ErbA with Smad4 Disrupts TGF-β Signaling 
Molecular Biology of the Cell  2009;20(5):1509-1519.
Disruption of the transforming growth factor-β (TGF-β) pathway is observed in the majority of cancers. To further understand TGF-β pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-β responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-β–induced cell growth inhibition and that dysregulation of TGF-β signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-β–induced growth inhibition and that TGF-β sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-β signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.
doi:10.1091/mbc.E08-08-0836
PMCID: PMC2649266  PMID: 19144825
24.  A Concise Total Synthesis of Largazole, Solution Structure, and Some Preliminary Structure Activity Relationships 
Organic letters  2008;10(16):3595-3598.
A total synthesis of largazole that proceeds in 8 steps from commercial materials is reported, along with some structure-activity relationships. A combination of NMR studies and molecular modeling have also provided a preliminary picture of the conformation of largazole.
doi:10.1021/ol8013478
PMCID: PMC2664405  PMID: 18616341
25.  Regulation of Kinetochore Recruitment of Two Essential Mitotic Spindle Checkpoint Proteins by Mps1 Phosphorylation 
Molecular Biology of the Cell  2009;20(1):10-20.
Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.
doi:10.1091/mbc.E08-03-0324
PMCID: PMC2613107  PMID: 18923149

Results 1-25 (30)