Author Correction: The role of sentrin-specific protease 2 substrate recognition in TGF-ß-induced tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The role of sentrin-specific protease 2 substrate recognition in TGF-ß-induced tumorigenesis.

Smad4, a common-mediator of Smads, plays a central role in forming complexes with receptor-phosphorylated Smads, and then transduces transforming growth factor (TGF)-ß signals into the nuclei. Although many cellular factors are involved in TGF-ß induced epithelial-to-mesenchymal transition (EMT) and cell migration, very little is known with the mechanism of Smad4 regulation on pro-oncogenes response by TGF-ß. Herein, we demonstrate the interaction of Sentrin-specific protease 2 (SENP2) with Smad4 through SENP2 residue 363~400. The same segment is also important for desumoylation of Smad4, and able to relieve sumoylation-mediated TGF-ß repression. The SENP2363~400 segment is critical for TGF-ß-induced cell migration, which is correlated with SENP2363~400 deletion mutant failed to increase matrix metalloproteinase (MMP)-9 and EMT marker gene expression. Moreover, our results suggest that the interaction and desumoylation between SENP2 and Smad4 promote cell migration in triple-negative breast cancer cells. Altogether, our data show how SENP2 regulates its substrate for desumoylation, and also the role of SENP2 in TGF-ß induced cancer cell migration.

HIC1 interacts with and modulates the activity of STAT3.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene, expression of which is frequently suppressed in human cancers. Very little is known about the molecular basis of HIC1 in antagonizing oncogenic pathways. Here, we report that HIC1 forms complexes with the signal transducers and activators of transcription 3 (STAT3) and attenuates STAT3-mediated transcription. STAT3 was identified as a HIC1-interacting protein by affinity capture and followed by mass spectrometry analysis. Overexpression or depletion of HIC1 resulted in decreased or increased levels of interleukin-6 (IL-6)/oncostatin M (OSM)-induced STAT3-mediated reporter activity and expression of target genes such as VEGF and c-Myc, respectively. Furthermore, HIC1 suppressing the VEGF and c-Myc promoter activity and the colony formation of MDA-MB 231 cells were STAT3-dependent. Further studies showed that HIC1 interacts with the DNA binding domain of STAT3 and suppresses the binding of STAT3 to its target gene promoters. Domain mapping study revealed that HIC1 C-terminal domain binds to STAT3. HIC1 mutant defective in STAT3 interaction reduced its repressive effect on STAT3 DNA binding activity, the reporter activity and gene expression of the VEGF and c-Myc genes, and cell growth in MDA-MB 231 cells. Altogether, our findings not only provide a novel role of HIC1 in antagonizing STAT3-mediated activation of VEGF and c-Myc gene expression and cell growth, but also elucidate a molecular basis underlying the inhibitory effect of HIC1 on STAT3 transcriptional potential.

The PML isoform IV is a negative regulator of nuclear EGFR's transcriptional activity in lung cancer.

Epidermal growth factor receptor (EGFR) is a membrane-bound receptor tyrosine kinase, which can transduce intracellular signals responsible for cell proliferation. It is frequently overexpressed and/or constitutively activated in non-small cell lung cancer and thus is considered as a major cause of this disease. Recently, EGFR has been found in the nucleus where the nuclear EGFR (nEGFR) can function as a transcription factor activating the transcription of genes such as cyclin D1 gene (CCND1), which is essential for cell proliferation. Nevertheless, how nEGFR's transcriptional activity is regulated remains unclear. Promyelocytic leukemia protein (PML) is a tumor suppressor, which is lost in various cancers including lung cancer. However, the role of PML in the suppression of lung cancer growth is still unclear. When we investigated the role of PML in the regulation of lung cancer cell growth, we found that PML isoform IV (PMLIV) preferentially represses the growth of lung cancer cells bearing constitutively active EGFR. Besides, when growing in the EGFR activating conditions, the growth of EGFR wild-type bearing A549 cells has been repressed by PMLIV overexpression. We also found that PMLIV can interact physically with nEGFR and represses the transcription of nEGFR target genes. We showed that PMLIV is recruited by nEGFR to the target promoters and reduces the promoter histone acetylation level via HDAC1. Together, our results suggest that PMLIV interacts with nEGFR upon EGFR activation and represses the transcription of nEGFR target genes such as CCND1 and thus leading to inhibition of the lung cancer cell growth.

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response.

While numerous small ubiquitin-like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid-dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ-K-X-E/D consensus motif, such as CBP and Elk-1, but not substrates with core ψ-K-X-E/D motif alone or SUMO-interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia-elicited modulation of sumoylation and target gene expression of CBP and Elk-1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.

Structural and functional roles of Daxx SIM phosphorylation in SUMO paralog-selective binding and apoptosis modulation.

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

Cdh1 controls the stability of TACC3.

Transforming acidic coiled-coil protein 3 (TACC3) was reported to be important for regulating mitotic spindle assembly and chromosome segregation. While the protein level of TACC3 was shown to be altered during cell cycle progression, the molecular mechanism in controlling TACC3 level is unclear. Here, we show that TACC3 protein level can be regulated by Cdh1, a well known activator of anaphase-promoting complex/cyclosome. We identified Cdh1 as an interacting partner of TACC3 by a yeast array screen. Both in vitro and in vivo binding studies indicated that TACC3 can form complexes with Cdh1. Depletion of endogenous Cdh1 prolonged TACC3 protein level during mitotic exit. Alteration of Cdh1 level by ectopic overexpression or siRNA knockdown correlated well with an increase or decrease of ubiquitinated TACC3, respectively. Furthermore, the domain mapping studies of TACC3 revealed that multiple domains are involved in Cdh1-regulated degradation of TACC3. Altogether, our findings suggest that Cdh1 controls TACC3 protein stability during mitotic exit.

SUMO modification modulates the activity of calpain-2.

Small ubiquitin-like modifier (SUMO) modification has been shown to be involved in the regulation of various cellular processes including gene transcription, nucleocytoplasmic transport, cell cycle, DNA repair, stress response, and signal transduction. However, very little is known about the process of cell migration being modulated by SUMO modification. Here, we show that calpain-2, a protease involved in cell motility, can be SUMO modified at lysine residue 390. Converting the SUMO acceptor lysine residue to arginine residue significantly attenuated calpain-2 activity, correlating well with a loss of calpain-2-elicited cell motility. Accordingly, expression of SENP1 could abrogate calpain-2 sumoylation, causing an inhibition on calpain-2-dependent activity and cell motility. These results not only identify calpain-2 as a substrate for sumoylation but also provide an important role of sumoylation in regulating cell migration.

Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression.

As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein posttranslational modifications, including phosphorylation and sumoylation. However, gaps exist in our knowledge of how KAP1 phosphorylation cross-talks with KAP1 sumoylation and what the biological consequence is. Here, we show that doxorubicin (Dox) treatment induces KAP1 phosphorylation at Ser-824 via an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21WAF1/CIP1 and Gadd45alpha. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 sumoylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 sumoylation and stimulation of p21 transcription before the exposure of Dox. We further provide evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21 and Gadd45alpha transcription. Moreover, KAP1 and differential sumoylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its sumoylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses.

Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

SUMO modification negatively modulates the transcriptional activity of CREB-binding protein via the recruitment of Daxx.

Small ubiquitin-like modifier (SUMO) modification is emerging as an important control in transcription regulation. Here, we show that CREB-binding protein (CBP), a versatile transcriptional coactivator for numerous transcription factors in response to diverse signaling events, can be modified by SUMO-1 at lysine residues 999, 1034, and 1057 both in vitro and in vivo. Mutation of the SUMO acceptor lysine residues either individually or in combination enhanced CBP transcriptional activity, and expression of a SUMO protease SENP2 potentiated the transcriptional activity of CBP wild-type but not its sumoylation mutant, indicating that SUMO modification negatively regulates CBP transcriptional activity. Furthermore, we demonstrated an interaction of SUMO-1-modified CBP with the transcriptional corepressor Daxx and an essential role of Daxx in mediating SUMO-dependent transcriptional regulation of CBP through histone deacetylase 2 recruitment. Together, our findings indicate that SUMO modification and subsequent recruitment of Daxx represent a previously undescribed mechanism in modulating CBP transcriptional potential.

Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase.

The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%-90% of recombinant proteins should be produced in authentic form by our engineered MetAPs.

Regulation of ribonuclease expression by estradiol in Rana catesbeiana (Bullfrog).

Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.