Hongliang Zong

Elevated β1,4-Galactosyltransferase I in Highly Metastatic Human Lung Cancer Cells IDENTIFICATION OF E1AF AS IMPORTANT TRANSCRIPTION ACTIVATOR

The elevated levels of β1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5′-region flanking the transcription start point of the GalT I gene (–1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides –205 and –200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the β1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

Cyclin D3/CDK11p58 complex is involved in the repression of androgen receptor

ABSTRACT Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function.

β1,4-Galactosyltransferase V Functions as a Positive Growth Regulator in Glioma

β1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcβ1→6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

HSP70 protects BCL2L12 and BCL2L12A from N-terminal ubiquitination-mediated proteasomal degradation

MINT‐7026304: BCL2L12A (uniprotkb:Q9HB09‐2) physically interacts (MI:0218) with Hsp70 (uniprotkb:P08107) by anti tag coimmunoprecipitation (MI:0007)

Functional Interaction of E1AF and Sp1 in Glioma Invasion

ABSTRACT Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for β1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

Cdc34-mediated degradation of ATF5 is blocked by cisplatin

ATF5, a member of activating transcription factor (ATF)/cAMP-response element-binding protein (CREB) family of b-ZIP transcription factors, contributes to neural cell differentiation and is involved in cell apoptosis in response to cisplatin and a number of environment factors. However, the mechanisms governing the regulation of ATF5 protein during apoptosis are largely unknown. In this study we reported that ATF5 protein was a substrate of the ubiquitin-proteasome pathway. Interestingly, the ubiquitin-dependent degradation of exogenous ATF5 protein was independent of lysine residues. Instead, the addition of a large N-terminal enhanced green fluorescence protein tag increased the stability of ATF5 protein, and the free amino acid group of the N-terminal methionine of ATF5 protein was a site for ubiquitinylation, indicating that exogenous ATF5 was degraded via the ubiquitin-proteasome system through N-terminal ubiquitinylation. Furthermore, cisplatin increased ATF5 protein expression via preventing its ubiquitin-dependent degradation, which might be associated with its promoting the nucleus-to-cytoplasm translocation of E2 ubiquitin-conjugating enzyme Cdc34 and reducing the interaction between ATF5 and Cdc34. In summary, a down-regulation of proteasome-mediated degradation of ATF5 might contribute to cisplatin-induced apoptosis, providing a new mechanism of cisplatin-induced apoptosis.

CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin–proteasome degradation

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11(p58) as a novel protein involved in the regulation of VDR. CDK11(p58), a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11(p58) interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11(p58) decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11(p58) is involved in the negative regulation of VDR.

Thr-370 Is Responsible for CDK11p58 Autophosphorylation, Dimerization, and Kinase Activity

CDK11p58, a member of the p34cdc2-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11p58 interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11p58 was autophosphorylated, and the main functions of CDK11p58, such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11p58 was autophosphorylated at Thr-370. By mutagenesis, we created CDK11p58 T370A and CDK11p58 T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11p58, respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11p58 and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivition of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11p58. Moreover, Thr-370 mutants might affect CDK11p58-mediated signaling pathways.

Repression of Estrogen Receptor alpha by CDK11p58 Through Promoting its Ubiquitin–Proteasome Degradation

Estrogen receptor alpha (ERalpha) is a ligand-dependent transcription factor that mediates physiological responses to 17beta-estradiol (E(2)). These responses of cells to estrogen are regulated in part by degradation of ERalpha. In this report, we found that CDK11(p58) repressed ERalpha transcriptional activity. And we further demonstrated that ERalpha protein level was down-regulated by CDK11(p58) in mammalian cells in a ligand independent manner. This effect could be abrogated by treatment with proteasome inhibitor MG132. Our results indicated that the ubiquitin/proteasome-mediated degradation of ERalpha was promoted by CDK11(p58). Furthermore, the interaction between ERalpha and CDK11(p58) was detected. This interaction was necessary for the polyubiquitination and degradation of ERalpha. On the contrary, the other isoform of CDK11, CDK11(p110) and the kinase dead mutant of CDK11(p58), D224N, did not associate with ERalpha and failed to reduce the ERalpha protein level. These data identified a new negative regulatory protein of ERalpha and provided a new pathway by which CDK11(p58) negatively regulated cells.

Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP)

Human Papilloma virus E6‐associated protein (E6‐AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6‐AP interaction protein and a target of E6‐AP‐mediated degradation. Overexpression of E6‐AP results in degradation of TH1 in a dose‐dependent manner, whereas knock‐down of endogenous E6‐AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6‐AP. Wild‐type E6‐AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6‐AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6‐AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6‐AP‐catalyzed polyubiquitination. J. Cell. Biochem. 101: 167–180, 2007.