Dan Zheng

UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins.

Ubiquitin and ubiquitin‐like proteins are known to be covalently conjugated to a variety of cellular substrates via a three‐step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin‐activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin‐like proteins. Ubiquitin‐activating enzyme E1‐domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin‐like protein, ubiquitin‐fold modifier 1 (Ufm1), by forming a high‐energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin‐like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin‐like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated‐SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm. J. Cell. Biochem. 104: 2324–2334, 2008.

Dual specificity phosphotase 18, interacting with SAPK, dephosphorylates SAPK and inhibits SAPK/JNK signal pathway in vivo.

The SAPK/JNKs play important roles in numerous cellular processes, and for this reason they have become putative drug targets. Most dual-specificity protein phosphatases (DSPs) play important roles in the regulation of mitogenic signal transduction and cell cycle control in response to extracellular stimuli. Dual-specificity phosphatase 18 (DUSP18), a newly recognized SAPK/JNK phosphatase, is widely expressed. This expression is modulated in response to extracellular stimuli. By phosphorylation assay, pull down and coimmunoprecipitation experiments, it is shown here that DUSP18 interacts with SAPK/JNK and dephosphorylates it both in vitro and in vivo. DUSP18 does not dephosphorylate p38 or p44ERK1. Furthermore, DUSP18 inhibits SAPK/JNK pathway in vivo. Based on these findings, DUSP18 appears to serve an important role by regulation of SAPK/JNK pathway.

LNX (Ligand of Numb-protein X) interacts with RhoC, both of which regulate AP-1-mediated transcriptional activation

LNX (Ligand of Numb-protein X) was originally isolated as a binding partner to the cell-fate Determinant Numb during development, and then identified to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of Numb. LNX contains 4 PDZ domains which are proved to play a central role in organizing diverse cell signaling assemblies. A yeast two-hybrid screening was used to identify LNX as a potential binding partner for RhoC. RhoC, a member of the Ras family of small GTPases, promotes reorganization of the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between LNX and RhoC in mammalian cells was identified by co-immunoprecipitation assays, and the efficient binding required the first PDZ domain of LNX. LNX and RhoC were further colocalized with each other in mammalian cells, in which RhoC changed its sublocalization from cytoplasm to nucleus when co-transferred with LNX. Furthermore, co-expression of RhoC reduced the transcriptional activity of AP-1, which was up-regulated by over-expression of LNX alone. These results suggest that LNX and RhoC might be part of a larger protein complex that would have important functions in signaling transduction about regulating the transcriptional activities of AP-1.

Cellular Retinaldehyde-Binding Protein-Like (CRALBPL), a Novel Human Sec14p-Like Gene That Is Upregulated in Human Hepatocellular Carcinomas, May Be Used as a Marker for Human Hepatocellular Carcinomas

Sec14p-like lipid-binding domain (SEC14 domain) is an evolutionarily conserved protein domain often found in secretory proteins, such as Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p, and in lipid-regulated proteins, such as GTPase-activating proteins, guanine nucleotide exchange factors, and neurofibromin. We have cloned a novel human gene, cellular retinaldehyde-binding protein-like (CRALBPL), containing SEC14 domain from the cDNA library of human fetal brain. The RT-PCR expression pattern of 16 adult human tissues indicated that CRALBPL was only expressed in brain, while it was expressed in all of seven human carcinoma cell lines we used, especially in human gastric adenocarcinoma cell line, human rhabdomyoma cell line, human hepatocellular carcinoma (HCC) cell line, and human prostatic carcinoma cell line. Further, we found that CRALBPL has a remarkably more abundant RT-PCR expression pattern in human HCC cell lines than in normal human liver cell line, and the same result was gained when RT-PCR expression patterns between human HCC specimens and normal human liver specimens were compared. We also found that CRALBPL is located mainly in cytoplasm in human liver cell line L-02, which is consistent with the common function of Sec14p-like domain family. Our results show that CRALBPL may be used as a marker for human HCCs.

Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2

This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56–1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney.

[Identification of a novel human MAST4 gene, a new member of the microtubule associated serine-threonine kinase family].

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here, we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64, 63, 59, and 39% identical amino acid residues with MAST1, MAST2, MAST3, and MASTL, respectively. RT-PCR analysis revealed a relatively high expression level of MAST4 in most normal human tissues, with the exception of in testis, small intestine, colon, and peripheral blood leukocyte.

A global genomic view on LNX siRNA-mediated cell cycle arrest

LNX protein is the first described PDZ domain-containing member of the RING finger-type E3 ubiquitin ligase family. Studies have approved that LNX could participate in signal transduction, such as Notch pathway, and play an important role in tumorigenesis. In this study, we found that down-regulation of LNX resulted in G0/G1 cell cycle arrest in G0/G1 phase in HEK293 cells. To explore the molecular mechanism of this phenomenon, we employed expression microarray to comparatively analyze the genome-wide expression between the LNX-knockdown cells and the normal cells. We also used quantitative real-time PCR to further confirm the differential expression patterns of 25 transcripts involved in cell cycle. Combined with known information about genic functions, signal pathways and cell cycle machinery, we analyzed the role of endogenous LNX in cell cycle. The results suggest that down-regulation of LNX could result in cell cycle arrest in G0/G1 phase through inhibition of β-catenin, MAPK, NFκB, c-Myc-dependent pathway and activation of p53, TGF-β-dependent pathway. This study provides new perspectives on LNX’s pleiotropic activities, especially its essential role in cell proliferation and cell cycle.

A Novel Zinc Finger Gene ZNF468 with Two Co-Expressional Splice Variants, ZNF468.1 and ZNF468.2

A novel human zinc finger protein encoding gene ZNF468 was obtained from a fetal brain cDNA library. By BLAST-N analysis we found two different splice variants. We termed the two splice variants ZNF468.1 and ZNF468.2. By BLAST search against the human genome database, ZNF468 was mapped to 19q13.4. The ZNF468.1 cDNA has four exons, and the ZNF468.2 cDNA has one more, between the third and fourth exon. This extra exon creates a difference between the deduced protein N-termini of the two splice variants. The ZNF468.1 cDNA is 3906 bp in length, encoding a 522a a protein, and ZNF468.2 is 4024 bp, encoding a 469-aa-protein. Both proteins contain 11 C2H2-type zinc finger motifs at their C-termini. The N-terminus of the deduced protein of ZNF468.1 has a well-conserved Krüppel-associated box (KRAB) domain that consists of KRAB boxes A and B, whereas the protein of ZNF468.2 does not have the {KRAB} domain. Tissue distribution of the ZNF468 gene indicates that the two splice variants are widely expressed in normal human tissues, except in heart and brain, and they are also co-expressional.

Gene expression profile of NFκB repressing factor (NKRF) knockdown cells by microarray analysis

Human NFB repressing factor (NKRF) is a negative regulation transcription factor, which is able to repress transcription by binding to the negative regulatory element (NRE) near the NFκB binding site in certain genes’ promoters. Current researches reveals that NKRF represses the activation of IFN-, IL-8, hiNOS and HIV-1 by NFκB. We used optical fiber beadchip to analysis the different gene expression patterns of RNAi mediated NKRF knockdown HEK293 cells and found that several genes showed significant change of expression levels. Real-time PCR was performed to verify the changes of expression of candidate genes. We analyzed the function of candidate genes by searching the gene ontology databases and publications and revealed that these genes functioned in cell cycle, cell proliferation, apoptosis, cell migration, DNA repair, transcription, metabolism, response to stimulus and signal transduction. This study provides new perspectives on NKRF’s potential multiple functions.