Fangyan Dai

Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues

Digestion and detoxification are the two most important functions of the liver, and liver cells always keep a high metabolism level and active vesicular traffic. The malfunction of the vesicular traffic system might be a cause of the abnormal biological behavior of cancerous liver cells. The Ras superfamily is known to regulate various steps of vesicular traffic in eukaryotic cells. It would be significant to determine the change of vesicular transport molecules such as the members of Ras superfamily in carcinogenesis of liver cells. In the present study, we have cloned nine novel genes encoding human small GTPases: RAB1B, RAB4B, RAB10, RAB22A, RAB24, RAB25 ARL5, SARA1, and SARA2, among which the former six belong to the RAB family and the latter three belong to the ARF/SAR1 family. The identification of these new genes has greatly enlarged the pool of the Ras superfamily. It is interesting to find that they are upregulated in most of the 11 hepatocellular carcinoma and 1 cholangiohepatoma cases. Furthermore, the expression in 16 normal human adult tissues, the chromosome loci, and the gene structures of the nine genes are also described. The above findings could be valuable for understanding the vesicular transport system and elucidating the molecular basis of liver cancer carcinogenesis.

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3β) at serine-9 through direct interaction

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.

Molecular cloning, genomic organization, and mapping of β4GalT-VIb, a brain abundant member of β4-galactosyltransferase gene family, to human chromosome 18q12.1

In the present study, a brain abundant member of g 4-galactosyltransferase gene family with an open reading frame encoding 343 amino acids was cloned and identified from a human leukemia cell cDNA library. The putative protein sequence is about 94.8 and 94.2% identical to the 382-amino-acid mouse and rat g 4-galactosyltransferase respectively and also contains cysteine residues previously shown to be important for the function of the gene family members. This cDNA (tentatively termed g 4GalT-VIb) is identical to a recently reported cDNA (tentatively termed g 4GalT-VIa) of human g 4-galactosyltransferase except lacking one exon, suggesting that these two cDNAs are two different alternative transcripts of the same gene. Northern hybridization showed that the new alternative transcript, g 4GalT-VIb, is expressed in all 16 human tissues tested with highest level in brain and rich level in testis, thymus and pancreas, whereas weak expression was detected in lung. The g 4GalT-VIb gene was located to human chromosome 18q12.1 between markers WI-9180 and SGC35630 by radiation hybrid mapping. The genomic organization and adjacent gene content of g 4GalT-VIb were identified by comparing its cDNA sequence with three genomic sequences AC017100, AP002474 and AP001336, which showed that g 4GalT-VIb spans an ~58 u kb region and is composed of 8 exons. In addition, the most conserved motif composed of 41 residues, LXYX 3 FGGVSXL(T/S)X 2 QFX 2 INGFPNX(Y/F)WGWGGEDDDX 2 NR, was defined according to 17 sequences of g 4GalTs from seven different organisms for the first time.

Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF A NOVEL HUMAN CDNA FRAGMENT ENCODING A PUTATIVE SER/THR PROTEIN KINASE

A novel full-length cDNA encoding a putative serine/threonine kinase has been isolated from a human testis cDNA library. A nucleotide sequence of 1225 bp length has been determined containing an open reading frame of 1 044 nucleotides (encoding 348 amino acids). In view of its degree of homology to members of the Ser/Thr protein kinase family and the closest relationship toMus musculus STK-1, the predicted product was designated by the name of HUMSTK-1. Its mRNA is present in large amounts in thymus, and small amounts in testis, small intestine and colon.