Molecular Cloning And Characterization Of Human ASB-8 Gene Encoding A Novel Member Of Ankyrin Repeat And SOCS Box Containing Protein Family

In the course of isolating novel genes possibly related to the development of cancer, a new member of human ankyrin repeat and SOCS box containing protein family (ASB), designed as ASB-8, was isolated from a human placenta cDNA library. After combination of 5( and 3( RACE, the full-length cDNA was isolated with 2,545 bp in length, a predicted open reading frame (ORF) encoding a protein of 288 amino acids, which was 96% identical to mouse ASB-8 protein. InterProScan analysis predicted that this protein had four ankyrin repeats of 131 amino acid residues located at middle portion and a SOCS box of 42 amino acids at C-terminus. It also shared 96% amino acid identity with mouse ASB-8. The RH mapping data obtained from the 93 RH clones in the GeneBridge 4 RH panel showed that human ASB-8 was located 12.33 cR from WI-3757 and 4.0 cR from WI-6377, which was mapped to chromosome 12q13, in the order cen-WI-3757-ASB-8-WI-6377. BLAST search with nucleotide acid sequences of ASB-8 cDNA against the unfinished high throughput genomic sequences (HTGS) database indicated the alignment of ASB-8 to one genomic sequence from chromosome 12 clone RP11-474C8. We examined the tissue mRNA expression pattern of ASB-8 by Northern blot and detected a transcript of ~ 2.6 Kb in human heart, brain, placenta, liver, skeletal muscle, kidney and pancreas. The results also showed a high level of expression in skeletal muscle, a varied level of expression in heart, brain, placenta, liver, kidney and pancreas. No expression was found in normal lung tissue. Furthermore, ASB-8 was found to be expressed in various tumor cell lines, including several lung cancer lines by using RT-PCR. To determine the subcellular localization of ASB-8, an expression vector ASB-8-EGFP was transiently transfected in BEL-7402 human hepatocellular carcinoma cells. The fusion protein was predominantly distributed in cytoplsm of BEL-7402 cancer cells. To investigate whether the ASB-8 protein could interact with Elongin B C complex, the Myc-His tagged ASB-8 , twoASB-8 mutants and the HA-tagged Elongin B and Elongin C were constructed. The ASB-8 mutant (pASB-8(SB) was a carboxy-terminal deletion mutant that lacked the entire SOCS box; the other ASB-8 mutant (pASB-8-M1) contained point mutations at position 249(T?N) and 250 (L?P) of the BC box. In vitro association and co-immunoprecipitation (Co-IP ) assay showed that ASB-8 could interact with Elongin B and C complex, and the interaction was dependent on the presence of an intact ASB-8 BC-box , since combination of point mutations at position 249(T?N) and position 250 (L?P), or deletion of the complete SOCS box could disrupt the interaction. The expression and biological effects of ASB-8 were also studied. Using immunohistochemical staining, we found that expression of ASB-8 protein was up-regulated in non-small cell lung carcinoma tissues when compared with the matched noncancerous tissues. The difference of ASB-8 expression between normal lung and lung carcinoma led us to hypothesize that ASB-8 might have a regulatory effect on the cell growth of lung cancer cell. The colony formation experiment showed that ASB-8(SB could reduce the numbers of colony formation of SPC-A1 cells, as compared with Neo-vector transfected cells. The stable transfetants of SPC-A1 lung cancer cell lines expressing ASB-8 or ASB-8(SB were also established by G418 selection, respectively. The results of growth curve showed significant growth inhibition observed in SPC-A1 cell line overexpressing ASB-8(SB when compared with Neo-vector transfected cells. However, the growth properties of ASB-8 -overexpressing cell line had no difference from that of Neo-vector transfected cells. In vivo tumorigenicity assay also showed that over-expression of ASB-8(SB could suppress the tumor growth. These results implied that this mutant might function as a dominant negative regulator to interfere with the function of intrinsic wild-type ASB-8. We assume that ASB-8 may play an important role in the development of lung cancer as a putative positive regulat...