Department of Biology, Hope College, Holland, MI, USA.
APMIS. 2011 Jul;119(7):421-30. doi: 10.1111/j.1600-0463.2011.02747.x.
VACM-1, a cul-5 gene product, functions via an E3 ligase complex and when overexpressed, has an antiproliferative effect in many cell types. Overexpression of VACM-/cul5 cDNA mutated at the PKA-specific phosphorylation site at Ser730 reversed this phenotype. These effects are associated with the appearance of larger M(r) species subsequently identified as a Nedd8-modified VACM-1/cul5. Although decreased levels of VACM-1 mRNA detected in several cancers and cancer cell lines may explain the progression of cell growth, possible genetic and epigenetic changes in its sequence have not been analyzed. We hypothesized that in rapidly proliferating cells, VACM-1/cul5 may be mutated at either the PKA-specific phosphorylation site or the consensus neddylation site. We used RT-PCR and PCR, to amplify and to sequence mRNA and genomic DNA, respectively. To date we have sequenced all 19 coding exons of the VACM-1/cul5 gene in T47D breast cancer cells, U138MG glioma cells, ACHN renal cancer cells, and OVCAR-3 ovarian cancer cells. Our results indicate that in those cells VACM-1/cul5 is not mutated at the putative phosphorylation or the neddylation site. We have found one silent mutation in the genomic DNA isolated from U138MG, ACHN, and OVCAR-3 cell lines, but not from T47D cells. Our work suggests that in T47D breast cancer cells biologic activity of VACM-1/cul5 may be regulated by posttranslational modifications.
VACM-1 是一个 cul-5 基因产物，通过 E3 连接酶复合物发挥作用，过量表达时，对许多细胞类型具有抗增殖作用。在 PKA 特异性磷酸化位点 Ser730 处突变的 VACM-/cul5 cDNA 的过表达逆转了这种表型。这些作用与随后鉴定为 Nedd8 修饰的 VACM-1/cul5 的更大分子量 (Mr) 物种的出现有关。尽管在几种癌症和癌细胞系中检测到 VACM-1 mRNA 水平降低，但可能解释细胞生长的进展，但尚未分析其序列中的可能遗传和表观遗传变化。我们假设在快速增殖的细胞中，VACM-1/cul5 可能在 PKA 特异性磷酸化位点或公认的 Neddylation 位点发生突变。我们使用 RT-PCR 和 PCR 分别扩增和测序 mRNA 和基因组 DNA。迄今为止，我们已经对 T47D 乳腺癌细胞、U138MG 神经胶质瘤细胞、ACHN 肾癌细胞和 OVCAR-3 卵巢癌细胞中的 VACM-1/cul5 基因的所有 19 个编码外显子进行了测序。我们的结果表明，在这些细胞中，VACM-1/cul5 未在假定的磷酸化或 Neddylation 位点发生突变。我们在从 U138MG、ACHN 和 OVCAR-3 细胞系中分离的基因组 DNA 中发现了一个沉默突变，但在 T47D 细胞中没有发现。我们的工作表明，在 T47D 乳腺癌细胞中，VACM-1/cul5 的生物学活性可能受到翻译后修饰的调节。
