Mutational analysis of VACM-1/cul5 exons in cancer cell lines
STEVEN P. LEWIS
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Contributed equally to the project.
Search for more papers by this authorANGELICA N. Willis
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Contributed equally to the project.
Search for more papers by this authorALYSSA E. JOHNSON
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Search for more papers by this authorMARIA A. BURNATOWSKA-HLEDIN
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Search for more papers by this authorSTEVEN P. LEWIS
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Contributed equally to the project.
Search for more papers by this authorANGELICA N. Willis
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Contributed equally to the project.
Search for more papers by this authorALYSSA E. JOHNSON
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Search for more papers by this authorMARIA A. BURNATOWSKA-HLEDIN
Department of Biology, Hope College, Holland, MI
Department of Chemistry, Hope College, Holland, MI
Search for more papers by this authorAbstract
Lewis SP, Willis AN, Johnson AE, Resau J, Burnatowska-Hledin MA. Mutational analysis of VACM-1/cul5 exons in cancer cell lines. APMIS 2011; 119: 421–30.
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 Mr 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.
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