Open AccessExpression and cell transformation activity of dynactin‐associated protein isoforms
Author(s) -
Yin Xiaobo,
Yamada Shota,
Kobayashi Hiroaki,
Tanaka Ryota,
Togo Yuki,
Hosoi Miho,
Tsuchida Mie,
Kunoh Tatsuki,
Wada Shuichi,
Nakamura Toshinobu,
Sasaki Ryuzo,
Mizukami Tamio,
Hasegawa Makoto
Publication year - 2021
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.13202
Subject(s) - gene isoform , transmembrane protein , alternative splicing , biology , microbiology and biotechnology , transmembrane domain , subcellular localization , transformation (genetics) , hek 293 cells , cell culture , proximity ligation assay , golgi apparatus , protein isoform , cell , biochemistry , cytoplasm , gene , receptor , genetics
Overexpression of human dynactin‐associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single‐pass transmembrane protein with a carboxy‐terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino‐terminal residues (NH 2 ‐MVA in dynAPa and NH 2 ‐MEYQLL in dynAPc). DynAPb has the same amino‐terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy‐terminal region, including the transmembrane domain. Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki‐1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy‐terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U‐bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.