Expression and cell transformation activity of dynactin‐associated protein isoforms. Issue 8 (16th July 2021)

• Record Type: Journal Article
• Authors: 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
• Is Part Of: FEBS open bio. Volume 11:Issue 8(2021)
• Page Start: 2110
• Page End: 2117
• Publication Date: 2021-07-16
• Subjects: alternative splicing -- cell transformation -- dynactin‐associated protein -- spheroid formation -- subcellular localization; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1002/2211-5463.13202
• Languages: English
• ISSNs: 2211-5463

Abstract

Abstract : 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 (NH2 ‐MVA in dynAPa and NH2 ‐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. Abstract : Human dynactin‐related protein dynAP and its splicing variants were found to have different amino‐terminal residues or to lack residues in the intracellular region. Spheroid formation showed that dynAPa has the highest cell transformation activity, which was significantly diminished in dynAPb and completely lost in dynAPc. The results will assist future identification of the amino acid residues required for cell transformation activity.