2. Academic Validation
  3. Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis

Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis

  • Biochem Biophys Res Commun. 2020 Dec 17;533(4):732-738. doi: 10.1016/j.bbrc.2020.08.093.
Kosuke Kasai 1,  Yoshiyuki Kuroda 2,  Yutaro Takabuchi 3,  Akihide Nitta 3,  Takashi Kobayashi 4,  Hiroyuki Nozaka 5,  Tomisato Miura 1,  Toshiya Nakamura 6
Affiliations

Affiliations

  • 1 Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan; Research Center for Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
  • 2 Division of Glyco-signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai, Miyagi, 981-8558, Japan.
  • 3 Department of Medical Technology, Hirosaki University School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
  • 4 Department of Glycotechnology, Center for Advanced Medical Research, Graduate School of Medicine, Hirosaki University, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
  • 5 Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
  • 6 Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan; Research Center for Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan. Electronic address: toshiyan@hirosaki-u.ac.jp.
PMID: 32993960 DOI: 10.1016/j.bbrc.2020.08.093
Abstract

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the Casein Kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.

Keywords

Casein kinase 1; Hyaluronan synthase 2; Phosphorylation.

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