RRC ID |
63076
|
著者 |
Kasai K, Kuroda Y, Takabuchi Y, Nitta A, Kobayashi T, Nozaka H, Miura T, Nakamura T.
|
タイトル |
Phosphorylation of Thr328 in hyaluronan synthase 2 is essential for hyaluronan synthesis.
|
ジャーナル |
Biochem Biophys Res Commun
|
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.
|
巻・号 |
533(4)
|
ページ |
732-738
|
公開日 |
2020-12-17
|
DOI |
10.1016/j.bbrc.2020.08.093
|
PII |
S0006-291X(20)31693-4
|
PMID |
32993960
|
MeSH |
Animals
COS Cells
Casein Kinase I / genetics
Chlorocebus aethiops
HEK293 Cells
Humans
Hyaluronan Synthases / genetics
Hyaluronan Synthases / metabolism*
Hyaluronic Acid / biosynthesis*
Hymecromone / pharmacology*
Mass Spectrometry
Mutagenesis, Site-Directed
Phosphorylation
Point Mutation
Protein Processing, Post-Translational
Tetradecanoylphorbol Acetate / pharmacology*
Up-Regulation
|
IF |
2.985
|
リソース情報 |
ヒト・動物細胞 |
COS-7(RCB0539) |