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KMID : 0390320080180020458
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Chungbuk Medical Journal
2008 Volume.18 No. 2 p.458 ~ p.465
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Phosphorylation of Amphiphysin 2 in PC12 Cell Line
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Kim Seung-Ryul
Lee Sang-Jin
Jin Yulian
Seong Nak-Kyund
Kim Kyung-Yong
Kim Young-Chul
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Abstract
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PURPOSE: This study was designed to investigate the pathways for phosphorylation of amphiphysin-2 in PC12 cells.
MATERIALS and METHODS: To explore the phosphorylation of amphiphysin-2, PC12 cells were treated with various growth factors (100 ng/ml EGF, 50 ng/ml NGF, 10 ng/ml bFGF, 10 uM insulin and 50 uM PDGF). Phosphorylation and dephosphoeylation of amphiphysin-2 were determined by alteration in the mobility shift on SDS-PAGE. To study the phosphorylation of amphiphysin-2, various protein kinase inhibitors were used. Western blot and immumoprecipitation analyses were employed to identify the proteins interacting with amphiphysin-2.
RESULTS: bFGF, NGF, and EGF all induced the phosphorylation of endogenous amphiphysin-2 as determined by the mobility shift on electrophoresis. Interestingly, PD98059, a specific MEK inhibitor, blocked the mobility shift of amphiphysin-2, suggesting the involvement of the Ras/Erk cascade. LY294002 and wortmannin, PI 3-kinase inhibitors, did not exert any effect on the phosphorylation of amphiphysin-2. The pretreatment of KCl decreased bFGF or PMA-induced phosphorylation of amphiphysin-2, and this effect was inhibited by cyclosporin A, a specific inhibitor of calcineurin. Staurosporine blocked the phosphorylation of amphiphyisn-2 in response to bFGF and PMA, but GF109203x was effective only on PMA-induced phosphorylation of amphiphysin-2. Finally, the immunoprecipitation analysis showed that the interaction of amphiphysin-2 with unknown protein is dependent on the phosphorylation status of amphiphysin-2.
CONCLUSIONS: bFGF-induced phosphorylation of amphiphysin-2 in PC12 cells is mediated via the Ras/MEK/Erk cascade. Calcineurin is considered a potential phosphatase to dephosphorylate phospho-amphiphysin-2. Interaction of amphiphysin-2 with clathrin and dynamin is dependent on the phosphorylation in PC12 cells.
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KEYWORD
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amphiphysin, phosphorylation, dephosphorylation, Ras/MEK/ERK signal pathway, bFGF
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