Department of Physiology I, University of Tübingen, Germany.
J Membr Biol. 2010 Feb;233(1-3):35-41. doi: 10.1007/s00232-009-9222-0. Epub 2009 Dec 30.
The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca(2+) channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active (S422D)SGK1, constitutively active (T308D,S473D)PKB, wild-type PIKfyve, and (S318A)PIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I(Ca)) resulting from TRPV6-induced Ca(2+) entry and subsequent activation of Ca(2+)-sensitive endogenous Cl(-) channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I(H) was increased by coexpression of (S422D)SGK1 and by (T308D,S473D)PKB. Coexpression of wild-type PIKfyve further increased I(H) in TRPV6 + (S422D)SGK1-expressing oocytes but did not significantly modify I(Ca) in oocytes expressing TRPV6 alone. (S318A)PIKfyve failed to significantly modify I(Ca) in the presence and absence of (S422D)SGK1. (S422D)SGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.
血清和糖皮质激素诱导的激酶 SGK1 和蛋白激酶 PKB/Akt 可能会磷酸化并激活哺乳动物磷脂酰肌醇-3-磷酸-5-激酶 PIKfyve（PIP5K3），PIKfyve 反过来又被证明可以调节转运体和通道。SGK1 调节的通道包括 Ca(2+)通道 TRPV6，它在多种上皮细胞和非上皮细胞中表达，包括肿瘤细胞。SGK1 和蛋白激酶 B PKB/Akt 促进肿瘤生长。因此，本研究探讨了 TRPV6 是否受 PIKfyve 调节。TRPV6 在非洲爪蟾卵母细胞中表达，无论是否额外共表达组成性激活的 (S422D)SGK1、组成性激活的 (T308D,S473D)PKB、野生型 PIKfyve 和缺乏 SGK1 磷酸化位点的 (S318A)PIKfyve。通过 TRPV6 诱导的 Ca(2+)内流和随后激活 Ca(2+)敏感的内源性 Cl(-)通道产生的电流 (I(Ca)) 来确定 TRPV6 的活性。利用免疫组织化学和 Western blot 测定细胞膜中 TRPV6 蛋白的丰度。在表达 TRPV6 的卵母细胞中，(S422D)SGK1 和 (T308D,S473D)PKB 的共表达增加了 I(H)。野生型 PIKfyve 的共表达进一步增加了在表达 (S422D)SGK1 的 TRPV6 卵母细胞中的 I(H)，但在单独表达 TRPV6 的卵母细胞中 I(Ca)没有显著改变。(S318A)PIKfyve 在存在和不存在 (S422D)SGK1 的情况下均未能显著改变 I(Ca)。(S422D)SGK1 增加了细胞膜中 TRPV6 蛋白的丰度，这种效应在额外表达野生型 PIKfyve 时增强。我们得出结论，PIKfyve 参与 TRPV6 的调节。
