Evidence for the direct involvement of βTrCP in Gli3 protein processing
Evidence for the direct involvement of βTrCP in Gli3 protein processing
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DOI：
10.1073/pnas.0509927103
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发表时间：
2006-01-03
影响因子：
11.1
通讯作者：
Li, YY
中科院分区：
文献类型：
--
作者：
Wang, BW;Li, YY
果蝇中转录因子 cubitus interruptus (Ci) 的 Hedgehog 调节加工取决于 cAMP 依赖性蛋白激酶以及随后的酪蛋白激酶 1 和糖原合酶激酶 3 对 Ci C 末端区域的磷酸化。Ci 加工还需要 Slimb、SCF (Skp1/Cullin/F-box 蛋白) 复合物的 F-box 蛋白和蛋白酶体，但磷酸化与 Slimb 和 Slimb 活性之间的相互作用蛋白酶体仍不清楚。在这里，我们表明，Gli3 蛋白（Ci 的同源物）的加工也取决于一组四个 cAMP 依赖性蛋白激酶位点的磷酸化，这些位点引发相邻酪蛋白激酶 1 和糖原合酶激酶 3 的后续磷酸化。我们在培养细胞中的功能获得和丧失分析进一步表明，β TrCP（Slimb 的脊椎动物同源物）是 Gli3 加工所必需的，并且我们证明 β TrCP 可以结合磷酸化的 Gli3体外和体内。我们还发现 Gli3 蛋白在细胞中被多泛素化，并且其加工取决于蛋白酶体活性。我们的研究结果为 Gli3/Ci 蛋白磷酸化与 β TrCP/Slimb 作用之间的直接联系提供了证据，从而支持了 Gli3/Ci 加工受蛋白酶体影响的假设。
Hedgehog-regulated processing of the transcription factor cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by casein kinase 1 and glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. Here we show that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Our gain- and loss-of-function analyses in cultured cells further reveal that beta TrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and we demonstrate that beta TrCP can bind phosphorylated Gli3 both in vitro and in vivo. We also find that the Gli3 protein is polyubiquitinated in the cell and that its processing depends on proteasome activity. Our findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and beta TrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome.