Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing.
Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing.
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发表时间：
2006
影响因子：
11.1
通讯作者：
Baolin Wang;Yanyun Li
中科院分区：
文献类型：
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作者：
Baolin Wang;Yanyun Li
在果蝇中，刺猬调节的转录因子cubitus interruptus （Ci）的加工依赖于camp依赖性蛋白激酶对Ci c端区域的磷酸化，随后由酪蛋白激酶1和糖原合成酶激酶3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和蛋白酶体活性之间的相互作用尚不清楚。在这里，我们发现Gli3蛋白（Ci的同系物）的加工也依赖于一组四个camp依赖性蛋白激酶位点的磷酸化，这些位点随后会引发相邻的酪蛋白激酶1和糖原合成酶激酶3的磷酸化。我们对培养细胞的功能增益和功能丧失分析进一步表明，脊椎动物的sllimb同源物betaTrCP是Gli3加工所必需的，并且我们证明betaTrCP可以在体外和体内结合磷酸化的Gli3。我们还发现Gli3蛋白在细胞中多泛素化，其加工依赖于蛋白酶体的活性。我们的研究结果为Gli3/Ci蛋白磷酸化与betaTrCP/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 betaTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and we demonstrate that betaTrCP 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 betaTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome.