Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.
Methods Mol Biol. 2022;2374:213-229. doi: 10.1007/978-1-0716-1701-4_19.
Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (Ci/Gli) into an activator form (Ci/Gli). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates Ci/Gli, sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of Ci/Gli. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.
刺猬 (Hh) 信号转导最终导致潜在转录因子 Cubitus interruptus (Ci)/Gli 从抑制形式 (Ci/Gli) 转变为激活形式 (Ci/Gli)。蛋白激酶 A(PKA)、糖原合成酶激酶 3 (GSK3) 和酪蛋白激酶 1 (CK1) 对 Ci/Gli 的蛋白水解加工至关重要，该过程会生成 Ci/Gli，而 Ci/Gli 的顺序磷酸化由 Fused (Fu)/Unc-51 样激酶 (Ulk) 家族激酶 Fu/Ulk3/Stk36 和 CK1 完成，有助于 Ci/Gli 的形成。Hh 刺激 Fu/Ulk3/Stk36 介导的 Ci/Gli 磷酸化，导致 Ci/Gli 与 Hh 通路抑制因子 Sufu 之间的相互作用发生改变。在这里，我们描述了体外和体内测定法，以确定 Fu/Ulk 家族激酶对 Ci/Gli 的磷酸化及其对 Hh 的调节。
