Zadorozny Eva V, Little Jamie C, Kalderon Daniel
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Department of Biological Sciences, Columbia University, New York, NY 10027, USA
Development. 2015 Mar 1;142(5):931-42. doi: 10.1242/dev.112904. Epub 2015 Jan 29.
The Drosophila kinesin-family protein Costal 2 (Cos2) and its mammalian ortholog Kif7 play dual roles in Hedgehog (Hh) signaling. In the absence of Hh, Cos2 and Kif7 contribute to proteolytic processing and silencing of the Hh-regulated transcription factors, Drosophila Cubitus interruptus (Ci) and mammalian Gli proteins. Cos2 and Kif7 are also necessary for full activation of full-length Ci-155 and Gli transcription factors in response to Hh proteins. Here, we use classical fused alleles and transgenic Cos2 products deficient for Fused (Fu) association to show that Cos2 must bind to Fu to support efficient Ci-155 processing. Residual Ci-155 processing in the absence of Cos2-Fu interaction did not require Suppressor of Fused, which has been implicated in processing mammalian Gli proteins. We also provide evidence that Cos2 binding to the CORD domain of Ci-155 contributes to both Ci-155 processing and Ci-155 silencing in the absence of Hh. In the presence of Hh, Ci-155 processing is blocked and Cos2 now promotes activation of Ci-155, which requires Fu kinase activity. Here, we show that normal Ci-155 activation by Hh requires Cos2 binding to Fu, supporting the hypothesis that Cos2 mediates the apposition of Fu molecules suitable for cross-phosphorylation and consequent full activation of Fu kinase. We also find that phosphorylation of Cos2 by Fu at two previously mapped sites, S572 and S931, which is thought to mediate Ci-155 activation, is not required for normal activation of Ci-155 by Hh or by activated Fu.
果蝇驱动蛋白家族蛋白Costal 2（Cos2）及其哺乳动物同源物Kif7在刺猬信号通路（Hh）中发挥双重作用。在没有Hh的情况下，Cos2和Kif7有助于Hh调节的转录因子——果蝇分节异常（Ci）和哺乳动物Gli蛋白的蛋白水解加工和沉默。Cos2和Kif7对于全长Ci-155和Gli转录因子响应Hh蛋白的完全激活也是必需的。在这里，我们使用经典的融合等位基因和缺乏与融合蛋白（Fu）结合的转基因Cos2产物，以表明Cos2必须与Fu结合才能支持有效的Ci-155加工。在没有Cos2-Fu相互作用的情况下，残留的Ci-155加工不需要融合抑制因子，而融合抑制因子与哺乳动物Gli蛋白的加工有关。我们还提供证据表明，在没有Hh的情况下，Cos2与Ci-155的CORD结构域结合有助于Ci-155的加工和Ci-155的沉默。在有Hh的情况下，Ci-155的加工被阻断，Cos2现在促进Ci-155的激活，这需要Fu激酶活性。在这里，我们表明Hh对Ci-155的正常激活需要Cos2与Fu结合，支持了Cos2介导适合交叉磷酸化的Fu分子并列从而使Fu激酶完全激活的假说。我们还发现，Fu在两个先前定位的位点S572和S931对Cos2的磷酸化，被认为介导Ci-155的激活，但Hh或激活的Fu对Ci-155的正常激活并不需要这种磷酸化。
