Contributions of Costal 2-Fused interactions to Hedgehog signaling in Drosophila.

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.