The Role of Costal2 and its Collaborators in Regulation of Ci Processing and in Mediation of Response to Hedgehog in Drosophila

Hedgehog (Hh) family proteins specify many cell fates in flies and mammals that depend on Hh concentration. Hh achieves differential expression of target genes by regulating the activity and stability of Cubitus interruptus (Ci) / Glioblastoma (Ci / Gli) transcription factors. Both modes of regulation require kinesin-like protein Costal2 (Cos2) (Kif7 in mammals) that acts in a complex with Fused (Fu) kinase. We used a group of fused (fu) alleles with truncations in the Fu-regulatory domain to confirm the importance of physical association between Cos2 and Fu for processing of Ci-155 to Ci-75 repressor in the absence of Hh. By specifically disrupting Cos2 interaction with Fu using Cos2deltaFu transgenes, we confirmed the importance of this interaction for stability of the Fu protein and for Ci processing when the transgene was expressed at natural levels (under control of genomic regulatory sequences). It is possible that Cos2/Fu interaction helps recruit processing-promoting kinases to Ci. We found no evidence for the importance of Suppressor of fused (Su(fu)), a protein known to limit Ci activity, in regulating Ci processing. Using a group of Cos2 variants under the control of genomic sequences, we found that expression of the wild type genomic Cos2 transgene (gCos2) or gCos2 deficient in Fu-dependent phosphorylation of Ser572 and Ser931 rescued animals to adulthood. Contrary to past observations of over-expressed phosphorylation variants, these gCos2 transgenes supported normal responses to Hh at the anterior-poster (AP) border of wing discs, a region of active Hh signaling. As shown previously, Ser572 had a role in stabilization of Ci-155 by activated Fu kinase. gCos2deltaFu greatly reduced the response to Hh and was also unable to rescue animal viability. The transgene allowed for induction of ptc-lacZ in response to activated Fu (GAP-Fu) but did not respond to activated Smo (SmoD123), suggesting that it fails to mediate Fu activation in response to activated Smo . Normal accumulation of Smo in the regions of active Hh signaling in cos2-null wing discs expressing Cos2deltaFu also suggested that Cos2deltaFu does not affect the accumulation component of Smo activation. We propose that Cos2deltaFu is defective in promoting Fu activation through cross-phosphorylation of Fu molecules that requires physical interaction with Cos2. Similarly, a gCos2-S182N variant with impaired binding to Ci failed to rescue animal viability and was defective in Ci processing, but gCos2-S182N supported almost normal responses to Hh at the AP border. Ectopic induction of ptc-lacZ in anterior cos2 clones with gCos2-S182N indicated a defect in limiting Ci activity possibly through physical interaction.