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

Abstract

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.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused

“…Our results provide a new output for FU as they show that it is likely to upregulate the levels of SMO directly at the cell surface and its signaling activity by phosphorylation. It is noteworthy that the FU-dependent phosphorylation of COS2 has been recently shown not to be essential for its activity as SMO and COS2 interact (Zadorozny et al, 2015); it is thus possible that the effect of FU on COS2 activity could in fact occur mainly via SMO. Our work also reveals that the FU downstream activity [i.e.…”

Dual functions of Rack1 in regulating Hedgehog pathway

“…To date, the mechanisms by which Hh dissociates Ci-Cos2-kinases association and promotes Smo cell surface expression are still unclear. The previous studies have showed that Hh stimulates Cos2 differential phosphorylation on S572 and S931 [17], but the phosphorylation is not required for normal activation of Ci by Hh [18], suggesting that other mechanism is involved in this process.…”

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing

“…Previously, such experiments showed that activated Fu alone was sufficient to elicit strong Hh target gene induction, suggesting that Ci-155 activation can be effective even without the normal inhibition of processing that occurs at the AP border (Zhou and Kalderon, 2011). Fu kinase activity is not required for Hh to block Ci-155 processing at the AP border (Alves et al, 1998;Ohlmeyer and Kalderon, 1998;Zadorozny et al, 2015). Nevertheless, synthetically activated Fu was observed to increase Ci-155 levels in anterior clones and further tests suggested this likely resulted from partial inhibition of Ci-155 processing mediated by Cos2 phosphorylation (Zhou and Kalderon, 2011).…”