TI - Hedgehog-stimulated phosphorylation of the kinesin-related protein Costal2 is mediated by the serine/threonine kinase fused . AB - The Hedgehog ( Hh ) signaling molecule is required for the development of numerous tissues in Drosophila . Within the cell , Hh signal transduction utilizes a large protein complex consisting of the Fused ( Fu ) , Costal2 ( Cos2 ) , and Cubitis interruptus ( Ci ) proteins , but the functional interactions between these proteins are still largely uncharacterized . Using a baculovirus system , we demonstrate that the serine/threonine kinase Fu phosphorylates the kinesin-like protein Cos2 when coexpressed with Cos2 . Coexpression of Cos2 and a kinase -inactive version of Fu eliminates the majority of Cos2 phosphorylation . We then show that the primary Fu -induced phosphorylation site of Cos2 is serine 572 , whereas serine 931 is phosphorylated to a lesser extent . Mutation of serine 572 to alanine eliminates most , but not all , specific phosphopeptides of Cos2 when coexpressed with Fu . We also demonstrate that the phosphorylation pattern of Cos2 produced by baculovirus coexpression with kinase -dead Fu is almost identical to the phosphorylation pattern of Cos2 isolated from unstimulated S2 cells . Finally , the phosphorylation pattern of Cos2 produced by baculovirus coinfection with wild-type Fu is almost identical to that of Cos2 isolated from S2 cells stimulated by Hh , indicating that phosphorylation of serines 572 and 931 is a genuine Hh signaling event . This study clarifies the unique functions of Fu and Cos2 in Hh signal transduction and identifies only the second known phosphorylation site of a kinesin-like molecule .