Hedgehog (Hh) signaling is mediated by binding of Hh to the 12-transmembrane protein Patched (PTCH), which alleviates PTCH-mediated inhibition of the seven-transmembrane protein Smoothened (Smo). A member of the G protein–coupled receptor kinase (GRK) family has been implicated in Hh signaling through its phosphorylation of Smo. Jiang et al. report that GRK2 also has a kinase-independent function in Hh signaling through an interaction with PTCH. Human GRK2 and PTCH1 interacted in a yeast two-hybrid assay. The two proteins coimmunoprecipitated when heterologously expressed in cultured cells, and addition of an N-terminal fragment of Sonic hedgehog (ShhN) decreased this interaction. A peptide with GRK2 residues 262–379 (BP) was sufficient for the interaction, but kinase activity was not, because a kinase-dead mutant also bound PTCH1. When activated forms of cyclin B1 and PTCH1 were cotransfected into cells, PTCH1 sequestered cyclin B1 in the cytosol. Also, the addition of ShhN promoted the release of cyclin B1 from PTCH1 and its nuclear translocation. Knockdown of GRK2 prevented this ShhN-stimulated nuclear translocation of cyclin B1, and nuclear translocation was restored by expression of either the wild-type GRK2 or kinase-dead GRK2. When BP was overexpressed in PTCH1- and cyclin B1-transfected cells, the amount of cyclin B1 that coimmunoprecipitated with PTCH1 was decreased. ShhN triggered an increased coimmunoprecipitation between GRK2 and PTCH1 and reduced the coimmunoprecipitation of PTCH1 and cyclin B1. Transfection of PTCH1 inhibited cell proliferation, and this was alleviated by the coexpression of any GRK2 with the BP region or just BP. Zebrafish embryos in which GRK2 was knocked down exhibited defects related to disrupted Hh signaling and showed an early growth arrest phenotype likely related to decreased cell proliferation. The early growth arrest phenotype was rescued by GRK2, kinase-dead GRK2, BP, or cyclin B1. Thus, the authors proposed that GRK2 has two functions in Hh signaling, one as a kinase that phosphorylates Smo and one as a kinase-independent binding partner for PTCH1.
X. Jiang, P. Yang, L. Ma, Kinase activity-independent regulation of cyclin pathway by GRK2 is essential for zebrafish early development. Proc. Natl. Acad. Sci. U.S.A. 106, 10183–10188 (2009). [Abstract] [Full Text]