Fig. 1: SMO binds PKA-C as a pseudosubstrate.
a, CLUSTAL alignment of the mouse SMO pCT with the PKIɑ pseudosubstrate region. Additional PKA-C pseudosubstrate and substrate sequences are provided for comparison32,65,98. P-site is yellow; other key conserved residues are green. Spiral cartoon above alignment indicates predicted SMO helical region. Standard (615–638) and extended (615–652) SMO peptides used for in vitro assays are colored red or black, respectively. Inset, structure of PKA-Cɑ bound to PKIɑ(5–24) (PDB: 3FJQ), with ATP colored orange and key PKI residues colored as described above. b, Top, fluorescence polarization assay employing FAM-labeled SMO peptide, 1 mM ATP, and varying concentrations of human PKA-Cɑ. Points represent the mean from two separate experiments composed of quadruplicates. Bottom, the same assay except with 3 μM PKA-Cɑ and varying concentrations of ATP. Quadruplicates representative of two independent trials are plotted. c, Overlay of purified mouse PKA-Cɑ onto an array of SMO peptides containing the indicated substitutions in the P−13, P−3, and P−2 positions. d, SPR sensorgram for binding of PKA-Cɑ, in concentrations ranging from 5 nM to 2.5 μM, to GST-tagged wild-type SMO pCT. Buffer contained 1 mM ATP and 10 mM MgCl2. e, As d, but with SMO pCT harboring the WRR mutation. Note that the negative signal results from the subtraction of the non-specific binding of PKA-Cɑ to a GST control surface.