Fig 1

(A) Schematic of assay to report PKA activity toward soluble substrates. PKA-C phosphorylates CREB which binds CRE, inducing expression of luciferase. SMO can inhibit PKA-C by decreasing cAMP via inhibitory G proteins and AC (route “1”). Alternatively, SMO may inhibit PKA-C via a G protein–independent mechanism (route “2”). (B) Wild-type HEK293 cells were transfected with CRE-luciferase reporter plasmid and GFP (as a negative control) or PKA-C, either alone, with SMO674 (see cartoon above), or with a canonical Gα_i/o-coupled GPCR, M2AchR. Transfected cells were treated with the indicated drugs (vehicle control, M2AchR ligand carbachol (3 μM), or SMO inverse agonist KAADcyc (1 μM)). Following drug treatment, cells were lysed and luminescence measured. Note that transfected SMO is constitutively active in HEK293 cells because its inhibitor PTCH1 is present at minimal levels [32,33,41,42], whereas M2AchR requires carbachol for activity. For the sake of clarity, the SMO constructs utilized in each experiment are indicated in the corresponding figure panel. (See S1B Fig for additional information.) (C) Wild-type HEK293 cells were transfected with GFP (as a negative control) or PKA-C, either alone or with SMO674. Lysates were separated via SDS-PAGE and probed with antibodies against phospho-CREB (top) or total CREB (bottom). Molecular masses are in kDa. (D) HEK293 Gα-null cells were transfected with PKA-C, either alone or with SMO674, and treated with vehicle or KAADcyc (1 μM). All CREB reporter data are normalized to 100%, which reflects reporter activation from PKA-C-transfected cells treated with vehicle (n = 3 biological replicates per condition; error bars = SEM). The underlying data for this figure can be found under S1 Data. The uncropped westerns are included in S8 Data. See S1 Table for statistical analysis. AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic AMP; CRE, cyclic AMP response element; CREB, cyclic AMP response element binding protein; dCT, distal segment of the cytoplasmic tail; GPCR, G protein–coupled receptor; KAADcyc, KAAD-cyclopamine; M2AchR, M2 acetylcholine receptor; pCT, proximal segment of the cytoplasmic tail; PKA, protein kinase A; PKA-C, PKA catalytic subunits; SMO, Smoothened.