FIGURE 5.

Phosphorylation of Ser¹⁹ modulates TPH2 expression. A, a Western blot from HEK293 cells expressing HA-TPH1, HA-TPH2, or the HA-TPH2-(1–41)-TPH1 chimera with and without treatment with 2.5 μm forskolin for 4 h demonstrates a PKA-mediated increase in TPH2 and TPH2-(1–41)-TPH1 expression with no appreciable effect on TPH1 expression. B, bar graph showing the result of n = 5 experiments. ^*, p < 0.05 compared with untreated control; ^**, p < 0.001 compared with untreated control. The data in graph B are normalized to neomycin expression and compared with their respective untreated controls. C, a Western blot from HEK293 cells expressing HA-TPH2(S19A) to remove the putative PKA phosphorylation site or S19D to create a pseudophosphorylated protein supports the hypothesis that phosphorylation of Ser¹⁹ leads to an increase in protein expression. The responses to forskolin treatment (described above) support this, since the pseudophosphorylated S19D construct is not appreciably affected by forskolin-mediated PKA activation, and the S19A response to forskolin is blunted compared with wild type TPH2. The graph shown in D represents n = 3 experiments. †, p < 0.05 compared with HA-TPH2; ‡, p < 0.01 compared with HA-TPH2. The data in graph D are normalized to neomycin expression and compared with untreated HA-TPH2 expression. E and F, to remove the possible confound of additional PKA phosphorylation sites, the S19A mutation was generated in the HA-(1–58)-GFP construct. Although the expression of the HA-(1–58)-GFP construct increased in response to the previously outlined forskolin treatment, expression of the S19A mutant remained unchanged. The results shown in F represent n = 3 experiments with treated samples normalized to neomycin and compared with the untreated partner sample. #, p < 0.01 compared with HA-(1–58)-GFP; %, p < 0.01 compared with S19A + forskolin. G, phosphorylation of a peptide corresponding to amino acids 10–20 and that same peptide with the S19A mutation confirm the status of Ser¹⁹ as a PKA phosphorylation site (n = 3). ^***, p < 0.01 compared with “no enzyme,” “no substrate,” and “S19A peptide” controls. H, in vitro translation performed on cDNA in a modified pcDNA vector shows that the S19D mutation does not dramatically alter TPH2 translational efficiency (n = 4). All results are means ± S.E.