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. 2016 Dec 1;27(24):3841–3854. doi: 10.1091/mbc.E16-06-0379

FIGURE 5:

FIGURE 5:

S54 and S334 are PKA target sites in vitro and in vivo. (A) Sequence alignment of conserved amino acid within the N-terminus and the C-terminus between TIS11b, TIS11d, and TTP showing PKA consensus motifs (highlighted in red, RRHS and RLS). These motifs are also conserved between species in TIS11b sequence and harbor S54 S334 (hs, Homo sapiens; Bt, Bos taurus; Rt, Rattus norvegicus; mm, Mus musculus; xl, Xenopus laevis). (B) Dose-dependent in vitro phosphorylation of synthetic N-terminal and C-terminal peptides of TIS11b by the catalytic subunit of PKA. Both peptides contain the PKA consensus motifs RRHS or RLS (S54 and S334 are shown in bold; aa, amino acids). Phosphorylated peptides were resolved by SDS–PAGE (15%) and visualized by autoradiography. (C) In vitro phosphorylation of recombinant Flag-WT TIS11b and Flag-TIS11b mutants S54A and S334A (1 μg purified proteins). Protein extracts from E. coli transformed with empty vector (pET15b) served as control (Vect). (D) PKA-mediated phosphorylation of recombinant TIS11b was significantly impaired when S54 and S334 were replaced by an alanine. Ratios of phosphorylated protein/total protein are reported (n = 4 independent experiments, mean ± SEM). Asterisks: significantly different from the WT with **p < 0.01 and ***p < 0.001. (E) Characterization of the phosphospecific antibodies in vitro. Unphosphorylated control peptides were run alongside phosphorylated peptides to determine whether the antibodies could detect the phospho-S34 (pS54) or phospho-S334 (pS334). (F) Characterization of the phosphospecific antibodies in forskolin-stimulated cells. COS7 cells were transfected with empty pTarget Vector (Vect), pTarget-WT TIS11b (WT), pTarget-TIS11b-S54A, or pTarget-TIS11b-S334A plasmids and then stimulated or not (basal) with 25 μM of forskolin for 60 min in the presence or absence of H89. Cell lysates were analyzed by Western blot to assess the specificity of the anti-pS54 or anti-pS334 antibodies. Blots were imaged simultaneously with the Chemidoc Imaging system for 5 s to accurately detect strong and weak bands for all conditions.