Fig. 1
From: Phosphorylation of Bok at Ser-8 blocks its ability to suppress IP3R-mediated calcium mobilization
Bok is phosphorylated by PKA at Ser-8 in vitro and in vivo. (A) Amino acid sequences of mouse (m) and human (h) Bok (accession numbers O35425 and Q9UMX3, respectively) with amino acid differences labeled with asterisks. The PKA consensus sequence, RRSS, is highlighted in yellow and Ser-8 is labeled with a red dot. The epitopes for anti-BokA (BokA) and anti-BokB (BokB), as well as the approximate epitope for anti-BokC (BokC) are indicated. In mammalian cells, Bok can be expressed as two forms: full-length Bok and varying levels of a shorter version, which results from alternative translation initiation at Met-15 (highlighted in blue). These forms migrate at 23/21 kDa in mouse cells and 22/20 kDa in human cells [8]. BokA recognizes both forms, while BokB preferably recognizes the full-length form. BokC only recognizes human Bok, most likely due to amino acid differences between mouse and human Bok in the BokC epitope region. (B) Left panel, Coomassie blue stain of purified, bacterially expressed HS-BokΔTM incubated without or with the catalytic subunit of PKA (40 kDa). HS-BokΔTM migrates at 36 kDa and a cleavage fragment (BokΔTM) migrates at 18 kDa. Right panel, immunoblot of the same samples probed with BokA and a PKA substrate antibody (RRXpS), with a contaminating phospho-protein at ~ 125 kDa in lane 6 labeled with an asterisk. (C) Immunoblots of lysates or immunoprecipitated Bok (using BokB or IP3R1) or PKA substrates (using RRXpS) from WT αT3 cells treated with 100 nM CalA and/or 20 µM Fsk for 10 min, probed for the proteins indicated (lanes 1–4). BKO αT3 cells treated with CalA and Fsk (lane 5) and each IP antibody only (lane 6) are negative controls, with a non-specific, phospho-protein at 30 kDa in the BokB IP labeled with an asterisk. (D) Immunoprecipitated Bok (using BokB) from WT αT3 cells, treated as in Fig. 1C, lane 4, was incubated with 0.25 µM of PP1 and/or PP2A for 30 min at 37 °C. Samples were probed for the proteins indicated, with untreated (lane 5) and CalA and Fsk-treated (lane 6) BKO αT3 cells serving as negative controls. The 30 kDa phospho-protein, present in both WT and BKO cells (lanes 1 and 6, asterisk), is also dephosphorylated by the protein phosphatases (lanes 2–4) and serves as a control for the methodology. (E) Summary of MS data from HS-BokΔTM phosphorylated by PKA in vitro and Bok purified via co-IP with IP3R1 from CalA and Fsk-treated WT αT3 cells