Fig 2: 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

Fig 3: IP3R1-dependent ER Ca2+ leak is suppressed by endogenous Bok. (A) WT, BKO, and IP3R1 KO αT3 cells were transfected to express R-CEPIAer with representative images of R-CEPIAer fluorescence shown. (B) Left, [Ca2+]ER (F/F0) in R-CEPIAer expressing cells exposed to 30 nM GnRH, added at t = 60 s. The lack of response to GnRH in IP3R1 KO cells (dotted line) shows that decreases in [Ca2+]ER are due to endogenous IP3R1 expression. Right, minimal F/F0 values (Fmin) (mean ± SEM, n = 5, ns = not significant, p > 0.05). (C) Left, Post-minimal recovery in [Ca2+]ER graphed as F/Fmin. Right, post-minimal area under the curve (AUC) and time to F/Fmin = 1.3 (mean ± SEM, n = 5, ** designates p < 0.005). (D–F) Parallel analysis of BKO αT3 cells stably transfected to express BokWT, with vector as a control (mean ± SEM, n = 4, ** designates p < 0.005, ns = not significant, p > 0.05). (G) [Ca2+]ER (F/F0) in R-CEPIAer expressing cells exposed to 30 nM GnRH or vehicle, added at t = 60 s, and 1 µM Tg, added during the recovery phase of [Ca2+]ER at t = 300 s (when [Ca2+]ER partially recovered). (H) Left, decline in [Ca2+]ER after 1 µM Tg addition normalized to Ft=300 and graphed as F/Ft=300. Right, rate of ER Ca2+ leak graphed as time to F/Ft=300 = 0.6 (mean ± SEM, n = 6, ** and *** designates p < 0.005 and P < 0.0005, respectively, ns = not significant, p > 0.05). (I–J) Parallel analysis of BKO cells stably transfected to express BokWT, with vector as a control (mean ± SEM, n = 5, * designates p < 0.05, ns = not significant, p > 0.05)

Fig 4: Phosphorylation of endogenous Bok, measured through changes in Bok immunoreactivity, is triggered by GPCR activation. (A) HeLa, SH-SY5Y, and αT3 cells were treated with DMSO, 100 nM CalA and/or 20 µM Fsk for 10 min and lysates were probed in immunoblots as indicated. HeLa BKO cells serve as a negative control for the Bok antibodies, probes for Mcl-1 and phospho-proteins serve as controls for the methodology, and p97 serves as a loading control. Histograms show Bok immunoreactivity seen with BokA, BokB, and BokC, normalized to DMSO-treated cells (mean ± SEM, n = 5, * and ** designates p < 0.05 and p < 0.005 compared to DMSO-treated cells, respectively). (B) HeLa cells were treated with 10 µM isoproterenol, and SH-SY5Y and αT3 cells were treated with 100 nM PACAP for the times indicated. Lysates were probed in immunoblots as indicated, with Mcl-1 and phospho-proteins serving as controls for the methodology, and p97 serves as a loading control. Graphs show Bok immunoreactivity seen with BokA, BokB, and BokC, normalized to 0 min levels (mean ± SEM, n = 5, * and ** designates p < 0.05 and p < 0.005 compared to 0 min levels, respectively)

Fig 5: Bok phosphorylation at Ser-8 slows the post-maximal decline in exogenous IP3R1-mediated [Ca2+]C responses in HEK-3KO cells. (A) HEK-3KO cells were transfected to express IP3R1HAWT (2 µg cDNA) and either BokWT, BokS8A, or BokS8E (0.25 µg cDNAs). Lysates were probed in immunoblots as indicated, with p97 serving as a loading control. (B) [Ca2+]C (F/F0) in transfected HEK-3KO cells exposed to 100 µM CCh, added at t = 0. Cal6 fluorescence intensity (F) was normalized to the basal (initial) F value (F0) and graphed as F/F0. (C) Maximal F/F0 values (Fmax) (mean ± SEM, n = 5, ns = not significant, p > 0.05). (D) Post-maximal decline in [Ca2+]C graphed as F/Fmax. (E) and (F) Post-maximal area under the curve (AUC) and time to F/Fmax = 0.6 (mean ± SEM, n = 5, * designates p < 0.05, ns = not significant, p > 0.05). (G–K) Parallel analysis of cells pretreated with 1 µM PGE1 for 2.5 min prior to CCh addition (mean ± SEM, n = 5, * designates p < 0.05, ns = not significant, p > 0.05)