Calcium-dependent dephosphorylation of brain mitochondrial calcium/cAMP response element binding protein (CREB)

Abstract

Calcium-mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium-dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium-dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium-dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore-forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium-dependent phosphatase regulate its phosphorylation state.

Fig. 1

pCREB immunoreactivity in rat forebrain homogenates, isolated mitochondria. (a) Brain homogenates and isolated non-synaptosomal brain mitochondria were treated with lysis buffer. Samples (25 μg protein) were applied to each lane of the electrophoresis gel. Immunoblots for pCREB (top), the nuclear marker histone H3 (middle), and the mitochondrial inner membrane marker cytochrome c oxidase subunit I (COX, bottom) were performed. (b) Isolated non-synaptosomal brain mitochondria, mitoplasts and outer membrane fractions (25 μg protein) were electrophoretically separated then blotted for pCREB (top), COX (middle) and the intermembrane marker cytochrome c (Cyt c, bottom).

Fig. 2

pCREB and total CREB Immunoreactivity in brain mitochondria following incubation in the absence and presence of Ca²⁺. (a) Representative immunoblots of mitochondrial lysates probed for pCREB (upper panel), then stripped and re-probed for total CREB (lower panel). (b) Optical density of bands from immunoblots (Fig. 2a) of mitochondrial lysates exposed to 50 μM EGTA or Ca²⁺ (0.8–1.6 μmol/mg mitochondrial protein). Data are expressed as the ratio of the pCREB : CREB optical densities and are representative of four separate experiments. (c) pCREB (ng/mg mitochondrial protein) as assessed by ELISA following exposure of isolated mitochondria to EGTA (1–101 μM). The medium free [Ca²⁺] was determined from Fura 6F fluorescent measurements according to Grynkiewicz et al. (1985).

Fig. 3

Enrichment of pCREB by mitochondrial osmotic lysis. Mitochondria were incubated for 5 min in the presence of either EGTA (10 μM) or Ca²⁺ (20 μM) in the absence or presence of the pore-forming molecule alamethicin (80 μg/mL). Following centrifugation, mitochondrial pellets were analyzed for pCREB using ELISA. Data are expressed as the mean percentage of control values on each ELISA plate ± SE for n = 3–5 separate experiments. ⁺Significantly different (p < 0.01) from Ca²⁺ treatment alone; *significantly different (p < 0.05) from EGTA with alamethicin treatment.

Fig. 4

Calcium-induced pCREB dephosphorylation following EGTA-induced phosphorylation. Mean pCREB levels as assessed by ELISA following exposure of isolated mitochondria to EGTA (10 μM) or Ca²⁺ (20 μM) alone, or addition of Ca²⁺ after 5 min of EGTA treatment. Data are expressed as a percentage of the control values (EGTA) on each ELISA plate. Each bar is the mean of four to five separate experiments; error bars are SEM. *Significantly different (p < 0.05) from control.

Fig. 5

Effect of mitochondrial calcium uptake inhibition on pCREB. (a) Fura 6F fluorescent recordings of the ambient free [Ca²⁺] in the standard reaction medium containing isolated rat brain mitochondria (0.25 mg/mL), in the presence of the oxidizable substrates malate (5 mM) plus glutamate (5 mM) plus ATP (3 mM). The medium contained either EGTA (10 μM) or Ca²⁺ (20 μM) ± the mitochondrial Ca²⁺ uptake inhibitor RU360 (500 nM). (b) Typical immunoblot of pCREB in mitochondrial pellets obtained following centrifugation of suspensions as shown in (a), representative of three separate experiments.

Fig. 6

Effect of phosphatase inhibitors on mitochondrial pCREB. Mitochondria were incubated for 5 min in the presence of either EGTA (10 μM) or Ca²⁺ (20 μM) in the absence or presence of okadaic acid (10 nM) or FK506 (100 nM). Following centrifugation, mitochondrial pellets were analyzed for pCREB using ELISA. Data are expressed as the mean percentage of control values on each ELISA plate ± SE for n = 3–12 separate experiments.