Phospho-CREB

FIGURE 26-8 Immunohistochemical localization of cAMP response element-binding protein (CREB) in rat hippocampal neurons. Using a polyclonal antibody which recognizes both CREB and phospho-CREB protein, it is apparent that CREB protein is enriched in the nucleus of pyramidal cells of the CA1 region of the hippocampus. Scale bar is 30 pm. (Courtesy of Dr Stephen Ginsberg, Department of Pathology, University of Pennsylvania.)... 

Clem, B. F., Hudson, E. A., and Clark, B. J. (2005). Cyclic adenosine 3 ,5 -monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter. Endocrinology 3, 1348-1356. 

Figure 6 Effect of hypoxia on the phosphorylation of Akt. PC12 cells were pretreated with either wortmannin (100 nM), a specific inhibitor of PI3-Kinase, or vehicle (0.01% DMSO) for 1 hr prior to exposure to either normoxia (21% O2) or hypoxia (5% O2) for 6 hr. Whole-cell lysates were then immunoblotted for either phospo-Akt (a), total Akt (b), phospho-GSK3 (c), phospho-CREB (d), or EPASl (e). Note that CREB phosphorylation and EPASl phosphorylation and accumulation persist in the presence of wortmannin.

Figure 7 Effect of hypoxia on CREB phosphorylation. PC12 cells were exposed to either normoxia or hypoxia (5% O2) for the indicated time. Whole-cell lysates were immunoblotted with an antibody to Ser phospho-CREB (a) or total (phosphorylation-state-independent) CREB (b).

Figure 8 CREB phosphorylation by hypoxia does not require Ca, PCK, RSK-2, MAPK, or p38. Cells were pretreated with various drugs or vehicle (0.1% dimethyl sulfoxide) as indicated. Cells were then exposed to either normoxia (C, 21% O2) or hypoxia (H, 5% O2) for 6 hr, and whole-cell lysates were immunoblotted with an antibody specific for Ser phospho-CREB. (a) Cells were preincubated for 40 min in serum-fi ee medium in the presence of Ca and vehicle (—) or in serum-iree medium formulated without Ca and supplemented with 1 mM EGTA-I-100 pM BAPTA-AM (-h). The medium was then replaced (minus drug or vehicle) and cells were exposed to either normoxia or hypoxia, (b) Cells were pretreated for 40 min in serum-free medium with either vehicle (—) or 20 pM chelerythrine chloride, an inhibitor of PKC (CHL -P), and exposed to either normoxia or hypoxia, (c) Cells were pretreated for 40 min in serum-fi ee medium with either vehicle (—) or 0.3 pM Ro 31-8220, an inhibitor of RSK and p70 S6 kinase (-P), and exposed to either normoxia or hypoxia, (d) Cells were pretreated for 40 min in serum-lree medium with either vehicle (—) or 50 pM PD098059, an inhibitor of MEKl and the downstream MAPKS (-P), and exposed to either normoxia or hypoxia, (e) Cells were pretreated for 1 hr in serum-fiee medium with either vehicle (—) or 10 nM rapamycin, an inhibitor of p70 S6 kinase (-P), and exposed to either normoxia or hypoxia, (f) Cells were pretreated for 1 hr in serum-fiee medium with either vehicle (—) or 20 pM SB203580, an inhibitor of p38a kinase and MAPKAP kinase (-P), and then exposed to either normoxia or hypoxia. In all of these experiments, hypoxia did not alter the total levels of CREB.

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