FIGURE 4:

ACTH induces a cAMP-dependent expression and phosphorylation of TIS11b. (A) BAC cells were preincubated in the absence or presence of H89 (5 μM) for 30 min before addition of 10 nM of ACTH for the indicated periods of time. TIS11b and VEGF protein levels of whole-cell extracts (20 μg) were analyzed by Western blot. The blot was subsequently probed with an anti–β-actin to assess equal loading of samples. (B–D) Quantification of TIS11b, VEGF mRNA, and protein levels from independent experiments (n = 5, means ± SEM). Protein-level values were normalized to actin and are expressed as percentage of control values at time 0 (unstimulated cells). VEGF mRNA levels were measured by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). (E) Time-course of TIS11b phosphorylation in BAC cells stimulated with 10 nM of ACTH in the presence of [³²P]orthophosphate and in the presence or absence of H89. TIS11b was immunoprecipitated (IP) from cell extracts, resolved by SDS–PAGE, and then visualized by autoradiography. One representative experiment of four is shown. (F) Quantification of phospho-TIS11b/total TIS11b ratio in ACTH-stimulated BAC cells (n = 4, means ± SEM). (G) Phosphorylation of recombinant TIS11b by the catalytic subunit of PKA. Purified GST-TIS11b fusion protein was produced as described previously (Ciais et al., 2004). Increasing doses of GST-TIS11b were subjected to in vitro phosphorylation as described in Materials and Methods. Protein extract from Escherichia coli (30 μg) transformed with empty pGEX vector was used as control in the phosphorylation assay (first lane, 0 μg).