Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase Cγ (PLCγ) and phosphatidylinositol (PI) 3-kinase. While it is well established that tyrosine phosphorylation of PLCγ is necessary for its activation, we show here that PLCγ is regulated additionally by the lipid products of PI 3-kinase. We demonstrate that the pleckstrin homology (PH) domain of PLCγ binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P 3 ], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P 3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLCγ PH domain-mediated membrane targeting and PLCγ activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLCγ membrane targeting by overexpression of the PLCγ PH domain prevents growth factor-induced PLCγ activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

The mitogen-activated protein kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun Nterminal kinase), and p38 phosphorylate and activate transcription factors that promote proliferative and inf lammatory responses, whereas glucocorticoid receptor (GR) activation inhibits cell growth and inf lammation. We demonstrate that JNK and ERK but not p38 phosphorylate GR in vitro primarily at Ser-246. Selective activation of either ERK or JNK in vivo inhibits GR-mediated transcriptional activation, which depends on receptor phosphorylation at Ser-246 by JNK but not ERK. Thus, JNK inhibits GR transcriptional activation by direct receptor phosphorylation, whereas ERK does so indirectly. We propose that phosphorylation of GR by JNK or of a GR cofactor by ERK provides mechanisms to ensure the rapid inhibition of GR-dependent gene expression when it conf licts with mitogenic or proinf lammatory signals.