Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9 as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKC). Prevention of Ser144 phosphorylation by inhibition of PKC or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKC and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKC restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.

E-cadherin is a transmembrane protein that maintains intercellular contacts and cell polarity in epithelial tissue. The down-regulation of E-cadherin contributes to the induction of the epithelial-to-mesenchymal transition (EMT), resulting in an increased potential for cellular invasion of surrounding tissues and entry into the bloodstream. Loss of E-cadherin has been observed in a variety of human tumors as a result of somatic mutations, chromosomal deletions, silencing of the CDH1 gene promoter, and proteolytic cleavage. To date, no compounds directly targeting E-cadherin restoration have been developed. Here, we report the development and use of a novel high-throughput immunofluorescent screen to discover lead compounds that restore E-cadherin expression in the SW620 colon adenocarcinoma cell line. We confirmed restoration of E-cadherin using immunofluorescent microscopy and were able to determine the EC50 for selected compounds using an optimized In-Cell Western assay. The profiled compounds were also shown to have a minimal effect on cell proliferation but did decrease cellular invasion. We have also conducted preliminary investigations to elucidate a discrete molecular target to account for the phenotypic behavior of these small molecules and have noted a modest increase in E-cadherin mRNA transcripts, and RNA-Seq analysis demonstrated that potent analogues elicited a 10-fold increase in CDH1 (E-cadherin) gene expression.