PubMed · 13035597
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C L OAKLEY, A J FULTHORPE. 1953. Antigenic analysis by diffusion.. https://doi.org/10.1002/path.1700650105
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In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.
The HePTP (haematopoietic protein tyrosine phosphatase) is a negative regulator of the ERK2 (extracellular signal-regulated protein kinase 2) and p38 MAP kinases (mitogen-activated protein kinases) in T-cells. This inhibitory function requires a physical association of HePTP through an N-terminal KIM (kinase-interaction motif) with ERK and p38. We previously reported that PKA (cAMP-dependent protein kinase) phosphorylates Ser-23 within the KIM of HePTP, resulting in dissociation of HePTP from ERK2. Here we follow the phosphorylation of this site in intact T-cells. We find that HePTP is phosphorylated at Ser-23 in resting T-cells and that this phosphorylation increases upon treatment of the cells with agents that elevate intracellular cAMP, such as prostaglandin E2. HePTP phosphorylation occurred at discrete regions at the cell surface. Phosphorylation was reduced by inhibitors of PKA and increased by inhibitors of protein phosphatases PP1 and PP2A, but not by inhibitors of calcineurin. In vitro, PP1 efficiently dephosphorylated HePTP at Ser-23, while PP2A was much less efficient. Activation of PP1 by treatment of the cells with ceramide suppressed Ser-23 phosphorylation, as did transfection of the catalytic subunit of PP1. Phosphorylation at Ser-23 is also increased in a transient manner upon T-cell antigen receptor ligation. In contrast, treatment of cells with phorbol ester had no effect on HePTP phosphorylation at Ser-23. We conclude from these results that HePTP is under continuous control by PKA and a serine-specific phosphatase, probably PP1, in T-cells and that this basal phosphorylation at Ser-23 can rapidly change in response to external stimuli. This, in turn, will affect the ability of HePTP to inhibit the ERK and p38 MAP kinases.
G protein-coupled receptors (GPCRs) are activated by a wide array of signals, which include light, neurotransmitters, hormones, cytokines, and drugs. Knowledge of the subcellular distribution of GPCRs is required in many experimental situations. Most GPCR signaling occurs in response to activators that interact with receptors localized on the cell surface. GPCRs must move from their site of synthesis to the plasma membrane, often undergoing redistribution in response to ligand binding. Endocytosis and recycling of receptors are important for desensitization and resensitization. Furthermore, mutations in GPCRs can alter receptor trafficking and prevent normal receptor function. This chapter describes a widely applicable method for visualizing a GPCR by indirect immunofluorescence microscopy.