Phospholipase C-γ1 (PLC-γ1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. We demonstrate that PLC-γ1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-γ1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-γ1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-γ1, but knockdown of PLC-γ1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-γ1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-γ1-dependent proliferation.

Several studies have sought systematically to identify protein subcellular locations, but an even larger task is to map which of these proteins conditionally relocates in disease (the mislocalizome). Here, we report an integrative computational framework for mapping conditional location and mislocation of proteins on a proteome-wide scale, called a conditional location predictor (CoLP). Using CoLP, we mapped the locations of over 10,000 proteins in normal human brain and in glioma. The prediction showed 0.9 accuracy using 100 location tests of 20 randomly selected proteins. Of the 10,000 proteins, over 150 have a strong likelihood of mislocation under glioma, which is striking considering that few mislocation events have been identified in this disease previously. Using immunofluorescence and Western blotting in both primary cells and tissues, we successfully experimentally confirmed 15 mislocations. The most common type of mislocation occurs between the endoplasmic reticulum and the nucleus; for example, for RNF138, TLX3, and NFRKB. In particular, we found that the gene for the mislocating protein GFRA4 had a nonsynonymous point mutation in exon 2. Moreover, redirection of GFRA4 to its normal location, the plasma membrane, led to marked reductions in phospho-STAT3 and proliferation of glioma cells. This framework has the potential to track changes in protein location in many human diseases.

No abstract

No abstract

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-⑀. The overexpression of PLC-⑀ led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-⑀ RA2 domain independently of Ras. The interaction of PLC-⑀ with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-⑀ led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-⑀ in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-⑀ could promote EGF-dependent cell growth by suppressing receptor down-regulation.