Abstract
Endosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.
Highlights
IntroductionVesicles that showed significant co-localization with HA-tagged PEA-15, and this was demonstrated when co-staining for Rab[11] (Fig. 4A)
Pre-immune Serum IP: PEA-15 Dots per cellClathrin Heavy Chain Input MW PRKACA PEA-15 SGTA AHNAK AAK1 DLGAP4 RABEP1vesicles that showed significant co-localization with HA-tagged PEA-15, and this was demonstrated when co-staining for Rab[11] (Fig. 4A).To compliment this approach we expressed the constitutively active Rab[5] Q79L mutant, which prevents Rab[5] dependent endosomal fission and increases the rate of endosomal fusion[40]
Proteomic analysis identified β1 integrin and focal adhesion proteins complexed with PEA-15 suggesting that PEA-15 interaction with integrin complexes may be important for the effects on migration (Table 1)
Summary
Vesicles that showed significant co-localization with HA-tagged PEA-15, and this was demonstrated when co-staining for Rab[11] (Fig. 4A). To compliment this approach we expressed the constitutively active Rab[5] Q79L mutant, which prevents Rab[5] dependent endosomal fission and increases the rate of endosomal fusion[40]. In U87MG cells expressing both HA-tagged PEA-15 and mCherry-tagged Rab5Q79L, we observed co-localization on these ring-like structures (Fig. 4B). Taken together, these data support subcellular localization of PEA-15 at endosomes
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