Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

Abstract

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.Both constitutive endocytosis and activated endocytosis are highly regulated events by which cells take up nutrients and internalize receptors for recycling or degradation (47). Endocytosed molecules are delivered to early endosomes, where the components are sorted to the cell surface for recycling back to the plasma membrane, or to late endosomes to be degraded in lysosomes (17). The molecular mechanisms regulating these events are not fully understood.One of the major protein families involved in the trafficking of membrane compartments is sorting nexins (SNXs), which are characterized by the presence of phox homology (PX) domains (8,65). The PX domain is a protein module which consists of approximately 130 amino acids with three ␤-strands followed by three ␣-helices forming a helical subdomain, and the general function of this module is to interact with the head groups of inositol phospholipids through which parental proteins are targeted to specific cellular compartments. Most of the SNXs examined to date specifically recognize phosphatidylinositol 3-phosphate [PtdIns(3)P], which is found predominately in early endosomes (11). The founding member of the SNX family, SNX1, was initially identified as an interaction partner of epidermal growth factor receptor (EGFR), and the expression of SNX1 enhanced lysosomal degradation of EGFR (38); therefore, SNXs are most likely to be involved in the trafficking of many different families of receptors which are recycled to the cell surface or sent to the lysosome for degradation (19). On the other hand, PX domain-containing proteins have also been reported to bind to phosphoinositides other than PtdIns(3)P and to have functions independent of receptor trafficking (54). For example, phosp...

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

“…All four oligonucleotides resulted in more than a 90% reduction of mRNA or protein levels corresponding to a moderate, but significant reduction of EGF degradation for both candidates ( Figure S1). During the validation period, a publication was released addressing the role of PXK in EGF degradation (22). This report justified our approach and contributed to the validation of our siRNA screen, as did the results from our positive controls, where HRS depletion strongly affected EGF degradation and TSG101 depletion gave a moderate effect, due to a moderate knock-down efficiency (not shown).…”

Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy

“…Both were classified as resistors in both the loss-of-function and gain-of-function screens. The IP-MS screen identified partners linked to endocytosis for these kinases, confirming previous reports that they play a role in this process (43)(44)(45). Endocytosis of TRAIL receptors occurs through a clathrin-dependent pathway, similar to receptor tyrosine kinases (RTKs), in which c-CBL mediates the monoubiquitination of the intracellular region of the receptor, leading to their recognition by the endocytosis machinery and then degradation to down-regulate TRAIL responsiveness (78)(79)(80).…”

“…PXK, a sorting nexin, promotes the internalization and degradation of EGFR (43), and AAK1 promotes clathrin assembly through phosphorylation of the adaptor-related protein complex 2 m1 (AP2M1) component of the AP2 complex (44,45). As expected, members of the endocytotic pathway coimmunoprecipitated with both kinases-AAK1 with AP2 family proteins and PXK with clathrin subunits (Fig.…”

PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain

“…Domain-We first examined phospholipid binding of the PRIP-C2 domain in a lipid-protein overlay assay (31). The isolated PRIP-C2 domain fused to GST did not show more binding than GST alone to phospholipids tested in the presence of Ca 2ϩ (Fig.…”