Abstract
BackgroundEpidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.Methodology/Principal FindingsHere we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.Conclusions/SignificanceCortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.
Highlights
Ligand-induced endocytic regulation of Epidermal growth factor receptor (EGFR) trafficking is utilized as a key mechanism for modulating growth factor signaling by controlling levels of EGFR surface expression [1]
Evidence to date indicates that regulated actin polymerization at all points of the internalization pathway is vital in driving the membrane dynamics necessary for EGFR internalization, with cortactin serving as an important scaffold by coupling Arp2/3 activity to cytoplasmic membrane surfaces during internalization [28,34,62]
Our present findings provide evidence that Associated Kinase 1 (ACK1) directly phosphorylates cortactin at tyrosine sites that provide enhanced Arp2/3-based actin polymerization, which may be utilized to facilitate EGFR internalization
Summary
Ligand-induced endocytic regulation of EGFR trafficking is utilized as a key mechanism for modulating growth factor signaling by controlling levels of EGFR surface expression [1]. Endocytic internalization of activated EGFR is regulated by multiple interacting factors, including receptor ubiquitination by Cbl, binding to the adaptor proteins Grb and AP-2, and receptor acetylation [3,4,5]. In addition to CME, EGFR can be internalized by recruitment into cholesterol-rich caveolae [6], macropinocytosis [7] or by circular dorsal ruffles generated from cortical actin polymerization [8]. These CMEindependent mechanisms are typically employed by cells either exposed to high experimental EGF concentrations or in cells with increased levels of surface EGFR expression [1,2]. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
