Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

<div><h3>Background</h3><p>Epidermal 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.</p> <h3>Methodology/Principal Findings</h3><p>Here 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.</p> <h3>Conclusions/Significance</h3><p>Cortactin 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.</p> </div

ACK1 directly phosphorylates cortactin at Y421/466/482.

<p>(A) Characterization of purified ACK1. Recombinant 6X-histidine tagged recombinant ACK1 was purified from SF9 cells and 5 micrograms stained with either Coomassie blue (<i>left</i>) or analyzed by Western blotting with anti-ACK antibodies (<i>middle</i>). Kinase activity was assayed by analyzing autophosphorylation (Autophos) using 0.5 micrograms of 6X-His ACK1 incubated with gamma-³²P-ATP for 10 min at 30°C. The reaction was evaluated by SDS-PAGE and autoradiography (<i>right</i>). The position of molecular weight standards is shown on the left and 6X-His ACK1 on the right. (B) Identification of cortactin tyrosine residues phosphorylated by ACK1. Purified GST-cortactin murine wild type (WT) or Y421/466/482F (triple tyrosine mutant; TYM) (0.5 micrograms) were incubated in kinase assays without or with 3U of purified Src (<i>left</i>) or with the indicated amounts of purified 6X-His ACK1 (<i>middle</i> and <i>right</i>) in the presence of [gamma-³²P]ATP. Reactions were separated by SDS-PAGE and analyzed by audioradiography. Arrows indicate the positions of phosphorylated GST-cortactin and 6X-His ACK1 (<i>left</i>). (C) Determination of the K<i>_M</i> of ACK1 for cortactin. Graphical representation showing ACK1 phosphorylation kinetics for cortactin calculated from the kinase assay in <i>B</i> (<i>right</i> panel). Percent of maximum phosphorylation signal measured by densitometry is represented on the ordinate versus concentration of ACK1 (in micrograms) on the abscissa. The calculated K<i>_M</i> for cortactin phosphorylation is shown. Data in B and C are representative from three independent experiments. (D) ACK1 knockdown reduces cortactin phosphorylation on tyrosine 421. 1483 cells transfected with scrambled control (Ctl) siRNA or ACK1-targeting siRNA were lysed and analyzed by Western blotting for ACK1 knockdown (ACK1) and cortactin pY421 phosphorylation (Cort pY421). Beta actin was blotted to verify equivalent protein loading. Blots are representative of two independent experiments.</p

Cortactin tyrosine phosphorylation is downregulated during EGFR degradation.

<p>1483 cells were serum starved for 16 h then stimulated with 100 nanograms/milliter EGF for the indicated times. Cell lysates were analyzed after stimulation by immunoblotting with anti-EGFR, anti-cortactin, anti-actin, and anti-cortactin pY241 (A) or anti-cortactin pS418 antibodies (B). Blots are representative of three independent experiments.</p

Cortactin localizes with ACK1 in vesicles containing ligand-bound EGFR.

<p>(A) 1483 cells serum starved for 16 h were stimulated with 100 nanograms/milliter Alexa Fluor-488 conjugated EGF (green) for 30 min. Cells were fixed and labeled with phalloidin (Actin; pseudocolored white) and anti-EGFR antibodies (pseudocolored red). Cells were evaluated by confocal microscopy and images rotated 45° and 90° as indicated to demonstrate EGF/EGFR colocalization throughout the z-plane. (B) Serum starved (No Tx) 1483 cells were stimulated with FITC-EGF (pseudocolored white) as in (A). Cells were fixed and labeled with anti-ACK1 (green) and cortactin (red) antibodies. Confocal images of labeled EGR in the apical (top) and ventral (bottom) cellular regions are shown. Dashed boxes in the merged images indicate the areas enlarged in the photos to the <i>right</i>. Scale bars, 20 micrometers.</p

ACK1 mediates Cdc42-induced cortactin tyrosine phosphorylation.

<p>(A) COS1 cells were cotransfected with FLAG-cortactin WT and Myc-tagged Cdc42 constructs as indicated. EV; empty vector. Cells were lysed 18 h after transfection, FLAG-cortactin immunoprecipitated and total cortactin tyrosine phosphorylation analyzed by Western blotting with a pan anti-phosphotyrosine antibody (pTyr). The blot was then stripped and reprobed for total cortactin levels (Cort). Equal amounts of total cell lysates were blotted in parallel with anti-Cdc42 antibodies to confirm GTPase expression. Ratios of tyrosine phosphorylated (pTyr) cortactin to total cortactin levels are indicated at the bottom. Blots are representative from ≥ three independent experiments. (B) ACK1 overexpression induces cortactin tyrosine phosphorylation. COS1 cells transfected with empty HA vector (EV), HA-ACK1 wild-type (WT), kinase dead (KR) (K-R mutation at amino acid 158 in the kinase domain) and a Cdc42 binding-null mutant (BD) (H-A mutation of codons 464 and 467 in the CRIB domain) were immunoprecipitated from lysates with anti-FLAG or anti-HA antibodies. FLAG-cortactin and HA-ACK1 proteins were evaluated for phosphotyrosine levels by Western blotting (pTyr). Expression levels for each protein were evaluated by stripping and reblotting with anti-cortactin and anti-HA antibodies as indicated. Ratios of tyrosine phosphorylated (pTyr) cortactin to total cortactin levels are indicated at the bottom. Blots are representative from three independent experiments.</p

Identification of the ACK1 binding region in cortactin.

<p>(A) Endogenous cortactin was immunoprecipitated from 1483 cells transfected with empty Myc vector (EV) or with Myc-tagged ACK1. Non-transfected cells were mock precipitated (Bead Only) as background control. Immune complexes were assayed by Western blotting with anti-Myc and anti-cortactin and antibodies. Total cell lysates from transfected cells were probed with anti-Myc, Anti-ACK1 and anti-cortactin antibodies. (B) Clarified lysates from 293T cells coexpressing Myc-tagged ACK1 with either empty FLAG vector (EV), full-length (FL), N-terminal (NT), C-terminal (CT) or SH3-binding deficient (W525K) FLAG-cortactin constructs (<i>schematic diagram at top right</i>) were immunoprecipitated with anti-FLAG antibodies. Membranes were immunoblotted with anti-ACK1, anti-Myc, anti-FLAG and anti-cortactin antibodies (<i>left</i>). Expression of recombinant ACK1 and cortactin proteins for each transfection condition were confirmed by Western blotting of total cells lystes with the indicated antibodies (<i>bottom right</i>). The positions of molecular weight markers are shown on the right. All blots are representative of ≥ three independent experiments.</p

EGFR downregulation requires ACK1 and the cortactin SH3 domain.

<p>(A) 1483 cells were transfected with non-targeting (Ctl) or human-specific ACK1 siRNA (ACK1 Si) for 48 h. Murine Myc-ACK1 was subsequently transfected into ACK depleted cells to rescue ACK1 expression. Cells were serum starved for 16 h and then treated with EGF for the indicated times. Following stimulation, clarified lysates were immunoprecipitated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-ACK1 and anti-actin antibodies. (B) 1483 cells were transfected with a non-targeting (Ctl) or cortactin specific siRNA (CTTN Si) for 48 h. Cortactin expression was rescued by transfection with FLAG-cortactin wild type (WT) or with an SH3-null binding mutant (W525K). Cells were serum starved for 16 h prior to EGF stimulation for the indicated times. EGFR was immunoprecipated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-cortactin to verify knockdown and expression of the FLAG-cortactin rescue constructs. Western blotting with anti-actin antibodies was conducted to verify equal protein loading. Blots are representative of two independent experiments.</p

Use of High Frequency Ultrasound to Monitor Cervical Lymph Node Alterations in Mice

<div><p>Cervical lymph node evaluation by clinical ultrasound is a non-invasive procedure used in diagnosing nodal status, and when combined with fine-needle aspiration cytology (FNAC), provides an effective method to assess nodal pathologies. Development of high-frequency ultrasound (HF US) allows real-time monitoring of lymph node alterations in animal models. While HF US is frequently used in animal models of tumor biology, use of HF US for studying cervical lymph nodes alterations associated with murine models of head and neck cancer, or any other model of lymphadenopathy, is lacking. Here we utilize HF US to monitor cervical lymph nodes changes in mice following exposure to the oral cancer-inducing carcinogen 4-nitroquinoline-1-oxide (4-NQO) and in mice with systemic autoimmunity. 4-NQO induces tumors within the mouse oral cavity as early as 19 wks that recapitulate HNSCC. Monitoring of cervical (mandibular) lymph nodes by gray scale and power Doppler sonography revealed changes in lymph node size eight weeks after 4-NQO treatment, prior to tumor formation. 4-NQO causes changes in cervical node blood flow resulting from oral tumor progression. Histological evaluation indicated that the early 4-NQO induced changes in lymph node volume were due to specific hyperproliferation of T-cell enriched zones in the paracortex. We also show that HF US can be used to perform image-guided fine needle aspirate (FNA) biopsies on mice with enlarged mandibular lymph nodes due to genetic mutation of Fas ligand (Fasl). Collectively these studies indicate that HF US is an effective technique for the non-invasive study of cervical lymph node alterations in live mouse models of oral cancer and other mouse models containing cervical lymphadenopathy.</p></div

4-NQO exposure induces precancerous alterations in mouse mandibular lymph nodes.

<p><b>A–C.</b> Images of dissected H&E and whole animal HF US (ultrasound) mandibular lymph nodes from representative age-matched (AM) control (<b>A</b>), 4-NQO-treated (28 wk) (<b>B</b>) and Fasl (<b>C</b>) mice. Lymph node borders in the HF US images are indicated in yellow. Vascular flow identified by power Doppler imaging is shown in red. Power Doppler flow dynamics for each condition are visualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s004" target="_blank">Video S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s006" target="_blank">S4</a>. H&E scale bar  = 250 µm, ultrasound scale bar  = 1 mm. CP, Cheek Pouch. <b>D&E.</b> Analysis of lymph nodes by HF US. 4-NQO treated mice at 0 and 28 wk were imaged after 8 week 4-NQO treatment and study end point. B6 age-matched (AM) Ctl 0 and 28 wk mice were imaged at the same age as 4-NQO treated mice. The Fasl lymph node data is included for comparison. <b>D.</b> 4-NQO exposure induces increased mandibular lymph node volume. <b>E.</b> 4-NQO exposure increases vascular flow in mandibular nodes. N = 6 lymph nodes from 3 mice per group, except for the controls, where N = 8 lymph nodes were analyzed from 4 mice. Box and whisker plots show minimum, 25^th, median, 75^th and maximum values, respectively. *, p≤0.05.</p

Image-guided fine needle biopsy of Fasl mandibular lymph nodes.

<p><b>A.</b> Transverse section of a Fasl mouse neck imaged with HF US. The enlarged cervical mandibular node is evident as an oval hypoechoic region near the skin surface (circumscribed in yellow). Scale bar  = 1 mm. <b>B.</b> Frames from fine needle biopsy of a Fasl mandibular node guided by HF US. Images show the position of the sampling hyperechoic needle tip prior to cervical skin penetration (<i>left</i>), position of the needle during tissue removal (<i>middle</i>), and following needle withdrawal (<i>right</i>). Note the break in the skin following needle withdrawal (arrow). The angle and trajectory of the dorsal needle surface is denoted by the yellow dotted line. Scale bar  = 1 mm. The entire procedure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s003" target="_blank">Video S1</a>. <b>C.</b> Examples of lymph tissue obtained by HF US guided FNA of a Fasl cervical mandibular node following staining and processing by cytospin. Scale bar  = 100 µm. LT; lymph tissue, RF; reticular fibers, L; individual lymphocytes.</p