Elsevier

Cellular Signalling

Volume 20, Issue 7, July 2008, Pages 1359-1367
Cellular Signalling

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

https://doi.org/10.1016/j.cellsig.2008.03.007Get rights and content

Abstract

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.

Introduction

c-Src is a member of a family of membrane-bound tyrosine kinases that regulate cell growth, differentiation and adhesion [1], [2]. c-Src is initially synthesized on soluble ribosomes in the cytosol and is co-translationally modified with myristate on its N-terminus. Fatty acylation of c-Src influences its ability to bind cellular membranes and consequently affects its intracellular distribution and signaling capabilities [3].

c-Src is a critical signal transducer for the EGF receptor tyrosine kinases (EGFR) [4]. Considerable biological synergy exists between c-Src and the EGFR [5]. In many human cancers, particularly breast cancer, c-Src and members of the human epidermal growth factor receptor (HER) tyrosine kinases, specifically HER1/EGFR and HER2/neu, are both highly overexpressed [6]. Dual overexpression of c-Src and EGFR occurs in some breast cancer cell lines and this has been correlated with Src-dependent increases in activation of and/or phosphorylation of EGFR downstream effectors. c-Src activity is required for ErbB2-mediated induction of cell growth and motility [7], [8]. Overexpression of both c-Src and EGFR leads to synergistic activation of EGF induced mitogenesis and tumor formation [9], [10]. c-Src binds to and phosphorylates EGFR on Tyr845; this phosphorylation event is required for EGF-mediated mitogenesis [9], [11]. The synergism between c-Src and EGFR serves to upregulate the mitogenic activity of EGFR downstream effectors involved in tumorigenesis, as well as increasing EGFR internalization. Thus, obtaining a clearer understanding of c-Src subcellular localization and its relationship with EGFR signaling is pivotal in understanding the mechanisms underlying malignant cell signaling.

Ligand addition induces EGFR endocytosis [12]. Although it was originally assumed that only plasma membrane localized receptors would signal, it is now known that internalized EGFR can continue to signal from endosomes [13]. Given that endosomally localized EGFR is capable of signaling, and that c-Src phosphorylation of EGFR is important for EGFR signaling, it is logical to hypothesize that c-Src may share a trafficking pathway with EGFR that allows the activated receptor to signal from endosomes. Several lines of evidence indicate that c-Src interacts with the endosomal machinery. First, c-Src associates with endosomal membranes in fibroblasts [14]. Other Src family kinases have also been shown to interact with endosomes and/or lysosomes in other cell types [15], [16], [17], [18], [19]. Second, c-Src co-localizes with dynamin and γ-adaptin, proteins involved in clathrin-coated pit endocytosis [20]. c-Src phosphorylates dynamin and clathrin in EGF stimulated cells; phosphorylation promotes endocytosis and increases the pool of activated, internalized receptors in endosomes [21], [22]. Third, c-Src regulates the rate of EGFR down-regulation [23]. Upon EGF binding, EGFR is ubiquitinated by the ubiquitin ligase Cbl and degraded. Overexpression of c-Src increases Cbl ubiquitination and degradation, thereby resulting in less Cbl-mediated EGFR degradation and increased levels of EGFR [24]. Thus, it is possible that c-Src endocytoses along with internalized receptors, and that this event promotes receptor-mediated signaling from internal organelles.

Recent studies have indicated that different trafficking pathways are used by different Src family kinases en route to the plasma membrane. For example, Lck, Lyn and Hck traffic through the Golgi and the secretory pathway, whereas Src and Fyn do not [15], [25], [26], [27], [28]. Surprisingly, the trafficking pathway of native c-Src remains largely unknown. Imaging studies have revealed the presence of c-Src in both endosomes and the plasma membrane [14], but the mechanisms regulating the distribution between these two locations are only starting to be understood. It was recently shown that upon PDGF stimulation of cells, an internal pool of c-Src is activated and transported to the plasma membrane and cytoskeleton by RhoB containing endosomes [29]. c-Src kinase activity has also been shown to regulate RhoD-dependent endosome movement [30].

To date, little is known about the mechanism of c-Src trafficking from the plasma membrane to intracellular compartments. Multiple venues exist for internalization from the plasma membrane into the cell interior, including clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin and caveolin-independent endocytosis, phagocytosis and macropinocytosis [31], [32]. Previous studies have documented that v-Src expression induces constitutive macropinocytosis — an actin-driven, clathrin-independent endocytic process promoting the uptake of fluid-phase solute [33], [34], [35]. Here we show that c-Src internalizes via constitutive macropinocytosis from the plasma membrane and that this movement is dependent on c-Src kinase activity. c-Src and the activated EGFR co-localized and trafficked together during EGF stimulation, and EGF activation was prolonged in a c-Src kinase-dependent manner. We also report that c-Src can promote and sustain EGFR activation even in the absence of EGF ligand and that this results in enhancement of downstream EGFR signaling.

Section snippets

Antibodies and reagents

Antibodies were purchased from the indicated suppliers: polyclonal rabbit anti-EGFR, anti-c-Src (N-16), anti-ERK2 (C-14), anti-phospho-ERK (Y204), and anti-phospho-EGFR (Y1173) and mouse monoclonal anti-phospho ERK (E-4), Santa Cruz Biotechnology Inc. (Santa Cruz, CA); Rabbit anti-phospho-c-Src (Y416), and anti-phospho-EGFR (Y1173), Cell Signaling Technology (Danvers, MA); mouse monoclonal anti-Rac, anti-EEA1, BD Transduction Laboratories (San Diego, CA); anti-CD63, Cymbus Biotechnology Ltd.

c-SrcGFP is a bona fide reporter of c-Src

To facilitate the study of c-Src trafficking in cells, a c-Src GFP fusion construct was generated. Since the N-terminus of c-Src contains the myristoylation site that governs its association to membranes [3], GFP was fused to the C-terminus of the protein. A previous study revealed that fusion of GFP directly to the C-terminus of c-Src generates a constitutively activated c-Src that is no longer capable of undergoing negative regulation [38]. We therefore created a seven amino acid linker

Real time live imaging reveals that c-Src internalizes from the plasma membrane via macropinocytosis

In this study, we investigated the inter-relationship between c-Src trafficking and c-Src mediated signal transduction. A key element was the design of a c-Src-GFP fusion protein that retains the properties of wild-type c-Src. Others have reported that fusion of GFP directly to the c-Src C-terminus results in formation of a constitutively activated Src-GFP fusion protein [38]. Here we show that inclusion of a 7 amino acid linker between the C-terminus of c-Src and the N-terminus of GFP

Conclusions

In this study, we establish that c-Src can internalize into cells via macropinocytosis and that this process is dependent on c-Src tyrosine kinase activity. When cells are stimulated with EGF, c-Src and the activated EGFR co-localize and co-internalize. Overexpression of c-Src results in retention of activated EGFR within the cell and hyperactivation of downstream EGFR-mediated signaling. Moreover, even in the absence of EGF addition, EGFR is activated and basal signaling is elevated 2-fold in

Acknowledgements

We thank Dr. Katia Manova-Todorova and the staff of the Molecular Cytology Core Facility for expert assistance with microscopy, imaging and image analysis, and Raisa Louft-Nisenbaum for technical assistance. This work was supported by NIH grant GM57966 to MDR and by a Susan G. Komen Breast Cancer Foundation Postdoctoral Fellowship Award PDF0504316 to MD.

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      On the other hand, the subcellular location of c-Src critically affects its function (Dwyer et al., 2016), and c-Src localization and trafficking are not fully understood. c-Src can be found at the plasma, perinuclear, and endosomal membranes (Konitsiotis et al., 2017), and also in the cytoplasm (Donepudi and Resh, 2008) and nucleus (Honda et al., 2016). Endosomal recycling has been found to be crucial for the maintenance of c-Src enrichment at the plasma membrane (Konitsiotis et al., 2017).

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