Research ArticleEps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells
Introduction
The epidermal growth factor receptor pathway substrate 8 (Eps8) is a 97 kDa protein that was originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR). Early results demonstrated that Eps8 increases epidermal growth factor (EGF) responsiveness, thus contributed to malignant transformation in tumor cells [1], [2]. There is now growing evidence that Eps8 plays an important role in promotion of various solid malignancies, including colon, squamous cell, thyroid and cervical cancer [3], [4], [5], [6], [7]. We recently reported that Eps8 expression is enhanced in metastatic pancreatic cancer cells when compared to cells from the primary tumor [8]. Moreover, high Eps8 expression in metastatic cells was associated with increased cell motility and accumulation of Eps8 at actin dynamic sites, e.g. the leading edge of the cells.
The Eps8 molecule controls actin remodeling through multiple interactions. The C-terminal “effector region” directly binds F-actin and caps actin barbed ends [9]. In addition, Eps8 participates in the activation of the small GTPase Rac in a tri-protein complex with Abi1 and Sos1, and synergizes with IRSp53 to bundle actin filaments [10]. A number of recent evidences indicate that actin dynamics and endocytic machineries are intimately related. This notion appears to apply also in the case of Eps8. Rac-signaling through Eps8 can be hampered by binding of RN-tre, a GTPase activating protein (GAP) for Rab5, to the src homology-3 (SH3) domain of Eps8, leading to disruption of the Eps8-Abi1-Sos-1 complex and to the de-activation of Rab5, which in turns regulates the internalization of EGFR [11]. Intriguingly, Rab5-dependent endocytosis and early endosome trafficking have recently been shown to be required for Rac-mediated, spatially restricted actin remodeling at the cell periphery [12].
Within this context, Eps8 has all the features to posit that it might be an important regulatory element mediating the cross talk between membrane and actin dynamics, which ultimately is required to generate propulsive force at the leading cell edge. However, whether Eps8 directly participates in membrane internalization and/or endomembrane trafficking remains unclear. Since there is little known about the degradation of Eps8 [13], it might be possible that the regulatory function of Eps8 is controlled through its expression levels.
Chaperone-mediated autophagy (CMA) is a cellular process for the selective degradation of cytosolic proteins [14]. Unlike other forms of autophagy, CMA mediates the direct translocation of individual proteins across the lysosome membrane for their degradation in the lysosome [15], [16]. CMA activity decreases with age and has shown to be altered in different human pathologies [17], [18]. Malfunctioning autophagy has been closely linked to oncogenic signaling [19].
Here, we investigate a novel association of Eps8 to the lysosomal compartment in human cancer cells and describe its degradation through CMA.
Section snippets
Expression vectors, antibodies, reagents and siRNA
Vectors encoding for GFP fusion proteins were engineered in the pEGFP C1 plasmid (Clontech Laboratories, Inc.) by cloning the appropriate fragments, obtained by recombinant PCR, in frame with the GFP moiety as described recently [20]. All constructs were sequence verified. Details are available upon request. Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Eugene, OR) according to the manufacturer's instructions.
The antibodies used were: mouse monoclonal anti-Eps8
Results
We recently described that Eps8 localizes to dynamic actin structures, i.e. to the tips of F-actin filaments in filopodia, and to the leading edge of pancreatic cancer cells, and that various pancreatic cancer cell lines expressed different mRNA and protein levels of Eps8 according to the following order: PANC-1<Capan1<AsPC-1 [8].
In addition to the spot-like, F-actin-associated pattern (such as in PANC-1 cells, Figs. 1A, B), endogenous Eps8 strikingly localized also to large vesicular
Discussion
Here, we describe a novel and unexpected association of endogenous Eps8 with the lysosomal compartment in human cancer cell lines. Eps8 has been associated with dynamic actin sites and the early endocytic compartment [1], [9], [10], [11], [20], [26], [27]. These interactions are mediated by either the SH3-domain (Abi1 and RN-tre binding), or by the C-terminal “effector region” (Sos1 and F-actin binding), respectively. The protein region that was responsible for the lysosomal localization of
Conflict of interest
The authors state no conflict of interest in this study.
Acknowledgments
We would like to thank Sonja Bauer for her excellent technical assistance. This work was partly supported by the NIH Grant AG021904 (to AMC).
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