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Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN ; Międzynarodowy Instytut Biologii Molekularnej i Komórkowej
Contributor:Miączyńska, Marta : Supervisor ; Zdżalik-Bielecka, Daria : Assistant supervisor
Publisher:Nencki Institute of Experimental Biology PAS
Place of publishing: Date issued/created: Description:127 pages : illustrations ; 30 cm + 1 CD (suppl. tabl. S1 and S2) ; Bibliography ; Summary in Polish
Degree grantor:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Type of object: Subject and Keywords:AXL ; Endocytosis ; GAS6 ; Interactome ; TAM receptors
Abstract:AXL is a receptor tyrosine kinase (RTK) which together with TYRO3 and MER constitutes the TAM receptor subfamily. TAMs participate in the regulation of the immune system, phagocytic clearance of apoptotic cells and tumorigenesis. AXL and its ligand GAS6 were shown to be overexpressed in many types of human cancers, which correlated with increased tumor progression, metastasis and acquired resistance to anti-cancer therapies. In addition, AXL acts as an important receptor for the cellular entry of viruses, including ZIKA and SARS-CoV-2. Therefore, AXL is a promising therapeutic target, both for cancer treatment and anti-viral therapy, and one of its inhibitors is currently being tested in clinical trials for the treatment of cancer and COVID-19.Endocytosis facilitates uptake of fragments of the plasma membrane (PM) together with the extracellular content via endosomes. This process plays a crucial role in the regulation of RTK functions, since it may lead to degradation of RTKs in lysosomes or their recycling to the PM, which terminates or sustains RTK-mediated signaling, respectively.Despite numerous studies reporting the involvement of AXL in carcinogenesis as well as virus infections, the molecular mechanisms underlying these processes have been poorly characterized and AXL-binding proteins remained practically unknown. Additionally, none of TAM receptors have been studied so far with respect to their endocytosis. Thus, the aim of this thesis was the identification of AXL-interacting partners and the characterization of AXL endocytosis.To discover the interactome of AXL, the proximity-dependent biotin identification (BioID) was used. Its results showed that AXL interacted with proteins implicated in actin- related processes, axonogenesis, cell junction organization, signaling and endocytosis. The latter category indicated that intracellular trafficking is an important regulator of AXL function. Therefore, the mechanisms of AXL internalization have been examined in detail. It was demonstrated that, upon GAS6 stimulation, GAS6-AXL complexes were rapidly internalized into cells, and this uptake operated via multiple endocytic routes, both clathrin-mediated (CME) and clathrin-independent endocytosis (CIE). Interestingly, blocking a single endocytic route, except for clathrin-independent carriers/GPI-AP-enriched compartments (CLIC/GEEC) and ARF6-dependent endocytosis, was not sufficient to reduce endocytosis of GAS6-AXL complexes. In contrast, the inhibition of AXL kinase activity completely blocked internalization of the ligated receptor. These findings offer a mechanistic explanation for previous studies showing that AXL inhibitor treatment decreases AXL-mediated viral infections. They further provide a rationale for using pharmacological inhibition of AXL in anti-viral therapies.Subsequent analyses concerning the kinetics of AXL internalization revealed that this process operated faster than the uptake of other RTKs, such as EGFR and PDGFRβ. Moreover, in contrast to ligated EGFR, endocytosis of AXL did not lead to receptor degradation but most probably to its recycling back to the PM. The latter was associated with the prolonged phosphorylation of AXL and the sustained activation of its downstream effector AKT, which may contribute to AXL-driven cancer cell migration and invasion. Finally, the presented results revealed that depletion of AXL was sufficient to block GAS6 internalization, which supports a notion previously reported by our laboratory that AXL is a primary receptor for GAS6.Altogether, this study provides the first comprehensive analysis of the AXL interactome as well as a detailed characterization of endocytosis of AXL, the first TAM receptor studied in this respect. The results presented here shed light on the molecular mechanisms regulating AXL and AXL-mediated processes on the cellular level that significantly extends our current understanding of the role of AXL in cancer progression and viral entry
Resource type: Detailed Resource Type: Source: Language: Language of abstract: Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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