Structural and functional characterization of the intrinsically disordered Unique domain of c-Src

Abstract

Objectives:

Objective 1 – Effect of mutations perturbing the ULBR of the Unique domain of c-Src.

The first objective of this thesis was the in vitro characterization of the effect of mutations in the ULBR on the previously determined main interactions of the Unique domain: binding to lipids and to the SH3 domain.

Objective 2 - Phosphorylation of c-Src Unique domain in Xenopus laevis oocytes and mammalian cell extracts.

Phosphorylation of the Unique domain of c-Src is one of the modulation parameters affecting lipid binding and had previously been reported to affect c-Src activity. The second objective was to study the phosphorylation of the isolated Unique domain in Xenopus laevis oocytes, that enabled the study of phosphorylation in a living cell and in cell extracts, that allowed further manipulation of the phosphatase/kinase network.

Objective 3 - Functional studies of the Unique domain in the context of the full-length protein.

The last objective was the evaluation of the functional significance of the modifications investigated in the isolated Unique domain as objectives 1 and 2 in a human colorectal cell line and in the context of the full length protein.

The present thesis addressed fundamental aspects of the biological role of the intrinsically disordered Unique domain of c-Src that until now was poorly understood. We have first characterized through a structural biology approach the effects of sitedirected ULBR mutations on the previously described interactions with lipids and with the folded SH3 domain. Then, we have investigated the phosphorylation events taking place on the Unique domain by state-of-the-art in-cell NMR techniques. Finally, we have shown the functional role of USrc in the context of normal and cancer cell lines. All together, our results have demonstrated that the Unique domain of human c-Src actively participates in the regulation mechanism in which the tyrosine kinase c-Src is involved. This study represents a new essential milestone in the understanding of how c-Src can control a wide variety of signaling events inside cells. Furthermore, it potentially opens the way to the possibility of new applications for cancer therapy through the development of novel selective drugs targeting functional active residues locating in the Unique domain.

This thesis represents a “proof-of-concept” of how from the atomistic comprehension of a subject of interest (structural approach) it can be possible to understand and successively demonstrate the biological significance of the observed phenomena (functional approach).

Conclusions:

- Mutations in the Unique Lipid Binding Region affect the lipid binding capability of USrc but not the inter-domain interaction with the folded SH3. - Disruption of the conserved FGGF motif causes important local effects that affect the structure of the ULBR. - The SH3 domain of human c-Src loses its ability to interact with lipids in presence of a polyproline peptide. - Real-time NMR spectroscopy allowed the study of complex phosphorylation/dephosphorylation processes in USrc mediated by kinases and phosphatases in Xenopus laevis derived cells or cell extracts. - Three different phosphorylation sites present in the Unique domain that were unequivocally assigned to Ser 17, Ser 69 and Ser 75 have been identified in Xenopus laevis, COS-7, MEFs and HeLa cell systems. - It was the first time that phosphorylation of Ser 69 of c-Src was observed in those model systems. - The biological relevance of the ULBR has been firstly demonstrated in Xenopus laevis oocytes in the context of the full-length c-Src. - The functional roles of the described phosphorylation events together with ULBR mutations have been shown in HEK293T cells and in the human colorectal SW620 cancer cell lines in the context of the full-length protein. - A “positional regulation model” has been proposed as a new c-Src selectivity regulation mechanism.

Aquesta tesi, titulada “Caracterització estructural i funcional del domini únic intrínsecament desplegat de c-Src humana”, aborda els aspectes fonamentals de la funció biològica del domini Únic intrínsecament desordenat de c-Src (USrc) que fins ara ha sigut poc estudiat. Hem caracteritzat primer mitjançant tècniques de biologia estructural als efectes de les mutacions de la ULBR (Unique Lipid Binding Region) dirigides al lloc de les interaccions descrites anteriorment amb els lípids i amb el domini SH3. Llavors, hem investigat els esdeveniments de fosforilació que tenen lloc en el domini únic per tècniques de RMN en cèl•lules. Finalment, hem demostrat el paper funcional de USRC en el context de línies de cèl•lules normals i canceroses. En conjunt, els nostres resultats han demostrat que el domini únic de c-Src participa activament en el mecanisme de regulació en què la tirosina quinasa c-Src està implicada. Aquest estudi representa una nova fita essencial en la comprensió de com c-Src pot controlar una àmplia varietat d'esdeveniments de senyalització dins de les cèl•lules. A més, té el potencial d'obrir el camí a la possibilitat de noves aplicacions per al tractament del càncer mitjançant el desenvolupament de nous fàrmacs selectius dirigits residus actius funcionals localització en el domini únic. Aquesta tesi constitueix una "prova de concepte" de com des de la comprensió atomista d'un tema d'interès (enfocament estructural) pot ser possible entendre i successivament demostrar la importància biològica dels fenòmens observats (enfocament funcional).