N-Myristoylation-Dependent c-Src Interactions

Abstract

c-Src is the leading member of the Src family of non-receptor tyrosine kinases, which are involved in many signaling pathways. Its deregulation affects cell migration, proliferation and survival. c-Src is composed of the intrinsically disordered N-terminal SH4 and Unique domains, of the folded SH3, SH2, kinase domains and of a C-terminal tail. c-Src is myristoylated at its N-terminal region and anchored to membranes via cooperative electrostatic and hydrophobic interactions. Weak interactions with lipids in the Unique and SH3 domains and intramolecular interactions between them were recently found in the non-myristoylated form. These interactions involve the Unique Lipid Binding region (ULBR) in the Unique domain, and the RT and nSrc loops in the SH3 domain. Our objective consisted in obtaining and characterizing the myristoylated form of the Unique and SH3 domains (MyrUSH3).

Protocols for the efficient production of myristoylated proteins were developed. The incorporation of shorter acyl chains was characterized as a general problem in the preparation of myristoylated proteins, and conditions enabling to minimize their formation were found, in particular in the case of expression in minimum media. A well-defined myristoylation-induced cleavage site was identified and characterized in the Unique Lipid Binding Region of the Unique Domain of c-Src. Conditions to obtain degradation-free samples for structural studies were established. The kinetics of MyrUSH3 binding to liposomes was followed using surface plasmon resonance (SPR) and revealed two MyrUSH3 populations, a dominant form binding with relatively fast association and dissociation, and a minor persistently bound (PB) population not described earlier. This PB form was studied in an assay involving detection by a secondary antibody and the model better explaining the experimental results described these PB species to be dimer forms of MyrUSH3. In a construct in which the SH3 domain was replaced by the GFP protein, single molecule photobleaching experiments of these PB species bound to supported lipid bilayers were conducted. A major population of dimers over the bilayer surface was detected. When binding of the myristoylated SH4 (MyrSH4) peptide to liposomes by SPR, a PB population was also observed. Monitoring of the surface activity of MyrSH4 revealed the micelar behavior of the peptide at low concentrations.

Nuclear Magnetic Resonance (NMR) measurements permitted to study the effect of the myristoyl group on the intramolecular interactions between the Unique and SH3 domains, as well as on the binding of the ULBR and RT loops to liposomes when the protein was anchored in the bilayer. 1H-15N spectra of the myristoylated Unique domain (MyrUSrc) confirmed the propensity of the ULBR to bind liposomes, but in a different manner depending on the nature of the lipid in the bilayer. These measurements of MyrUSrc also pointed out some intermolecular propensities in the MyrSH4 domain. 1H-15N spectra of MyrUSH3 in solution revealed the presence of a myristoyl binding site has been found in the RT loop. Interaction of the myristoyl chain with lipids results in the loss of other lipid binding interactions in the Unique and SH3 domains that were observed in the non-myristoylated form. The interaction between the SH4 and the SH3 domains that restricts the conformational space of the Unique domain was preserved in the myristoylated forms and in the presence of lipids.

The SPR and single molecule fluorescence studies revealed the formation of self-associated complexes of limited size upon binding of MyrUSH3 or MyrUGFP to liposomes, possibly driven by the presence of the MyrSH4 domain. The NMR data highlighted the interplay between the lipid binding regions of the Unique and SH3 domains, in presence or absence of liposomes. Therefore, the myristoylated intrinsically disordered Unique domain may act in c-Src regulation at the lipid bilayer interface.

c-Src es miembro de una importante familia de tirosina quinasas, que está involucrada en la transducción de señales en las células. c-Src está formada por una región N-terminal desordenada (compuesta de los dominios SH4 y Único), por los dominios plegados SH3, SH2, SH1 (el dominio quinasa), y por una cola C-terminal. c-Src es una proteína miristoilada en su extremo N-terminal, lo cual permite su unión a membranas, unión reforzada por la interacción del dominio SH4 polibásico con los lípidos cargados negativamente. En ausencia del grupo miristoilo, se encontraron zonas de unión a lípidos y de interacciones intramoleculares en los dominios Único y SH3. El objetivo de este trabajo es la obtención y la caracterización de la forma miristoilada de los dominios Único y SH3 (MyrUSH3). Se desarrollaron protocolos que permitieron la producción de proteínas miristoiladas. La cinética de unión de MyrUSH3 a liposomas se estudió con resonancia de plasmones superficiales. Se observó una población mayoritaria con una asociación y una disociación relativamente rápidas, y una población minoritaria con una unión persistente a liposomas. Esta segunda especie se estudió por detección secundaria via un anticuerpo y se dedujo que estaba posiblemente formada por dímeros. El dominio SH3 se remplazó por una proteína verde fluorescente (MyrUGFP) y se estudió la unión de MyrUGFP a liposomas, mediante su observación con microscopía confocal, usando la técnica de fotoblanqueo de moléculas individuales. Se observó una población mayoritaria de dímeros. A continuación, se estudió la proteína MyrUSH3 con resonancia magnética nuclear, en solución o unida a liposomas. En solución, se encontró un sitio de unión al grupo miristoil en el dominio SH3. Tras la inserción del grupo miristoil en la bicapa lipídica, se perdió la capacidad de unión a lípidos en los dominios Único y SH3 (excepto el dominio SH4), y algunas interacciones intramoleculares fueron afectadas.