Colloids and Surfaces B: Biointerfaces. 2016 Feb;138:17-25.

Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding.

Anabel-Lise Le Roux,a,b Maria Antònia Busquets,c,e Francesc Sagués,d,e Miquel Ponsa

aBiomolecular NMR group, Inorganic and Organic Chemistry Department, University of Barcelona. bInstitute for Research in Biomedicine (IRB-Barcelona), cDepartment of Physicochemistry, Faculty of Pharmacy, University of Barcelona, dDepartment of Physical Chemistry, Faculty of Chemistry, University of Barcelona. eNanoscience and Nanotechnology Institute (IN2UB),



Cell signaling by the c-Src proto-oncogen requires the attachment of the protein to the inner side of the plasma membrane through the myristoylated N-terminal region, known as the SH4 domain. Additional binding regions of lower affinity are located in the neighbor intrinsically disordered Unique domain and the structured SH3 domain. Here we present a surface plasmon resonance study of the binding of a myristoylated protein including the SH4, Unique and SH3 domains of c-Src to immobilized liposomes. Two distinct binding processes were observed: a fast and a slow one. The second process lead to a persistently bound form (PB) with a slower binding and a much slower dissociation rate than the first one. The association and dissociation of the PB form could be detected using an anti-SH4 antibody. The kinetic analysis revealed that binding of the PB form follows a second order rate law suggesting that it involves the formation of c-Src dimers on the membrane surface. A kinetically equivalent PB form is observed in a myristoylated peptide containing only the SH4 domain but not in a construct including the three domains but with a 12-carbon lauroyl substituent instead of the 14-carbon myristoyl group. The PB form is observed with neutral lipids but its population increases when the immobilized liposomes contain negatively charged lipids. We suggest that the PB form may represent the active signaling form of c-Src while the labile form provides the capacity for fast 2D search of the target signaling site on the membrane surface.



Myristoylated c-Src forms dimers on the membrane surface

c-Src plays a critical role in multiple signaling pathways related to cell migration, invasion and survival, all of which contribute to its oncogenic potential. High levels of c-Src activity are associated to poor prognosis in breast and colorectal cancer. c-Src is the leading member of a family of kinases that includes at least 9 members (Src, Yes, Fyn, Lck, Lyn, Blk, Hck, Yrk, and Fgr) with the same domain structure formed by the SH4, Unique, SH3, SH2 and SH1 domains. All members are myristoylated at their N-terminus and most of them, but not c-Src, are palmitoylated. The lipid modifications contribute to membrane anchoring. In the case of c-Src, the affinity to negatively charged membranes is enhanced by the positive charge of the SH4 domain but membrane attachment is a reversible process (Figure 1).



Figure 1. Schematic representation of c-Src binding to the cytoplasmic side of the plasma membrane.


We have found that a fraction of a membrane-tethered myristoylated c-Src protein truncated after the SH3 domain (MyrUSH3) remains permanently bound to the membrane.1

The persistently bound form could be monitored by surface plasmon resonance (SPR) using secondary detection by an anti-SH4 antibody after extensive washings to remove the reversibly bound protein (Figure 2).



FIG2Figure 2. SPR sensograms and indirectly detected dissociation curves of persistent forms of MyrUSH3. Direct and antibody detected SPR responses of 60 s/350 s association/dissociation of MyrUSH3 on (a) DMPC (neutral) and (b) DMPC:DMPG (negatively charged) liposomes respectively.


Its formation followed a second order kinetics suggesting that the persistently bound form is a dimer.

Formation of c-Src dimers has been independently demonstrated using single molecules photobleaching experiments in a myristoylated proteins composed by the SH4 and Unique domains of c-Src fused to green fluorescent protein (GFP) (Figure 3)2.



Figure 3. Photobleaching of individual fluorescent spots of Myr-SH4-Unique-GFP deposited on a negatively charged supported lipid bilayer, showing A) one B) two and C) three discrete photobleaching steps. D) Distribution of the percentage of trajectories with one, two, three and more photobleaching steps at 200 pM (n = 159), 500 pM (n = 220), and 750 pM (n = 115); n is the number of trajectories. E-F) Predicted frequency distribution for a uniform population of non-interacting monomers E) or a mixed population containing a distribution of 25:70:5 of monomers, dimers, and trimers F). Reproduced from reference 2.


Possible implications of c-Src dimerization for signaling

In its resting state, c-Src is localized in the perinuclear region but, upon activation, it moves to the plasma membrane. Localization of c-Src at specific membrane sites is important for its role in cell migration. How c-Src is targeted to specific sites remains unclear. The finding that c-Src can switch from a reversible binding mode to a persistent one suggests a search mechanism similar to the one used by transcription factors to find a specific target site in the DNA molecule: transcription factors use a low affinity, non specific binding to DNA while searching using fast 1D diffusion for the target sequency, to which they bind with high affinity.

We suggest that the reversible lipid-binding mode of c-Src plays a similar role, tethering c-Src to the membrane surface but allowing for fast 2D diffusion to search for specific signaling sites where c-Src dimerization will result in irreversible binding.

Dimerization is enhanced by high local concentrations favored by the accumulation of negatively charged lipids. Thus, we suggest that local variations in phosphoinositide phosphates (PIP), that are known markers of membrane compartments, could determine c-Src dimerization and signaling sites.



  1. Le Roux, A.-L., Busquets, M.A., Sagués, F., Pons, M. 2016 Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding. Colloids and Surfaces B: Biointerfaces 138:17–25. 
  2. Le Roux, A.-L., Castro, B., Garbacik, E., Garcia Parajo, M.F., Pons, M. 2016. Single molecule fluorescence reveals dimerization of myristoylated Src N-terminal region on supported lipid bilayers. ChemistrySelect 10.1002/slct.201600117.


Acknowledgements:  This work was supported by funds from MINECO (BIO2013-45793-R, FIS2013- 41144-P and MAT2012-36270-C04-03), Fundació Marató TV3 and La Caixa



Prof. Miquel Pons. Biomolecular NMR group.

Department of Inorganic and Organic Chemistry. University of Barcelona. 

Cluster Building. Barcelona Science Park.

Baldiri Reixac, 10-12 08028 Barcelona. Spain.

E_mail: (





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