Visit our Expo - Redox and Inflammation signaling 2012

Session II : Receptor signaling and G proteins Poster II, 17
Functional overlapping between caspase cleavage site and CBL binding sequence in the
juxtamembrane domain of the MET receptor.
Julien DEHEUNINCK, Catherine LEROY, Zongling JI, Bénédicte FOVEAU, Vincent
VILLERET, David TULASNE and Véronique FAFEUR
CNRS UMR 8117, Institut de Biologie de Lille, Institut Pasteur de Lille, B.P.447, 59021
Lille, FRANCE. E-mail : veronique.fafeur@ibl.fr
Hepatocyte growth factor/ scatter factor (HGF/SF) binds and activates the MET tyrosine
kinase receptor to transduce cell survival. Nonetheless, we previously demonstrated that stress
stimuli cause a caspase-dependent cleavage in the MET juxtamembrane domain, which
converts MET into a pro-apoptotic p40 MET fragment. The caspase 3 cleavage occurs at
aspartic acid residue D1000. This residue is juxtaposed to tyrosine Y1001, which is rather
involved in binding CBL upon MET activation by its ligand, allowing MET trafficking and
recycling. We wished to examine the functional relationships between caspase-dependent
cleavage and CBL binding at the ESVDY sequence of the juxtamembrane domain of MET.
We first established that the caspase cleavage- and CBL binding- sites overlapped, since a
MET D1000N mutant lost the ability both to generate p40 MET and to recruit CBL. We then
individually mutated all amino acids of the ESVDY sequence of MET constituting the
caspase and CBL sites. While a MET V999A mutant was selectively defective in generating
p40 MET, a MET Y1001F mutant was selectively defective in recruiting CBL, demonstrating
that caspase-dependent cleavage and CBL recruitment are dissociable mechanisms. We
further examined the consequence of MET phosphorylation at CBL binding site on the
caspase-dependent cleavage. Using in vitro MET phosphorylation and dephosphorylation, we
demonstrated that generation of p40 MET is unfavored when MET is phosphorylated, and
vice versa. These data were strengthened by modeling studies using the MET peptide
sequence ESVDYR and the known structure of caspase 3 or CBL in complex with specific
peptide sequences. These studies show that a phosphorylated Y1001 is not compatible with
specific association of caspase-3 to MET and also exclude simultaneous fixation of both
caspase 3 and CBL to MET.
In conclusion, we demonstrated that the caspase-dependent cleavage of MET and binding of
CBL to the activated MET receptor are dissociable and incompatible mechanisms. As a
consequence, we propose that HGF/SF can protect MET against caspase-dependent cleavage
by activating autophosphorylation of the receptor.
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