Adhesion Molecule Phosphorylation in Cancer Chemotherapy - Adhesion Molecules - page 346

pY 418

Y 418

pY 530

Y 530

Fig. 3 Key tyrosine residues for Src kinase activity. h e protein domains of Src kinase protein
include an amino-(NH) terminal Src Homology 3 (SH3) domain, followed by a Src Homology
2 domain (SH2), the kinase domain (KD) and i nally the carboxyl-terminus (COOH).
Phosphorylation of Y 530 in the COOH-terminal end of the molecule creates a binding site for the
Src SH2 domain, causing an intra-molecular interaction and inhibition of the molecule's kinase
activity. Following Y 530 dephosphorylation, the intra-molecular interaction is lost, exposing Y 418 .
Auto-phosphorylation of this site then activates the kinase. Note that the amino acid designations
are based on the sequence of human c-Src. h e schematics (not to scale) are based on information
from Brunton and Frame (2008) and references therein.

role that FAK plays in cancer was revealed by studies demonstrating that FAK
expression is required for tumour formation and malignant progression (Brunton
et al. 2008). h us increasingly this protein is a target for novel therapeutic
approaches to cancer.
In response to integrin receptor stimulation, FAK undergoes auto-
phosphorylation at Y 397 (Ruest et al. 2001) creating a binding site for the Src
SH2 domain and other SH2 domain-containing proteins (Fig. 4) . An additional
5 tyrosine residues in FAK (Y 407 , Y 576 , Y 577 , Y 861 , Y 925 ) are subsequently
phosphorylated by Src. h ese phosphorylation events have been elegantly linked
to discrete signalling consequences. PY 407 , pY 576 , pY 577 signii cantly increase
in vitro kinase activity of FAK, while pY 407 , pY 861 or pY 925 create binding sites
for SH2 domain-containing proteins such as the adaptor molecule Grb2. Grb2 in
turn can activate cell proliferation via the Ras/mitogen-activated protein kinase
(MAPK) pathway (reviewed in Li et al. 2008). While FAK pY 397 can therefore
be used as a readout for FAK activity, FAK pY 576 and pY 577 levels are regularly
used as a measure of Src function. h ere are well-established phospho-specii c
antibodies to detect FAK phospho-modii cations on immunoblots of protein
extracts separated by SDS-PAGE (e.g., Cowell et al. 2006). Moreover, the FAK
phospho-specii c antibodies successfully stain formalin-i xed, parai n-embedded
tissue samples (e.g., Matkowskyj et al. 2003, Halder et al. 2005). When used for
immunol uorescence analysis, the phospho-specii c FAK pY 397 antibodies almost
exclusively localize to the focal adhesions (see example in Fig. 6 ) .