Rb. Birge et al., IDENTIFICATION AND CHARACTERIZATION OF A HIGH-AFFINITY INTERACTION BETWEEN V-CRK AND TYROSINE-PHOSPHORYLATED PAXILLIN IN CT10-TRANSFORMED FIBROBLASTS, Molecular and cellular biology, 13(8), 1993, pp. 4648-4656
The genome of avian sarcoma virus CT10 encodes a fusion protein in whi
ch viral Gag sequences are fused to cellular Crk sequences containing
primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Trans
formation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion
protein results in the elevation of tyrosine phosphorylation on specif
ic cellular proteins with molecular weights of 130,000, 110,000, and 7
0,000 (p130, p110, and p70, respectively), an event which has been cor
related with cell transformation. In this study, we have identified th
e 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10
-CEF) as paxillin, a cytoskeletal protein suggested to be important fo
r organizing the focal adhesion. Tyrosine-phosphorylated paxillin was
found to be complexed with v-Crk in vivo as evident from coimmunopreci
pitation studies. Moreover, a bacterially expressed recombinant glutat
hione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with
a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is s
ufficient for binding. Mapping of the sequence specificity of a GST-Cr
kSH2 fusion protein with a partially degenerate phosphopeptide library
determined a motif consisting of pYDXP, and in competitive coprecipit
ation studies, an acetylated A(p)YDAPA hexapeptide was able to quantit
atively inhibit the binding of GST-CrkSH2 to paxillin and p130, sugges
ting that it meets the minimal structural requirements necessary for t
he interaction of CrkSH2 with physiological targets. To investigate th
e mechanism by which v-Crk elevates the tyrosine phosphorylation of pa
xillin in vivo, we have treated normal CEF and CT10-CEF with sodium va
nadate to inhibit protein tyrosine phosphatase activity. Although many
additional cellular proteins became hyperphosphorylated on tyrosine i
n the vanadate-treated CT10-CEF, the GST-CrkSH2 fragment still bound p
referentially to the paxillin and 130-kDa proteins, suggesting a high
degree of specificity in the interaction of CrkSH2 with these proteins
. Paxillin phosphorylation was highly sensitive to vanadate treatment
in both normal CEF and CT10-CEF, and the elevation in tyrosine phospho
rylation resulted in increased binding to GST-CrkSH2. Moreover, bindin
g of full-length GST-v-Crk to tyrosine-phosphorylated paxillin in vitr
o protected paxillin from dephosphorylation by cellular protein tyrosi
ne phosphatase activity. These data suggest that paxillin is involved
in a highly dynamic kinase-phosphatase interplay in normal CEF and tha
t v-Crk binding may interrupt this balance to increase the steady-stat
e level of tyrosine phosphorylation. By contrast, the 130-kDa protein
was not tyrosine phosphorylated upon vanadate treatment of normal CEF
and only weakly affected in the CT10-CEF, suggesting that a different
mechanism may be involved in its phosphorylation.