Differential phosphorylation of the docking protein Gab1 by c-Src
and the hepatocyte growth factor receptor regulates different aspects
of cell functions
P-C Chan1,4, JN Sudhakar2,4, C-C Lai3,4and H-C Chen1,2
1Department of Life Science, National Chung Hsing University, Taichung, Taiwan;2Institute of Biomedical Sciences, National Chung
Hsing University, Taichung, Taiwan and3Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan
The docking protein Grb2-associated binder1 (Gab1) has
a central role in the integration of the growth-factor
signaling. In this study, we aimed to examine the
significance of Src-mediated Gab1 phosphorylation in
the hepatocyte growth factor (HGF) signaling. Using both
mutagenesis and mass spectrometry approaches, Y242,
Y259, Y317, Y373 and Y627 of Gab1 were identified to be
phosphorylated by c-Src. It is interesting to note that the
binding of the tyrosine phosphatase SHP2 to the Y627
antagonized the effect of c-Src on the phosphorylation of
the other four tyrosine residues. Moreover, the tyrosine
residues predominantly phosphorylated by c-Src were
different from those predominantly phosphorylated by the
HGF receptor. Gab1 overexpression potentiated both
mitogenic and motogenic activities of HGF. However, a
Gab1 mutant with substitutions of the Src phosphoryla-
tion sites (Y242, Y259, Y317 and Y373) failed to promote
HGF-induced DNA synthesis, but retained its ability to
facilitate HGF-induced chemotaxis. Taken together, our
results not only suggest that the phosphorylation of Gab1
by c-Src is important for HGF-induced DNA synthesis,
but also provide an example to illustrate how a docking
protein (for example, Gab1) is differentially phosphory-
lated by c-Src and a receptor tyrosine kinase to emanate
full spectrum of signals to the downstream.
Oncogene (2010) 29, 698–710; doi:10.1038/onc.2009.363;
published online 2 November 2009
Keywords: Gab1; phosphorylation; Src; Met; Crk; HGF
Grb2-associated binder 1 (Gab1) belongs to a family of
docking proteins that include Gab2 and Gab3 in
mammals, Dos in Drosophila melanogaster and SOC-1
in Caenorhabditis elegans (Gu and Neel, 2003). Gab1
has two isoforms; isoform B, which is a shorter variant,
has 694 amino acids, whereas isoform A has 30 extra
amino acids inserted between amino acid 528 and 529
of isoform B (Lehr et al., 1999). Gab1-null mice
die in uteruses with developmental defects in the heart,
the placenta and the skin, and show a phenotype
reminiscent of that of mice lacking functional hepato-
cyte growth factor (HGF), platelet-derived growth
factor and epidermal growth factor (EGF) signaling
pathways (Itoh et al., 2000; Sachs et al., 2000). Gab1
contains an NH2-terminal pleckstrin homology domain,
a Met-binding domain, 3 proline-rich sequences and
16 potential tyrosine phosphorylation sites that repre-
(Holgado-Madruga et al., 1996; Weidner et al., 1996).
Gab1 is phosphorylated upon stimulation of cells
with various growth factors (Holgado-Madruga et al.,
1996; Weidner et al., 1996; Kallin et al., 2004), as well
as upon activation of certain cytokines and antigen
receptors (Takahashi-Tezuka et al., 1998; Nishida et al.,
Gab1 has six tyrosine residues (Y242, Y259, Y307,
Y317, Y373 and Y406) in Tyr-X-X-Pro (YXXP) motifs,
which serve as the binding sites for the SH2 domain of
the adaptor protein Crk (Lamorte et al., 2000).
However, Crk is not the only molecule bound to those
residues in YXXP motifs in Gab1. For instance, Y307,
Y373 and Y406 of Gab1 have been shown to be the
binding sites for phospholipase Cg (PLCg) (Gual et al.,
2000). In addition, Y447, Y472 and Y589 of Gab1 in
YXXM motifs are the binding sites for PI3K (Rodrigues
et al., 2000; Schaeper et al., 2000), whereas Y627
and Y659 of Gab1 confer binding and activation
of the tyrosine phosphatase SHP2 (Schaeper et al.,
2000; Cunnicket al., 2001),
for extracellular signal-regulated kinase signaling in
response to growth-factor stimulation (Maroun et al.,
2000; Shi et al., 2000). Loss of SHP2-binding to Gab1
leads to early embryonic lethality with defects in
placental development and muscle progenitor cell
migration, but mice lacking PI3K-binding to Gab1 are
viable (Schaeper et al., 2007).
Gab1 is tyrosine phosphorylated not only by receptor
protein tyrosine kinases but also by the Src family
kinases (Chan et al., 2003; Podar et al., 2004). To
elucidate how Gab1 integrates the signals from Src and
receptor protein tyrosine kinases, it is important to
which is required
Received 28 May 2009; revised 19 August 2009; accepted 23 September
2009; published online 2 November 2009
Correspondence: Professor H-C Chen, Department of Life Science,
National Chung Hsing Univeristy, 250 Kuo-Kuang Road, Taichung
4These authors contributed equally to this work.
Oncogene (2010) 29, 698–710
& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00
know whether Src and receptor protein tyrosine kinases
have distinct preference for the tyrosine residues in
Gab1. Inthis study,we
c-Src phosphorylation sites in Gab1 and examine the
significance of this phosphorylation event in the HGF
c-Src phosphorylates Gab1 on multiple tyrosine residues
To identify the tyrosine residues of Gab1 preferentially
phosphorylated by c-Src, a mutagenesis approach was
first employed. Three Gab1 mutants, which had multiple
mutants with multiple substitutions of tyrosine (Y) with phenylalanine (F). The residues are numbered according to human Gab1
isoform B (NCBI accession no.: NP_002030). PH, the pleckstrin homology domain. (b) Myc-tagged wild-type Gab1 (myc-Gab1 wt) or
its mutants (YF3, YF4 and YF5) was co-expressed with (þ) or without (?) c-Src in HEK 293 cells for 36h. To measure the tyrosine
phosphorylation of Myc-tagged Gab1proteins, Myc-tagged Gab1 proteins were immunoprecipitated (IP) by anti-Myc antibody and
the washed immunocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-Gab1. To monitor the expression levels of
Myc-tagged Gab1 proteins and c-Src, an equal amount of whole cell lysates (WCL) was analyzed by immunoblotting with anti-Myc or
anti-Src. The tyrosine phosphorylation of Myc-tagged Gab1 proteins was quantified and expressed as the percentage relative to the
level of Gab1 wt in the presence of c-Src, which is defined as 100%. Values (means±s.d.) are from three independent experiments.
*Po0.05 (compared with the wt Gab1 in the presence of c-Src). (c) Myc-tagged Gab1 proteins were immunoprecipitated by the
polyclonal anti-Gab1 antibody and the immunocomplexes were analyzed by immunoblotting with anti-PY or anti-Gab1. To monitor
the expression of Myc-tagged Gab1 and c-Src, an equal amount of WCL was analyzed by immunoblotting with anti-Myc and anti-Src.
The tyrosine phosphorylation of Myc-tagged Gab1 proteins was quantified and expressed as the percentage relative to the level of
Gab1 wt in the presence of c-Src, which is defined as 100%. Values (means±s.d.) are from three independent experiments. *Po0.05
(compared with the wt Gab1 in the presence of c-Src).
c-Src phosphorylates Grb2-associated binder1 (Gab1) on multiple tyrosine residues. (a) Schematic presentation for the Gab1
Differential phosphorylation of Gab1 by Src and Met
P-C Chan et al
Coomassie blue staining. The in-gel digestion method used
in this study is modified from those described previously
(Gharahdaghi et al., 1999; Terry et al., 2004). Nanoscale
capillary liquid chromatography/tandem mass spectrometry
analysis was carried out using an Ultimate capillary LC system
(LC Packings, Amsterdam,
QSTARXLquadrupole-time of flight (Q-TOF) mass spectro-
meter (Applied Biosystem/MDS Sciex, Foster City, CA, USA).
A nano-electrospary interface was used for liquid chromato-
graphy/tandem mass spectrometry analysis. Ionization (2.0kV
ionization potential) was carried out with a coated nanoLC tip.
Data acquisition was carried out by automatic Information
Dependent Acquisition (IDA; Applied Biosystem/MDS Sciex).
The IDA automatically finds the most intense ions in a
spectrum of time-of-flight mass spectrometry, and then per-
forms an optimized tandem mass spectrometry analysis on
the selected ions. The product ion spectra generated by
nanoliquid chromatography/tandem mass spectrometry were
searched against NCBI databases for exact matches using the
MASCOT search program. A Homo sapiens (human) taxonomy
restriction was used, and the mass tolerance of both precursor
ion and fragment ions was set to 0.1Da. Carbamidomethyl
cysteine was set as a fixed modification, whereas phosphoryla-
tion was set as variable modification. Peptides were considered
identified if MASCOT score was over the 99% confidence limit
based on the significance threshold (Po0.01) of each peptide
and at least five successive y- or b-ions. All the spectra were
further verified by manual inspection. A false-positive rate was
determined by searching in a randomized decoy database within
the criteria. Phosphorylated sites were assigned with the criteria
that the phosphorylated sites are unambiguously determined
when y- or b-ions between which the phosphorylated residue
exists are observed in the peak lists of the fragment ions.
Netherlands) coupled toa
Chemotaxis assays were carried out in a 48-well chemo-
taxis chamber (Neuroprobe, Cabin John, MD, USA) as described
previously (Chen and Chen, 2006). Cells were allowed to migrate
for 5h and the migrated cells on the lower side of the membrane
were imaged under a microscope and enumerated by Image J
(NIH) software using ‘Cell Counter’ function.
BrdU incorporation assay
Cells were serum-starved for 24h and followed by a 6-h
labeling with 100mM BrdU in serum-free DMEM supplemen-
ted with or without 20ng/ml of HGF. Cells were washed thrice
with PBS, fixed in 4% paraformaldyhyde for 30min and
permeablized in 0.05% Triton X-100 in PBS for 10min. To
denature chromosomal DNA, cells were incubated in 2N HCl
for 10min and subsequently neutralized with 0.1 M borate
buffer, pH 8.5. Cells were rinsed with PBS and stained with the
monoclonal anti-BrdU antibody and a rhodamine-conjugated
secondary antibody. Nuclei were visualized by 2mg/ml of
Hoechst 33258 for 30min. BrdU-positive cells were counted
under a fluorescent microscopy.
Student’s t-test was used to determine whether there was a
significant difference between two means (Po0.05); statistical
differences are indicated with an asterisk.
Conflict of interest
The authors declare no conflict of interest.
We are indebted to Dr T Hirano (Osaka University, Osaka,
Japan) for Gab1 cDNA and Gab1-null MEFs and Dr DL
Wang (Academia Sinica, Taipei, Taiwan) for Flag-tagged
SHP2 and its CS mutant. This work is supported by grants
from the National Science Council, Taiwan and NHRI-
EX97-9730BI from the National Health Research Institutes,
tyrosine residues in Grb2-associated binder1 (Gab1), which is antagonized by the tyrosine phosphatase SHP2. In this model, the
tyrosine phosphorylation of Gab1 on Y242, Y259, Y317 and Y373 is coordinately regulated by both c-Src and SHP2, which then
transmits signals downstream through its binding to Crk and phospholipase Cg (PLCg). (b) Model illustrating that differential
phosphorylation of Gab1 by c-Src and the hepatocyte growth factor (HGF) receptor Met may contribute to HGF-elicited DNA
synthesis and cell motility, respectively. In this model, the phosphorylation of Gab1 by c-Src upon HGF stimulation may allow Gab1
to recruit Crk and PLCg, which then transmit signals to trigger DNA synthesis. On the other hand, the phosphorylation of Gab1 by
the HGF receptor may allow Gab1 to bind to SHP2 and/or PI3K, which then contributes to HGF-induced cell motility.
Illustration of the concepts proposed in this study. (a) Model illustrating that c-Src phosphorylates a unique cohort of
Differential phosphorylation of Gab1 by Src and Met
P-C Chan et al
Chan PC, Chen YL, Cheng CH, Yu KC, Cary LA, Shu KH et al.
(2003). Src phosphorylates Grb2-associated binder 1 upon hepato-
cyte growth factor stimulation. J Biol Chem 278: 44075–44082.
Chen SY, Chen HC. (2006). Direct interaction of focal adhesion kinase
(FAK) with Met is required for FAK to promote hepatocyte growth
factor-induced invasion. Mol Cell Biol 26: 5155–5167.
Cunnick JM, Mei L, Doupnik CA, Wu J. (2001). Phosphotyrosines
627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based
activation motif (BTAM) conferring binding and activation of
SHP2. J Biol Chem 276: 24380–24387.
Derman MP, Cunha MJ, Barros EJ, Nigam SK, Cantley LG. (1995).
HGF-mediated chemotaxis and tubulogenesis require activation of
the phosphatidylinositol 3-kinase. Am J Physiol 268: F1211–F1217.
Eulenfeld R, Schaper F. (2009). A new mechanism for the regulation
of Gab1 recruitment to the plasma membrane. J Cell Sci 122:
Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM.
(1999). Mass spectrometric identification of proteins from silver-
stained polyacrylamide gel: a method for the removal of silver ions
to enhance sensitivity. Electrophoresis 20: 601–605.
Gu H, Neel BG. (2003). The ‘Gab’ in signal transduction. Trends Cell
Biol 13: 122–130.
Gual P, Giordano S, Williams TA, Rocchi S, Van Obberghen E,
Comoglio PM. (2000). Sustained recruitment of phospholipase Cg
to Gab1 is required for HGF-induced branching tubulogenesis.
Oncogene 19: 1509–1518.
Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK,
Wong AJ. (1996). A Grb2-associated docking protein in EGF- and
insulin-receptor signaling. Nature 379: 560–564.
Itoh M, Yoshida Y, Nishida K, Narimatsu M, Hibi M, Hirano T.
(2000). Role of Gab1 in heart, placenta, and skin development and
growth factor- and cytokine-induced extracellular signal-regulated
kinase mitogen-activated protein kinase activation. Mol Cell Biol
Kallin A, Demoulin JB, Nishida K, Hirano T, Ronnstrand L, Heldin
CH. (2004). Gab1 contributes to cytoskeletal reorganization and
chemotaxis in response to platelet-derived growth factor. J Biol
Chem 279: 17897–17904.
Lamorte L, Kamikura DM, Park M. (2000). A switch from p130Cas/
Crk to Gab1/Crk signaling correlates with anchorage independent
growth and JNK activation in cells transformed by the Met receptor
oncoprotein. Oncogene 19: 5973–5981.
Lehr S, Kotzka J, Avci H, Sickmann A, Meyer HE, Herkner A et al.
(2004). Identification of major ERK-related phosphorylation sites in
Gab1. Biochemistry 43: 12133–12140.
Lehr S, Kotzka J, Herkner A, Klein E, Siethoff C, Knebel B et al.
(1999). Identification of tyrosine phosphorylation sites in human
Gab-1 protein by EGF receptor kinase in vitro. Biochemistry 38:
Lehr S, Kotzka J, Herkner A, Sikmann A, Meyer HE, Krone W et al.
(2000). Identification of major tyrosine phosphorylation sites in the
human insulin receptor substrate Gab-1 by insulin receptor kinase
in vitro. Biochemistry 39: 10898–10907.
Machide M, Kamitori K, Kohsaka S. (2000). Hepatocyte growth
factor-induced differential activation of phospholipase Cg1 and
phosphatidylinositol 3-kinase is regulated by tyrosine phosphatase
SHP-1 in astrocytes. J Biol Chem 275: 31392–31398.
Maroun CR, Naujokas MA, Holgado-Madruga M, Wong AJ,
Park M. (2000). The tyrosine phosphatase SHP-2 is required for
sustained activation of extracellular signal-regulated kinase and
epithelial morphogenesis downstream from the met receptor
tyrosine kinase. Mol Cell Biol 20: 8513–8525.
Montagner A, Yart A, Dance M, Perret B, Salles JP, Raynal P. (2005).
A novel role for Gab1 and SHP2 in epidermal growth factor-
induced Ras activation. J Biol Chem 280: 5350–5360.
Nishida K, Yoshida Y, Itoh M, Fukada T, Ohtani T, Shirogane T
et al. (1999). Gab-family adapter proteins act downstream of
cytokine and growth factor receptors and T- and B-cell antigen
receptors. Blood 93: 1809–1816.
Podar K, Mostoslavsky G, Sattler M, Tai YT, Hayashi T, Catley LP
et al. (2004). Critical role for hematopoietic cell kinase (Hck)-
mediated phosphorylation of Gab1 and Gab2 docking proteins in
interleukin 6-induced proliferation and survival of multiple myelo-
ma cells. J Biol Chem 279: 21658–21665.
Riordan SM, Lidder S, Williams R, Skouteris GG. (2000). The beta-
subunit of the hepatocyte growth factor/scatter factor (HGF/SF)
receptor phosphorylates and associates with CrkII: expression
of CrkII enhances HGF/SF-induced mitogenesis. Biochem J 350:
Rodrigues GA, Falasca M, Zhang Z, Ong SH, Schlessinger J. (2000).
A novel positive feedback loop mediated by the docking protein
Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor
receptor signaling. Mol Cell Biol 20: 1448–1459.
Sachs M, Brohmann H, Zechner D, Muller T, Hulsken J, Walther I
et al. (2000). Essential role of Gab1 for signaling by the c-Met
receptor in vivo. J Cell Biol 150: 1375–1384.
Schaeper U, Gehring NH, Fuchs KP, Sachs M, Kempkes B,
Birchmeier W. (2000). Coupling of Gab1 to c-Met, Grb2, and
Shp2 mediates biological responses. J Cell Biol 149: 1419–1432.
Schaeper U, Vogel R, Chmielowiec J, Huelsken J, Rosario M,
Birchmeier W. (2007). Distinct requirements for Gab1 in Met
and EGF receptor signaling in vivo. Proc Natl Acad Sci USA 104:
Shi ZQ, Yu DH, Park M, Marshall M, Feng GS. (2000). Molecular
mechanism for the Shp-2 tyrosine phosphatase function in promot-
ing growth factor stimulation of Erk activity. Mol Cell Biol 20:
Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y,
Nishida K et al. (1998). Gab1 acts as an adapter molecule linking
the cytokine receptor gp130 to ERK mitogen-activated protein
kinase. Mol Cell Biol 18: 4109–4117.
Terry DE, Umstot E, Desiderio DM. (2004). Optimized sample-
processing time and peptide recovery for the mass spectrometric
analysis of protein digests. J Am Soc Mass Spectrom 15: 784–794.
Weidner KM, Di Cesare S, Sachs M, Brinkmann V, Behrens J,
Birchmeier W. (1996). Interaction between Gab1 and the c-Met
receptor tyrosine kinase is responsible for epithelial morphogenesis.
Nature 384: 173–176.
Yu CF, Liu ZX, Cantley LG. (2002). ERK negatively regulates the
epidermal growth factor-mediated interaction of Gab1 and the
phosphatidylinositol 3-kinase. J Biol Chem 277: 19382–19388.
Yu CF, Roshan B, Liu ZX, Cantley LG. (2001). ERK regulates the
hepatocyte growth factor-mediated interaction of Gab1 and the
phosphatidylinositol 3-kinase. J Biol Chem 276: 32552–32558.
Differential phosphorylation of Gab1 by Src and Met
P-C Chan et al