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(Received for publication, March 8, 1996, and in revised form, March 29, 1996)
From the Departments of ABSTRACT The ret/ptc2 papillary thyroid cancer
oncogene, an oncogenic form of the c-Ret receptor tyrosine kinase, is
the product of a somatic crossover event fusing the dimerization domain
of the type I INTRODUCTION The c-ret proto-oncogene encodes a receptor tyrosine
kinase with a cadherin-like extracellular domain (1). Mutations of
c-ret are responsible for two distinct classes of genetic
disease. Germline loss of function mutations in c-ret result
in the developmental disorder Hirschsprung's disease (2, 3), while
activating mutations result in the multiple endocrine neoplasia family
of inherited cancers (4, 5, 6, 7). In addition to the germline alterations,
somatic mutational events lead to constitutively active forms of
c-ret, and these are found in nearly half of all papillary
type thyroid carcinomas (8).
Chromosome translocations or inversions in papillary thyroid carcinoma
(ptc)1 give rise to various fusion proteins
where the C-terminal tyrosine kinase domain of c-Ret is fused to an
N-terminal portion of another gene product. One of the resultant
transforming proteins, observed in multiple independent cases of ptc,
was the product of a crossover between the type I In the absence of a known ligand, studies of Ret signaling have been
done using activated forms of the Ret tyrosine kinase, like Ret/ptc2,
or a chimeric epidermal growth factor/Ret kinase receptor. It was
suggested from work with the EGFR/Ret chimera that Ret couples to a
novel mitogenic signaling pathway because, while growth stimulatory
effects were as strong as those of the EGF or platelet-derived growth
factor receptors, stimulation of mitogen-activated protein kinases and
PLC A yeast two-hybrid screen was used to identify the presumed Src
homology 2 (SH2) or phosphotyrosine binding domain that interacts with
Tyr-586 of Ret/ptc2. The SH2 domains of PLC EXPERIMENTAL PROCEDURES A yeast two-hybrid screen was performed
by the methods of Vojtek et al. (14), with reagents from
Stan Hollenberg. Ret/ptc2 cDNA was subcloned into the LexA-fusion
vector pBTM116 and coexpressed in the L40 strain of Saccharomyces
cerevisiae with an embryonic mouse random primed cDNA library.
From approximately two million co-transformants, seven interacted
specifically with the Ret portion of Ret/ptc2. The cDNA inserts of
these were sequenced by the dideoxy method (15), and sequences obtained
were compared with the contents of GenBank using the BLAST program
through the NIH/NCBI server on the World Wide Web. Three library
vectors encoded the following mouse sequences: the C-terminal 155
residues of Grb10 (16); 156 residues that share 97% identity with
residues 537-693 of rat PLC Two hybrid transformants were
assayed for Two-hybrid results were
verified using a stably transfected NIH3T3 cell line expressing an
EGFR/Ret chimeric protein (12). These cells were treated with 100
nM EGF for 10 min before resuspension in lysis buffer (50
mM HEPES, pH 7.4, 150 mM NaCl, 5 mM
KCl, 1 mM CaCl2, 1 mM
MgSO4, 10% glycerol, 1% Triton, 1 mM
benzamidine, 1 mM tosylphenylalanyl chloromethyl ketone, 1
mM
N The development of
this assay is described in detail elsewhere (11). Briefly, mouse 10T1/2
fibroblasts were plated on glass coverslips and grown to 70%
confluence in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum. The coverslips were then transferred to Dulbecco's
modified Eagle's medium containing 0.05% calf serum. After 24 h of
serum starvation, the cells were injected into their nuclei with
solutions of injection buffer (20 mM Tris, pH 7.2, 2
mM MgCl2, 0.1 mM EDTA, 20
mM NaCl) containing 100 µg/ml Ret/ptc2 expression plasmid
DNA and 8 mg/ml rabbit IgG (Sigma). For co-injection experiments, 200
µg/ml of a second expression plasmid was also present. All
microinjection experiments were performed using an automatic
micromanipulator (Eppendorf, Fremont, CA), with glass needles pulled on
a vertical pipette puller (Kopf, Tujunga, CA). Entry into S-phase was
assessed through incorporation of the thymidine analog
5-bromodeoxyuridine and its subsequent detection by immunostaining.
Injected cells were identified by immunostaining of the rabbit IgG
injection marker.
RESULTS To search for proteins that
interact with the Ret/ptc2 oncogenic protein, a mouse random-primed
cDNA library was screened using a yeast two-hybrid system (14).
Three sequences isolated from the library by interaction with Ret/ptc2
matched the SH2 domain of Grb10, the first SH2 domain of PLC Mutants of Ret/ptc2 were prepared and interactions were quantitated by
To verify the two-hybrid results,
affinity precipitation experiments were performed. The binding domains
from PLC Because the
LIM2 domain of Enigma bound at a site crucial for the mitogenic
activity of Ret, this interaction was investigated further. Using an
inducible two-hybrid system (21, 22), where higher expression levels
were achieved, it was possible to observe the phosphorylation state of
Ret using anti-phosphotyrosine antibodies. Enigma bound to both
Ret/ptc2 and to the Ret tyrosine kinase alone (Rettk) but
did not bind to either when the C-terminal 23 residues were deleted
(C
Based on optical densitometry, the extent of tyrosine phosphorylation
of Ret/ptc2 and the C-terminal deletion Ret/ptc2 C The interaction between LIM2 of Enigma and Ret was specific because LIM
domains from other proteins failed to interact with Ret (Fig.
2c). Another tyrosine kinase, EGFR, failed to interact with
the LIM domains of Enigma either in the two-hybrid system (Fig.
2c) or in GST-fusion binding reactions (data not shown).
The
functional significance of the association between Ret and Enigma was
investigated in vivo using microinjection. Various Ret/ptc2
expression constructs were injected into nuclei of serum-starved
fibroblasts, and the capacity of these constructs to induce DNA
synthesis was assayed by monitoring incorporation of the thymidine
analog 5-bromodeoxyuridine. Mutations in Ret/ptc2 that blocked
association with Grb10 (Y429F) or PLC
The strong correlation between mutants that failed to bind Enigma and
loss of mitogenic activity suggested that Enigma was either required
for the mitogenic signaling of Ret/ptc2 or that some other protein,
which was not detected in the two-hybrid screen, also binds at Tyr-586.
To discriminate between these two possibilities, co-injection
experiments were performed to attempt to block the Ret/ptc2 mitogenic
signal. Co-injection of Ret/ptc2 with a plasmid that expressed the
three LIM domains of Enigma (C275, Fig. 3b) blocked
Ret-induced DNA synthesis, while co-injection with full-length Enigma
had no effect. These results support the conclusion that Ret/ptc2
requires Enigma for mitogenic signaling. If Enigma was simply competing
for binding with some other signaling protein, then both full-length
Enigma and the LIM domains alone should block signaling, given that
both interact with Ret with an equivalent affinity.2 The
inhibition of mitogenesis was specific to the LIM domains of Enigma
because the LIM domains of Zyxin (23) were without effect. It was also
specific for Ret because the LIM domains of Enigma did not block the
mitogenic activity of the EGFR tyrosine kinase analog of Ret/ptc2
(RI/EGFR) previously shown to have mitogenic activity in this assay
(11).
DISCUSSION LIM domains contain approximately 50 amino acids, bind two atoms
of Zn2+, and are found in a variety of homeodomain proteins
(24), cytoskeleton-associated proteins (23, 25), protein kinases, and
proteins of unknown function (26). Enigma was originally discovered as
a protein that binds to exon 16 of the insulin receptor (13) at a
tyrosine-based sequence important for receptor internalization, and
that interaction is through the C-terminal LIM domain of Enigma, LIM3.
In the present study, Enigma bound to Ret via the LIM2 domain to a
sequence required for mitogenic signaling. LIM3 of Enigma is highly
specific for the Tyr-based motif in the insulin receptor whereas LIM2
is highly specific for the Tyr-586-based motif in Ret.2 The
N-terminal portion of Enigma is required for mitogenic signaling
because co-injection of only the LIM domains with Ret/ptc2 ablated the
mitogenic signal, while co-injection with full-length Enigma did
not.
As shown here, Enigma is required for Ret/ptc2 mitogenic signaling
while previous results established that Ret tyrosine kinase activity
was also required (11). Kinase activity, however, is not required for
recruitment of Enigma to Ret/ptc2. Enigma binds to a
tyrosine-containing sequence in an activation-independent manner, and
this is clearly different from SH2 or phosphotyrosine binding domain
interactions. Enigma might either become phosphorylated upon Ret
activation or serve to localize Ret to a subcellular position required
for kinase-mediated signaling. In either case, these results define a
novel mechanism for mitogenic signaling.
FOOTNOTES Acknowledgments We thank P. Paolo Di Fiore, European
Institute of Oncology, Milan, Italy, for cells expressing the EGFR/Ret
chimera, M. Pierotti, Institute Nationale Tumori, Milan, Italy, for the
Ret/ptc2 cDNA, and Tony Hunter and David Schlaepfer, Salk
Institute, La Jolla, CA, for the pGEX-Grb2 and pGEX-Src constructs.
REFERENCES
Volume 271, Number 22,
Issue of May 31, 1996
pp. 12691-12694
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
§,
,
Chemistry and Biochemistry,
¶ Biology, and '' Medicine, University of California,
San Diego, La Jolla, California 92093-0654
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
regulatory subunit of cyclic AMP-dependent
protein kinase (RI) with the tyrosine kinase domain of c-Ret. Mitogenic
activity of Ret/ptc2 required dimerization via the N terminus of RI and
a tyrosine residue located C-terminal to the kinase core of Ret,
Tyr-586 (Durick, K., Yao, V. J., Borrello, M. G., Bongarzone, I.,
Pierotti, M. A. and Taylor, S. S. (1995) J. Biol. Chem.
270, 24642-24645). Using the yeast two-hybrid system, Ret/ptc2
binding proteins were identified, and the sites of interaction with
Ret/ptc2 were mapped. The SH2 domains of phospholipase C
and Grb10
were both identified, and binding depended on phosphorylation of
Tyr-539 and Tyr-429, respectively. These interactions, however, were
not required for mitogenic signaling. The second of the three LIM
domains in Enigma (Wu, R. Y., and Gill, G. N. (1994) J. Biol.
Chem. 269, 25085-25090) was also identified as a Ret/ptc2
binding domain. Enigma, a 455-residue protein, was discovered based on
its interaction with the insulin receptor through the C-terminal LIM
domain. Although the association with Enigma required Tyr-586 of
Ret/ptc2, the interaction was phosphorylation-independent. In contrast
to the SH2 interactions, disruption of the interaction with Enigma
abolished Ret/ptc2 mitogenic signaling, suggesting that LIM domain
recognition of an unphosphorylated tyrosine-based motif is required for
Ret signal transduction.
regulatory subunit
of cyclic AMP-dependent protein kinase (RI) gene with
c-ret (9). This protein, Ret/ptc2, is 596 residues in length
and contains the N-terminal two-thirds of RI followed by the entire
tyrosine kinase domain of c-Ret (10). Using a microinjection-based
assay for mitogenic activity, we previously showed that the N-terminal
dimerization domain of RI was essential for constitutive activation of
Ret/ptc2 (11).
by Ret was, in comparison, very weak (12). Work with Ret/ptc2
indicated that both intrinsic protein tyrosine kinase activity and a
tyrosine residue (Tyr-586) located outside the kinase core were
absolutely required for Ret/ptc2-induced mitogenesis (11).
and Grb10 were both
found to interact with Ret, but neither bound at Tyr-586. Mutations in
Ret/ptc2, which interfered with these SH2 interactions, had no effect
on the mitogenic activity. A protein that interacted with Tyr-586 was
identified as Enigma (13). This interaction was found to be highly
specific, mediated by the second of the three LIM domains of Enigma and
independent of Ret phosphorylation or activity. Disruption of the
interaction of Ret/ptc2 with Enigma, either by mutation of Tyr-586 in
Ret/ptc2 or by co-expression with a dominant negative form of Enigma,
abolished the mitogenic activity of Ret/ptc2.
2 (17); and 131 residues with 95%
identity to the C-terminal 131 residues of human Enigma containing all
of LIM2 and LIM3 (13).
-Galactosidase Assay
-galactosidase activity by solution assay (18). Units of
activity were calculated as: activity =
1750(A420)/((time in min)(volume of culture in
assay)(A600 of culture)).
-p-tosyl-L-lysine
chloromethyl ketone, 1 mM phenylmethylsulfonyl fluoride, 1
mM NaVO4). Cleared lysates were incubated for 2
h with 2 µg of GST-fusion protein bound to glutathione-agarose beads
in a total volume of 300 µl. The beads were washed four times with
lysis buffer, resuspended in SDS-polyacrylamide gel electrophoresis
sample buffer, boiled, and run on 7% gels. Proteins were transferred
to polyvinylidene difluoride membranes and probed with either rabbit
anti-Ret (11) (1:100,000) or anti-phosphotyrosine (1:2500, Transduction
Laboratories) antibodies. The GST-fusion proteins used were bacterially
expressed from pGEX vectors coding for the following: GST, empty
vector; GST-Grb2, murine Grb2 SH2 domain; GST-Enigma, human Enigma LIM2
domain (residues 334-394); GST-Src, murine v-Src SH2 domain;
GST-PLC, murine PLC SH2 domain 1 obtained from the two-hybrid
screen; GST-Grb10, murine Grb10 SH2 domain obtained from the two-hybrid
screen.
, and a
C-terminal fragment of Enigma that contained LIM domains 2 and 3
(LIM2/3). The interaction of Ret with PLC and Grb10 has been
observed previously (12, 19). Using the two-hybrid system, the
interactions of these proteins with Ret were characterized.
-galactosidase activity (Fig. 1b). The SH2
domains failed to interact with a kinase-inactive mutant of Ret/ptc2
(K282R), indicating that these interactions depended on
autophosphorylated tyrosine residues. In contrast to the SH2 domains,
binding of the LIM2/3 was not diminished in the kinase-inactive mutant.
By testing for interaction between the SH2 domains and various Tyr to
Phe mutants of Ret/ptc2, residues required for Grb10 and PLC binding
were identified as Tyr-429 and Tyr-539, respectively. Both the Grb10
and PLC SH2 domains bound to the Y586F mutant, but binding to the
Enigma LIM domains was eliminated by this mutation and by a mutation in
which the C-terminal 23 residues of Ret were deleted (C
574).
Fig. 1.
Requirements for interaction of Grb10,
PLC, and Enigma with Ret. a, schematic representation
showing the location of tyrosines that are required for interaction
with the SH2 domains of Grb10 and PLC and the second LIM domain of
Enigma. The corresponding residues in c-Ret are indicated, and the
EGFR/Ret chimera used in panel c is shown. b,
analysis of mutant Ret/ptc2 interactions using the yeast two-hybrid
system. The indicated mutant of Ret/ptc2 and Grb10 SH2, PLC SH2, or
LIM 2/3 of Enigma were co-expressed in yeast, and interactions were
measured using solution assays of the reporter -galactosidase. Y586F
also contained R588T. The mean ± S.D. (n = 6) for each
interacting pair is shown. c, in vitro interactions between
GST-fusion proteins and the EGFR/Ret chimeric receptor. Clonal NIH3T3
cells expressing an EGFR/Ret chimeric receptor were treated (+) or not
treated (
) with EGF before lysis. Western blots of EGFR/Ret that
bound to the indicated GST-fusion proteins are shown. Gels were run in
parallel, blotted to polyvinylidene difluoride membranes, and probed
with anti-Ret (left) or anti-phosphotyrosine antibodies
(right).
, Grb10, and Enigma were expressed in Escherichia
coli as GST-fusion proteins. The three GST-fusion proteins were
incubated with lysates of NIH3T3 cells expressing the EGFR/Ret chimeric
receptor (Fig. 1a), where EGF-dependent
activation of EGFR/Ret has been characterized (12). In each case
binding to EGFR/Ret from lysates of EGF-treated cells was observed
(Fig. 1c). Results shown were using a GST-fusion protein of
only LIM2 from Enigma, because Enigma binding to Ret was determined to
be mediated by LIM2.2 GST-fusion proteins
with the SH2 domains of Grb2 and v-Src were also expressed and tested
for in vitro binding. Neither GST alone nor GST-Grb2 bound
to the EGFR/Ret chimera. The SH2 domain of v-Src, however, did interact
with EGFR/Ret, and interaction with all three GST-SH2 domains required
EGF-stimulated receptor autophosphorylation. In contrast, interaction
with GST-LIM2 of Enigma did not require receptor autophosphorylation.
Grb2 served as a negative control because Ret has two splice isoforms
(10). The long form binds to Grb2 (20) while the short form, used in
all of the constructs described here, does not contain the Grb2
consensus site. Both isoforms of Ret are mitogenic (12).
574, Fig. 2a). The interaction was not
dependent upon the phosphorylation state of Ret because
Rettk was not phosphorylated on tyrosine in the absence of
the dimerization domain of RI, whereas Ret/ptc2 underwent tyrosine
autophosphorylation (Fig. 2b, lanes
Rettk and Ret/ptc2).
Fig. 2.
Requirements for the association between
Ret/ptc2 and Enigma. a, mapping of binding determinants in
Ret/ptc2. Various fragments of Ret/ptc2, shown schematically, were used
in the yeast two-hybrid system to measure interaction with the product
of a plasmid expressing full-length Enigma. -Galactosidase activity
of transformants was measured by solution assay, and values shown are
averages of duplicate assays. The same pattern was observed in results
from three separate experiments with full-length Enigma, the C-terminal
half containing the LIM domains, or LIM2 alone. b, tyrosine
autophosphorylation of Ret/ptc2 mutants. Lysates of yeast transformed
with plasmids expressing fragments shown in panel a were run
on 10% SDS-polyacrylamide gel electrophoresis, blotted to
nitrocellulose, and probed as in Fig. 1c. Anti-PY,
anti-phosphotyrosine. c, specificity of the LIM domains of
Enigma for Ret/ptc2. pJG4-5 plasmids coding for full-length CRP, the
three LIM domains of Zyxin (residues 339-542), and the three LIM
domains of Enigma (residues 275-455) were co-transformed into yeast
with either Rettk, the pEG202 plasmid expressing the Ret
kinase with an intact C terminus, or EGFRtk, a pEG202
construct containing the intracellular domain of EGFR.
-Galactosidase activity was assessed by streaking transformants on
5-bromo-4-chloro-3-indoyl -D-galactoside plates. +++,
denotes dark blue after 12 h of incubation compared with white ( )
that was equivalent to background after 24 h.
574 were equivalent.
Because deletion of the C terminus of Ret/ptc2 did not decrease the
phosphotyrosine content by a detectable amount, Tyr-586, the only
tyrosine in the C terminus of this isoform of Ret, does not appear to
be a major site of autophosphorylation. Results using both the EGFR/Ret
chimera and Ret/ptc2 thus indicated that interaction with Enigma
required the C terminus containing Tyr-586 but was independent of
tyrosine autophosphorylation of Ret.
(Y539F) had no significant
effect on mitogenic activity, while mutation of Tyr-586 to Phe or
deletion of the C terminus of Ret/ptc2 completely blocked the ability
of Ret/ptc2 to induce DNA synthesis (Fig.
3a).
Fig. 3.
Effect of mutations in Ret that disrupt
association with Enigma and of co-expression with the LIM domains of
Enigma on mitogenic activity of RET/ptc2. a, effects of
mutations in Ret/ptc2. Serum-starved mouse fibroblasts (10T1/2) were
microinjected with plasmids expressing either wild-type Ret/ptc2 or
various mutants and then assessed for entry into S-phase by
immunofluorescent detection of 5-bromodeoxyuridine (BrdU)
incorporation. The fraction of injected cells positive for
5-bromodeoxyuridine incorporation is shown with error bars
displaying the 95% confidence interval calculated using the standard
error of proportion. The numbers in parentheses
are the total number of injected cells. Plasmids were injected at a
concentration of 100 µg/ml. Asterisk denotes a highly
significant difference between cells injected with Ret/ptc2 or Y586F
and Ret/ptc2 or C574 (p < 0.001). b, effects of
co-expression of Enigma and its fragments. Serum-starved fibroblasts
were microinjected with a mixture of two of the following expression
plasmids: Ret/ptc2 or RI/EGFR, a construct analogous to Ret/ptc2 with
the EGFR intracellular domain in place of the Ret kinase, plus either
con, control empty plasmid; C275, the C-terminal
275 residues of Enigma containing LIM domains 1, 2, and 3;
FL, full-length Enigma; N279, N-terminal 279
residues of Enigma lacking LIM domains; or Zyxin, LIM
domains from Zyxin, residues 339-452. In each case Ret/ptc2 and
RI/EGFR constructs were injected at 100 µg/ml, while the other
constructs were present at 200 µg/ml. Double asterisk
denotes a highly significant difference between cells injected with
Ret/ptc2+con or Ret/ptc2+C275 (p < 0.001).
§
Supported by the Markey Charitable Trust as a Fellow and currently
supported by National Institutes of Health Training Grant NCI T32
CA09523.
Supported by United States Army Breast Cancer Research
Fellowship DAMD 17-94-J-4124.
To whom correspondence should be addressed: 9500 Gilman Dr.,
0654, University of California, San Diego, La Jolla, CA 92093-0654.
Tel.: 619-534-3677; Fax: 619-534-8193; E-mail: staylor{at}ucsd.edu.
1
The abbreviations used are: ptc, papillary
thyroid carcinoma; RI, type I regulatory subunit of cyclic
AMP-dependent protein kinase; PLC, phospholipase C-;
GST, glutathione S-transferase; EGF, epidermal growth
factor; EGFR, epidermal growth factor receptor; SH2, Src homology
2.
2
Wu, R.-Y., Durick, R., Songyang, Z., Cantley, L.
C., Taylor, S. S., and Gill, G. N. (1996) J. Biol. Chem., in
press.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Chattopadhyay, M. Vecchi, Q.-s. Ji, R. Mernaugh, and G. Carpenter The Role of Individual SH2 Domains in Mediating Association of Phospholipase C-gamma 1 with the Activated EGF Receptor J. Biol. Chem., September 10, 1999; 274(37): 26091 - 26097. [Abstract] [Full Text] [PDF]
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J. Wang, H. Dai, N. Yousaf, M. Moussaif, Y. Deng, A. Boufelliga, O. R. Swamy, M. E. Leone, and H. Riedel Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis Mol. Cell. Biol., September 1, 1999; 19(9): 6217 - 6228. [Abstract] [Full Text] [PDF]
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K. K. Murthy, K. Clark, Y. Fortin, S.-H. Shen, and D. Banville ZRP-1, a Zyxin-related Protein, Interacts with the Second PDZ Domain of the Cytosolic Protein Tyrosine Phosphatase hPTP1E J. Biol. Chem., July 16, 1999; 274(29): 20679 - 20687. [Abstract] [Full Text] [PDF]
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Q. Zhou, P. Ruiz-Lozano, M. E. Martone, and J. Chen Cypher, a Striated Muscle-restricted PDZ and LIM Domain-containing Protein, Binds to alpha -Actinin-2 and Protein Kinase C J. Biol. Chem., July 9, 1999; 274(28): 19807 - 19813. [Abstract] [Full Text] [PDF]
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R. K. Goyal, P. Lin, J. Kanungo, A. S. Payne, A. J. Muslin, and G. D. Longmore Ajuba, a Novel LIM Protein, Interacts with Grb2, Augments Mitogen-Activated Protein Kinase Activity in Fibroblasts, and Promotes Meiotic Maturation of Xenopus Oocytes in a Grb2- and Ras-Dependent Manner Mol. Cell. Biol., June 1, 1999; 19(6): 4379 - 4389. [Abstract] [Full Text] [PDF]
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 R. M. Melillo, M. V. Barone, G. Lupoli, A. M. Cirafici, F. Carlomagno, R. Visconti, B. Matoskova, P. P. Di Fiore, G. Vecchio, A. Fusco, et al. Ret-mediated Mitogenesis Requires Src Kinase Activity Cancer Res., March 1, 1999; 59(5): 1120 - 1126. [Abstract] [Full Text] [PDF]
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L. Q. Dong, S. Porter, D. Hu, and F. Liu Inhibition of hGrb10 Binding to the Insulin Receptor by Functional Domain-mediated Oligomerization J. Biol. Chem., July 10, 1998; 273(28): 17720 - 17725. [Abstract] [Full Text] [PDF]
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K. Durick, G. N. Gill, and S. S. Taylor Shc and Enigma Are Both Required for Mitogenic Signaling by Ret/ptc2 Mol. Cell. Biol., April 1, 1998; 18(4): 2298 - 2308. [Abstract] [Full Text]
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S. Xing, T. L. Furminger, Q. Tong, and S. M. Jhiang Signal Transduction Pathways Activated by RET Oncoproteins in PC12 Pheochromocytoma Cells J. Biol. Chem., February 27, 1998; 273(9): 4909 - 4914. [Abstract] [Full Text] [PDF]
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L. J.-s. Huang, K. Durick, J. A. Weiner, J. Chun, and S. S. Taylor Identification of a Novel Protein Kinase A Anchoring Protein That Binds Both Type I and Type II Regulatory Subunits J. Biol. Chem., March 21, 1997; 272(12): 8057 - 8064. [Abstract] [Full Text] [PDF]
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S.'i. Kuroda, C. Tokunaga, Y. Kiyohara, O. Higuchi, H. Konishi, K. Mizuno, G. N. Gill, and U. Kikkawa Protein-Protein Interaction of Zinc Finger LIM Domains with Protein Kinase C J. Biol. Chem., December 6, 1996; 271(49): 31029 - 31032. [Abstract] [Full Text] [PDF]
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R.-y. Wu, K. Durick, Z. Songyang, L. C. Cantley, S. S. Taylor, and G. N. Gill Specificity of LIM Domain Interactions with Receptor Tyrosine Kinases J. Biol. Chem., July 5, 1996; 271(27): 15934 - 15941. [Abstract] [Full Text] [PDF]
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 J. Grimm, M. Sachs, S. Britsch, S. Di Cesare, T. Schwarz-Romond, K. Alitalo, and W. Birchmeier Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation J. Cell Biol., July 23, 2001; 154(2): 345 - 354. [Abstract] [Full Text] [PDF]
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