(Received for publication, July 26, 1995)
From the
Defects in the c-ret proto-oncogene, a member of the
protein tyrosine kinase receptor family, have recently been linked to
two types of genetic syndromes, Hirschsprung's disease and the
multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is
the product of a papillary thyroid carcinoma translocation event
between the genes coding for c-ret and the type I
regulatory subunit of protein kinase A (RI
) (Lanzi, C., Borrello,
M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M.,
Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M.(1992) Oncogene 7, 2189-2194). The resulting 596-residue
protein contains the first two-thirds of RI
and the entire
tyrosine kinase domain of c-ret (RET
). An in
vivo assay of growth stimulatory effects was developed, which
consisted of microinjecting a RET/ptc2 expression plasmid into the
nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of
5-bromodeoxyuridine. This assay was used to determine that only the
dimerization domain of RI
fused to RET
is required
for RET/ptc2's mitogenic activity. In addition, all of the
reported Hirschsprung's disease point mutations in the RET
(S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay,
confirming that these are loss of function mutations. Two tyrosines
outside the conserved kinase core were also identified that are
essential for full mitogenic activity of RET/ptc2. These two tyrosines,
Tyr-350 and Tyr-586, are potential sites for Src homology 2 and
phosphotyrosine binding domain interactions.
The ret oncogene was discovered in transfection experiments where DNA from human lymphomas was introduced into NIH3T3 cells(1) . This oncogene was not expressed in the original lymphoma, but instead had arisen from a rearrangement during the transfection, hence the name ret. The c-ret proto-oncogene encodes a novel receptor tyrosine kinase with a cadherin-like motif in its extracellular domain(2) .
The
c-ret proto-oncogene is responsible for four human disease
syndromes: Hirschsprung's disease, a developmental disorder, and
the dominantly inherited cancer syndromes FMTC, MEN2A, and
MEN2B. Hirschsprung's disease occurs at a rate of approximately 1
in 6000 live births and is characterized by a lack of parasympathetic
innervation of the lower intestine. Occurrence of this disease
corresponds to c-ret gene deletions and nonsense point
mutations leading to truncation of the expressed
protein(3, 4) . Also, three mutations resulting in
single amino acid substitutions in the kinase region of the c-ret gene have been reported in Hirschsprung's
patients(3, 4) . In addition to medullary thyroid
carcenoma, the unique pathologic feature of FMTC, MEN2A and MEN2B
patients display additional hyperplasias. These syndromes have been
linked to point mutations in the c-ret gene(5, 6, 7) . Recent evidence suggests
that, in the case of the MEN2A, these mutations result in the
expression of constitutively activated forms of
c-ret(8) .
It has also been demonstrated that
constitutively active forms of the c-ret proto-oncogene are
present in nearly half of papillary type thyroid
carcinomas(9) . These oncogenic forms of ret are the
result of somatic chromosome translocations or inversions, which fused
the c-ret tyrosine kinase domain (RET) with
different genes. One of the resultant transforming sequences, RET/ptc2
(papillary thyroid carcinoma), was the product of a crossover between
the genes coding for c-ret and the type I
regulatory
subunit of cyclic-AMP-dependent protein kinase (RI
)(10) .
The chimeric gene encodes a protein of 596 residues, which contains the
first two-thirds of RI
and the entire tyrosine kinase domain of
c-ret(11) . RET/ptc2 is transforming, presumably due
to constitutive tyrosine kinase activity, but the structural basis for
RET
activation via fusion to RI
remains unclear.
Here we report the development and use of a microinjection assay to
study the mitogenic activity of RET/ptc2. Deletion mutants were tested
in the assay to determine which portions of RI were required to
elicit the mitogenic activity of RET/ptc2. Only the amino-terminal
region of RI
, which encodes a dimerization domain(12) ,
fused to the RET
was necessary. This portion of RI
was also capable of activating the tyrosine kinase domain of the
epidermal growth factor receptor in this assay system. In addition, all
of the reported Hirschsprung's disease point mutations in the
RET
inactivated RET/ptc2 in our assay. To begin the search
for interaction sites with other signaling proteins, the mitogenic
activity of RET/ptc2 mutants lacking single tyrosines was tested. Two
tyrosines were found to be essential for the mitogenic activity of
RET/ptc2.
Site-directed mutagenesis was performed with the Kunkle method (14) using the Mutagene kit (Bio-Rad). Constructs expressing deletion mutants of RET/ptc2 were made by introducing NheI restriction sites flanking the segment of DNA to be deleted, digesting with NheI, and then ligating the new ends back together. All mutant constructs were sequenced to verify mutagenesis. Supercoiled plasmid DNA expressing various constructs were prepared by double banding in cesium chloride gradients(15) .
For microinjection, cells
were plated on glass coverslips and grown to 70% confluence in DMEM
+ 10% FBS. The coverslips were then transferred to DMEM containing
0.05% calf serum. After 24 h of incubation in the FBS-free medium, the
cells were injected into their nuclei with solutions of injection
buffer (20 mM Tris, pH 7.2, 2 mM MgCl,
0.1 mM EDTA, 20 mM NaCl) containing 100 µg/ml
expression plasmid DNA and 6 mg/ml either guinea pig or rabbit IgG
(Sigma). All microinjection experiments were performed using an
automatic micromanipulator (Eppendorf, Fremont, CA), with glass needles
pulled on a vertical pipette puller (Kopf, Tujunga, CA).
DNA synthesis was assessed through incorporation of the thymidine analog 5-bromodeoxyuridine (BrdUrd) and its subsequent detection by immunostaining(16) . Following nuclear microinjection, 0.1% BrdUrd labeling reagent (Amersham) was added to the starvation medium (DMEM + 0.05% calf serum), and the cells were incubated for an additional 24 h. Cells were fixed in 95% ethanol, 5% acetic acid for 30 min, and then washed with phosphate-buffered saline. Incorporation of 5-bromodeoxyuridine was visualized by successively incubating the fixed cells with mouse anti-bromodeoxyuridine (undiluted, Amersham), biotinylated donkey anti-mouse IgG (dilution 1:500, Jackson), Texas Red streptavidin (dilution 1:100, Amersham), and FITC anti-rabbit IgG (dilution 1:100, Jackson).
Figure 1:
Immunostaining for
RET/ptc2 expression and 5-bromodeoxyuridine (BrdUrd) incorporation in
10T1/2 cells injected with pRET/ptc2 (a and b, e and f) or pCAT (c and d, g and h) expression vectors. a, IgG injection marker
stained with FITC anti-guinea pig IgG. b, same field of cells
as a, but showing Texas Red anti-RET staining. c, IgG injection marker stained with FITC anti-guinea pig IgG. d, Same field of cells as c, but showing Texas Red
anti-RET
staining. e, IgG injection marker
stained with FITC anti-rabbit IgG. f, Same field of cells as e, but showing Texas Red anti-BrdUrd staining. g, IgG
injection marker stained with FITC anti-rabbit IgG. h, Same
field of cells as g, but showing Texas Red anti-BrdUrd
staining.
Cells injected with
pRET/ptc2 expressed protein detectable by anti-RET antibodies as early as 5 h post-injection (Fig. 1b). The expressed protein was cytoplasmic, and
the amount and distribution of expressed protein was indistinguishable
from that observed in pRET/ptc2-injected cells for all of the mutants
discussed in this paper (data not shown). A plasmid concentration of
100 µg/ml was chosen for injections. Under these conditions, over
30% of the pRET/ptc2-injected cells entered S phase, compared to less
than 6% of the pCAT-injected cells (Fig. 2).
Figure 2: Mitogenic activity of RET/ptc2 point mutants. The fraction of injected cells that stained positive for BrdUrd incorporation is shown for various plasmids expressing either wild-type RET/ptc2 or RET/ptc2 point mutants. In each case the plasmids were injected at a concentration of 100 µg/ml. The column labeled background represents uninjected cells under the assay conditions. The error bars show the 95% confidence interval calculated using the standard error of proportion. The number in parentheses is the total number of injected cells.
Figure 3:
Mitogenic activity of RET/ptc2 deletion
mutants. a, schematic representation of the deletion mutants
tested. In the RI subunit the dimerization domain includes
residues 1-84, the hinge region consists of residues 86-99,
and the cAMP binding domain extends from residues 100 through 236. b, the fraction of injected cells that stained positive for
BrdUrd incorporation is shown for various plasmids expressing either
wild-type RET/ptc2, RET/ptc2 deletion mutants, RI
/EGF receptor
kinase chimera, or RI
/EGF receptor kinase without its
carboxyl-terminal tail (see text). Plasmids were injected at a
concentration of 100 µg/ml. The error bars show the 95% confidence
interval calculated using the standard error of proportion. The number
in parentheses is the total number of injected
cells.
To investigate
whether RI-mediated dimerization is a general activating motif for
receptor tyrosine kinases, an analogous construct to RET/ptc2 was made
by substituting the epidermal growth factor receptor tyrosine kinase
(EGF
) for the RET
. The expression construct
encoding RI
residues 1-236 fused to the EGF
and
its COOH-terminal tail, residues 647-1186, was as active as pRET/ptc2
in our microinjection assay (Fig. 3b). The
carboxyl-terminal tail, residues 959-1186, contains all of the known
Src homology 2 domain (SH2) docking sites of the EGF
receptor(23) . Deletion of this tail in the RI
/EGF
construct eliminated its mitogenic response in our assay (Fig. 3b). COOH-terminal truncations of the holo-EGF
receptor have been shown to have increased transforming activity,
attributed to an inability to internalize and attenuate the EGF
signal(24, 25) . The complete loss of mitogenic
activity for our COOH-terminal EGF
deletion suggests that
SH2 domain interactions are more important for the mitogenic response
of RI
/EGF
than for COOH-terminal truncations of the
holo-EGF receptor.
Figure 4: Mitogenic activity of RET/ptc2 tyrosine to phenylalanine mutants. a, the fraction of injected cells which stained positive for BrdUrd incorporation is shown for various plasmids expressing either wild-type RET/ptc2 or RET/ptc2 Tyr to Phe point mutants. Plasmids were injected at a concentration of 100 µg/ml. The column labeled background represents uninjected cells under the assay conditions. The error bars show the 95% confidence interval calculated using the standard error of proportion. The number in parentheses is the total number of injected cells. b, schematic representation of RET/ptc2 showing the locations of the seven tyrosines mutated to phenylalanine. The kinase domain of RET extends from residues 237 to 545 of RET/ptc2, the COOH-terminal tail ends at residue 596, and the platelet-derived growth factor-like insert of RET encompasses residues 344-367. The corresponding tyrosines are shown on the c-Ret schematic(2) .
Tyrosines 586 and 350 are the best candidates for residues that provide SH2 or phosphotyrosine binding domain docking sites essential for the mitogenic response of RET/ptc2. Mutation of the other Tyr in the COOH-terminal tail, Tyr-553, had no effect on mitogenic activity. Results from the Y586F mutant indicate that kinase activity alone is not sufficient for the mitogenic response of RET/ptc2. Work is under way to investigate RET/ptc2 phosphorylation sites and to search for interacting proteins.