©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tyrosines outside the Kinase Core and Dimerization Are Required for the Mitogenic Activity of RET/ptc2 (*)

(Received for publication, July 26, 1995)

Kyle Durick (1)(§) Virginia J. Yao (1)(¶) Maria Grazia Borrello (2) Italia Bongarzone (2) Marco A. Pierotti (2) Susan S. Taylor (1)(**)

From the  (1)Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654 and (2)Divisione di Oncologia Sperimentale A, Istituto Nazionale Tumori, I20133 Milan, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 Ialpha regulatory subunit of protein kinase A (RIalpha) (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 RIalpha 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 RIalpha 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.


INTRODUCTION

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,^1 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 Ialpha regulatory subunit of cyclic-AMP-dependent protein kinase (RIalpha)(10) . The chimeric gene encodes a protein of 596 residues, which contains the first two-thirds of RIalpha 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 RIalpha 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 RIalpha were required to elicit the mitogenic activity of RET/ptc2. Only the amino-terminal region of RIalpha, which encodes a dimerization domain(12) , fused to the RET was necessary. This portion of RIalpha 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.


EXPERIMENTAL PROCEDURES

Construction of Mammalian Expression Plasmids

The cDNA coding for wild-type RET/ptc2 was excised from a pMAM-neo expression vector previously described (11) using the restriction enzyme XbaI. This fragment was then subcloned into the XbaI restriction site of the pRc/CMV mammalian expression vector (Invitrogen). Restriction digests were performed to screen for orientation, and then the entire cDNA sequence was verified using dideoxy sequencing (13) with Sequenase version 2.0 (U. S. Biochemical Corp.).

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) .

Cell Culture and Microinjection

Mouse 10T1/2 fibroblasts were plated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (DMEM + 10% FBS). The cells were maintained at 37 °C in a 10% CO(2) atmosphere and split just before reaching confluence.

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(2), 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).

Immunostaining

For detection of RET/ptc2 protein, cells were fixed in 3.7% formaldehyde 5 h after injection for 5 min, and then washed with phosphate-buffered saline. The cells were then incubated successively with rabbit anti-RET (dilution 1:500), biotinylated donkey anti-rabbit IgG (dilution 1:400, Jackson ImmunoResearch, West Grove, PA), Texas Red streptavidin (dilution 1:100, Amersham Corp.), and FITC anti-guinea pig IgG (dilution 1:100, Jackson). The anti-RET antibody is a rabbit polyclonal antibody raised against a synthetic peptide corresponding to residues 535-551 of RET/ptc2 conjugated to keyhole limpet hemocyanin (Calbiochem).

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).


RESULTS AND DISCUSSION

Development of Mitogenic Assay

To evaluate which structural features of RET/ptc2 are essential for its mitogenic activity, an expression plasmid microinjection assay was developed. This assay allows for both rapid screening of protein expression and evaluation of mitogenic activity. The expression vector, pRET/ptc2, was co-injected into nuclei of serum-starved 10T1/2 fibroblasts with rabbit or guinea pig IgG, which served as an injection marker. After incubation for 24 h in 5-bromodeoxyuridine (BrdUrd)-containing starvation medium, the cells were fixed and stained for the IgG injection marker and either for RET protein expression (Fig. 1, b and d) or DNA synthesis as assessed by BrdUrd incorporation (Fig. 1, f and h). An identical construct containing the chloramphenicol acetyltransferase gene (pCAT) was used as a negative control.


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.



Consequences of Mutations in ret

Using this assay, the mitogenic activity of various RET/ptc2 point mutants was tested (Fig. 2). To determine whether cAMP binding was important for RET/ptc2 activity, Arg-211 was mutated to Lys. This change eliminates high affinity cAMP binding in the first binding site of RIalpha(17) ; however, this point mutation had no significant effect on RET/ptc2 mitogenic activity. In contrast, elimination of a conserved Lys in the kinase domain(18) , K282R, eliminated in vivo mitogenic activity. Likewise, the three reported Hirschsprung's disease point mutations located in the c-Ret tyrosine kinase domain, S289P, R421Q, and R496G, all inactivated RET/ptc2 in this assay. These results support the model that Hirschsprung's disease results from a loss of c-Ret function and are consistent with recently obtained results using transfection assays on NIH3T3 and PC12 cell lines(19) . The MEN2B mutation (M442T) had no effect on this activated form of c-Ret, which does not contradict a recent report that this mutation alters substrate specificity rather than catalytic activity of c-ret(8) .

Role of RIalpha in RET/ptc2

The NH(2) terminus of RET/ptc2 comprises the first 236 residues of RIalpha, which includes the RIalpha dimerization domain, the cAMP-dependent protein kinase autoinhibitory site, and most of RIalpha's first cAMP binding domain(20) . This is fused to the cytoplasmic portion of the RET receptor, which consists of its tyrosine kinase domain (RET) followed by a short COOH-terminal tail. The wild-type RIalpha subunit is a disulfide-bonded dimer with the protomers aligned through residue 37 in an antiparallel orientation(12) . Constructs expressing deletion mutants were tested to determine the role of RIalpha in the activation of RET (Fig. 3). These mutants showed clearly that the only portion of RIalpha required for RET/ptc2 mitogenic activity was the dimerization domain (Fig. 3a) and support the hypothesis that the RET is activated in RET/ptc2 via the dimerization domain of RIalpha. One model for receptor tyrosine kinase activation is through ligand-induced dimerization, or oligomerization, followed by trans-phosphorylation (21, 22) . This would occur through a parallel orientation of dimerized receptors, since both monomers are membrane-bound. In contrast, the dimerization domain of RIalpha may provide an antiparallel orientation (12) for the linked monomers, and we are currently investigating the structural basis for this novel receptor tyrosine kinase activation mechanism in vitro.


Figure 3: Mitogenic activity of RET/ptc2 deletion mutants. a, schematic representation of the deletion mutants tested. In the RIalpha 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, RIalpha/EGF receptor kinase chimera, or RIalpha/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 RIalpha-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 RIalpha 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 RIalpha/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 RIalpha/EGF than for COOH-terminal truncations of the holo-EGF receptor.

Role of Tyrosines in RET/ptc2

To identify possible SH2 interaction sites in RET/ptc2, several tyrosines were mutated to Phe and the resultant mutants tested for mitogenic activity (Fig. 4). Of the seven Tyr mutants, two, Y350F and Y424F, exhibited significantly reduced mitogenic activity. Mutations at two other tyrosines, Y505F and Y586F, abolished the mitogenic effect. Tyr-350 is located in a variable insert region of receptor tyrosine kinases, and in the platelet-derived growth factor receptor this insert region contains two SH2 binding sites(26) . Tyr-424 in RET/ptc2 corresponds to Tyr-1158 in the insulin receptor(26) , and phosphorylation at Tyr-1158 is required for full activity(27) . Tyr-505 is in a sequence that aligns with the H-helix of the insulin receptor tyrosine kinase(18, 28) . This tyrosine is highly conserved in the receptor tyrosine kinases (26) and could serve a structural role rather than as an SH2 docking site. Tyr-586, which completely eliminated mitogenic activity when mutated to Phe, is in the carboxyl-terminal tail of the RET.


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.


FOOTNOTES

*
This research was supported in part by United States Army Grant AIBS1762 (to S. S. T.) and by the Italian Association for Cancer Research and Italian National Research Council Special Project ACRO (to M. A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Markey Charitable Trust as a Fellow; currently supported by National Institutes of Health Training Grant NCI T32 CA09523.

Supported by National Institutes of Health Research Service Award 5T32DK0704417.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: FMTC, familial medullary thyroid carcinoma; MEN, multiple endocrine neoplasia; RIalpha, type Ialpha regulatory subunit of cAMP-dependent protein kinase (protein kinase A); RET, tyrosine kinase domain of the c-Ret receptor; SH2, Src homology 2 domain; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; BrdUrd, bromodeoxyuridine; EGF, epidermal growth factor.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.