(Received for publication, November 10, 1995; and in revised form, December 19, 1995)
From the
The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.
The c-ret proto-oncogene is a member of the receptor tyrosine kinase superfamily and consists of an extracellular ligand binding domain with a cysteine-rich region, a single transmembrane domain, and an intracellular kinase domain(1) . Studies involving transgenic mice lacking the RET receptor have demonstrated a critical function for this receptor in kidney and enteric nervous system development(2) .
Mutations in the ret gene
result in several forms of human disease including familial medullary
thyroid carcinoma (FMTC), ()multiple endocrine neoplasia
types 2A and 2B (MEN2A and -2B), and Hirschsprung's
disease(3, 4, 5, 6) . FMTC is a
familial cancer syndrome characterized by medullary thyroid carcinoma
while MEN2A variably expresses pheochromocytomas, hyperparathyroidism
and rarely Hirschsprung's disease in addition to medullary
thyroid carcinoma(7, 8) . These cancerous states arise
by substitution of a cysteine residue in the extracellular domain of
RET resulting in ligand independent activation of the RET
receptor(9, 10) . MEN2B displays all the
characteristics of MEN2A but is additionally characterized by a more
rapid disease progression, skeletal abnormalities, ganglioneuromas of
the intestinal tract, and mucosal neuromas(7) . In MEN2B, a Met
Thr substitution occurs at position 918 in the kinase
domain(5) . Finally, Hirschsprung's disease is a loss of
function mutation characterized by decreased parasympathetic
innervation of the lower intestine(10) .
It is now well established that the phosphotyrosine residues of the receptor tyrosine kinases can serve as docking sites for molecules containing SH2 domains (11) or PTB domains(12) . In order to elucidate potential RET signaling pathways, we initiated efforts to map the phosphorylation sites of the wild-type, MEN2A, and MEN2B RET receptors. Both the wild-type RET and the MEN2A mutant were autophosphorylated at identical sites. In contrast, the MEN2B mutant showed both the creation of a novel autophosphorylation site and the loss of phosphorylation at other sites. This study suggests that the altered substrate specificity seen in the MEN2B mutant may be the result of the alterations in its pattern of autophosphorylation. Specifically, Grb2 was shown to bind less efficiently to the MEN2B mutant than to wild-type RET. Such changes in the association of RET with downstream signaling molecules suggest a molecular mechanism for the phenotype in the MEN2B versus the MEN2A mutant.
Figure 1: Expression and phosphorylation of RET. A, anti-HA (lanes 1 and 2) and anti-phosphotyrosine (lane 3) immunoblot of anti-HA immunoprecipitates from pRETHA (lanes 1 and 3) and mock-transfected (lane 2) COS cells. Autoradiograph of the wild-type RET (lane 4) and the kinase inactive mutant, K758M (lane 5) following in vitro phosphorylation. B, phosphoamino acid analysis performed on the in vitro labeled wild-type RET. The positions for the sample origin (O), phosphotyrosine (Y), phosphothreonine (T), phosphoserine (S), and free phosphate (Pi) are indicated.
In preparation for
phosphopeptide mapping, immunoprecipitated RET was autophosphorylated in vitro with [-
P]ATP. Consistent
with the Western blotting experiment, a 170-kDa band was labeled
strongly with
P (Fig. 1A, lane
4). To rule out the possibility that the observed phosphorylation
of RET resulted from other kinases that co-immunoprecipitated with RET,
a kinase-inactive form of the receptor was made by replacing lysine 758
with methionine (K758M). According to studies done on other receptor
tyrosine kinases, replacement of this lysine residue is predicted to
inactivate RET by disrupting the ATP binding site(18) . The
K758M mutant was expressed and immunoprecipitated and, as expected, no
phosphorylated band was visible on the SDS-PAGE after the in vitro kinase assay (Fig. 1A, lane 5). An
equivalent amount of RET receptor was produced in each experiment (data
not shown). This result confirmed that the tyrosine phosphorylation of
RET was due to autophosphorylation. To verify that RET was
phosphorylated due to its tyrosine kinase activity, a phosphoamino acid
analysis was performed on the labeled receptor. As shown in Fig. 1B, phosphorylated RET contained phosphotyrosine
but no phosphoserine or phosphothreonine.
Analysis of the two-dimensional map for the MEN2A mutant receptor
revealed 12 major P-labeled peptides (Fig. 2, A and C). The phosphopeptide map of the wild-type receptor
did not show any significant difference compared to the MEN2A receptor,
indicating that the MEN2A mutation does not affect the
autophosphorylation state of RET. In addition, an
NH
-terminal tagged receptor generated an identical
phosphopeptide map demonstrating that the COOH-terminal HA peptide does
not interfere with autophosphorylation (data not shown). In contrast,
the phosphopeptide map of the MEN2B mutation differed dramatically from
the wild-type RET pattern (Fig. 2B). Notably, the MEN2B
phosphopeptide map showed an absence of phosphopeptide 5, a diminished
phosphopeptide 1, and the appearance of an unidentified phosphopeptide
which runs very close to phosphopeptide 3. Phosphoamino acid analysis
confirmed that the MEN2B receptor was still phosphorylated exclusively
on tyrosine (data not shown).
Figure 2: Comparison of the phosphopeptide maps of MEN2A and MEN2B mutants of RET. Two-dimensional peptide maps of RET receptors bearing the MEN2A (C634S) (A) and MEN2B (M918T) (B) mutations. The numbered arrows in B indicate the positions of the diminished phosphopeptides in the peptide map caused by the MEN2B mutation. The arrow with the question mark indicates the position of the newly generated phosphopeptide. A diagram of the 12 major phosphopeptides in the two-dimensional map of the wild-type RET is shown (C) for comparison. Phosphopeptide map of the wild-type RET receptor labeled in vivo (D). New phosphopeptides are indicated by circles. The positions for the sample origin (O) and free phosphate (Pi) are indicated.
To demonstrate that the
phosphorylation obtained in vitro accurately represented
phosphorylation in vivo, COS cells were transfected with the
wild-type and K758M (kinase inactive) receptors and labeled with
[P]orthophosphate. The labeled receptors were
immunoprecipitated and subjected to phosphopeptide mapping as
described. As expected, the in vivo-labeled wild-type receptor
contained all the phosphopeptides obtained in the in vitro labeling experiments as well as additional labeled peptides (Fig. 2D). These additional peptides were also present
in the kinase inactive receptor's phosphopeptide map indicating
that they represent either background labeling or the phosphorylation
of the RET receptor by associated cellular kinases (data not shown).
Figure 3:
Identification of the
autophosphorylation sites in RET. Two-dimensional phosphopeptide
mapping was performed on the wild-type RET and individual Tyr
Phe mutants as described in the text. Compared to the two-dimensional
map of the wild-type RET (A), Y1096F mutant (B)
displayed a missing phosphopeptide number 5 (indicated by the arrow). The origin is labeled and free phosphate is indicated
by Pi.
Interestingly, mutation of Y1096F resulted in the absence of phosphopeptide 5, thereby allowing us to assign one of the phosphopeptides absent in the MEN2B receptor (Fig. 3B). Although this phosphopeptide contains two potential Tyr phosphorylation sites, mutation of Tyr-1090 does not result in the reduction or disappearance of phosphopeptide 5 (data not shown), suggesting that the major phosphorylated residue of this peptide is Tyr-1096. This has major repercussions on RET receptor signaling as this phosphotyrosine and adjacent amino acids represent a potential Grb2 binding site (pYXNX)(20) . Tyr-1096 is also not present in the short form of RET which is truncated near Tyr-1062(1) . It is possible that the disappearance of this putative Grb2 binding site in conjunction with the production of a new phosphopeptide and its alternative signal transduction pathway could result in the more aggressive MEN2B phenotype. Efforts are underway to determine the identity of this new phosphopeptide.
Mutations Y687F, Y826F, and Y1062F caused the absence of spots 1, 2, and 7, respectively (Table 1, data not shown), indicating that these mutations replaced the phosphorylated tyrosines in the corresponding tryptic peptides. We therefore deduce that Tyr-687, Tyr-826, and Tyr-1062 are phosphorylated in the wild-type receptor. On the contrary, we did not observe any altered phosphopeptide pattern with mutants Y660F and Y1090F (as mentioned above), indicating that these two sites are probably not phosphorylated in the activated receptor. We also noticed that mutations Y1015F and Y1029F both caused partial disappearance of spot 10, consistent with the fact that these two tyrosines are located in the same tryptic peptide (Table 1, data not shown).
Based on the phosphopeptide
maps, we can identify six tyrosine phosphorylation sites in RET:
Tyr-687, Tyr-826, Tyr-1062, Tyr-1096, Tyr-1015, and Tyr-1029. By
comparing the peptide map of each mutant to the wild-type receptor, we
can tentatively assign each tryptic peptide containing phosphorylation
sites to individual P-labeled spots on the two-dimensional
map (Table 1).
To confirm one of the assignments made by
site-directed mutagenesis, we synthesized a peptide with a sequence
identical with the tryptic peptide containing the predicted
phosphorylation site, Tyr-687, and tested the migration of the
phosphorylated synthetic peptide on the two-dimensional map. The P-labeled synthetic peptide migrated to the same position
as phosphopeptide 1, which was the assigned position for the tryptic
peptide containing Tyr-687 (data not shown).
Durick et al.(21) recently determined several of the phosphotyrosines
important for mitogenic activity of the RET/ptc2 oncogene. This version
of the RET receptor contains the tyrosine kinase domain fused to the
type I regulatory subunit of protein kinase A (22) . In
their assay, Tyr-826 and Tyr-900 were moderately important for
mitogenic activity, while mutation of Tyr-981 or Tyr-1062 completely
abolished mitogenic activity. Our analysis confirms that Tyr-826 and
Tyr-1062 are phosphorylated and are contained within phosphopeptides 2
and 7, respectively, on our map.
As mentioned previously,
phosphopeptide 5, which disappears in the MEN2B receptor, contains
potential Grb2 binding sites specified by the consensus sequence
pYXNX(20) . In order to demonstrate Grb2
binding, we expressed wild-type, K758M (kinase inactive), Y1090F,
Y1096F, the Y1090F/Y1096F double mutant, and M918T (MEN2B) in COS
cells. The various receptors were immunoprecipitated to equivalent
levels and allowed to interact with a GST fusion protein containing the
SH2 domain of Grb2 (Fig. 4). As anticipated, wild-type RET bound
the SH2 domain of Grb2 effectively. In contrast, the kinase inactive
RET mutant (K758M) displayed no Grb2 binding, confirming that
autophosphorylation of RET is required for Grb2 association. Analyses
of the RET tyrosine mutants showed that although the Y1090F mutant
bound to Grb2 in a manner similar to wild-type RET, the RET mutants
lacking Y1096 and the MEN2B mutant all demonstrated weak association
with Grb2. However, the Y1096 mutant was capable of binding to an SH2
domain of phospholipase C with the same affinity as the wild-type
receptor demonstrating that the mutant receptor is capable of
interacting with other SH2 domains through other phosphotyrosine
residues (data not shown). It is important to note that Grb2 binding
does not disappear completely, suggesting that other phosphotyrosine
residues may have some affinity for Grb2 binding. While it is clear
that the major binding site of Grb2 is Y1096 in vitro,
confirmation of this result in vivo awaits isolation of the
RET ligand.
Figure 4: Binding of Grb2 to wild-type RET and selected mutants. A, the wild-type and mutant RET receptors were immunoprecipitated from COS cells and autophosphorylated. In vitro binding assays with the GST-SH2 fusion protein containing the SH2 domain of Grb2 were performed as described in the text. The binding of the GST-SH2 fusion protein to RET immunoprecipitates was detected by Western blotting with anti-GST antibodies. B, Western blotting using anti-HA antibodies was performed to ensure that equal amounts of RET receptors were present in each case.
Our analyses have resulted in the assignment of several autophosphorylation sites located in the juxtamembrane, kinase insert, and COOH-terminal tail of the RET receptor. In addition, we have determined a major difference in the phosphopeptide maps between the MEN2A and MEN2B mutants. This change in enzymatic activity results in the loss of a Grb2 binding site while simultaneously creating a new phosphotyrosine peptide with presumably new signaling capabilities. We are pursuing the identity of this new phosphopeptide and continuing our determination of the phosphorylation status of the remaining tyrosines in the RET kinase domain. The successful completion of our experiments will provide us with a more complete picture of RET signaling and will put us in a position to address the different mechanisms by which the RET mutants affect various second messenger cascades.