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Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

Chen LI, Webster MK, Meyer AN, Donoghue DJ - J. Cell Biol. (1997)

Bottom Line: The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu.Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation.These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0367, USA.

ABSTRACT
The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

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Immunoprecipitation/kinase assay of consensus mutants. Lysates from transfected cells, labeled metabolically with  [35S]Cys and [35S]Met, were subjected to immunoprecipitation using monoclonal antibody 7.16.4, as described in Materials and  Methods. (A) Kinase assay. Immunoprecipitated lysates were  subjected to in vitro kinase reactions using γ-[32P]ATP. 32P-labeled  proteins were detected by SDS-PAGE and autoradiography. Exposure time was 21 h. (B) Expression. To demonstrate equivalent  levels of protein expression for different mutants, identical aliquots of immunoprecipitated lysates as used in A were analyzed  by SDS-PAGE followed by fluorography to detect 35S-labeled  proteins. Exposure time was 3 d. The samples shown in lanes 2, 4,  and 6 represent nontransforming clones, while the samples shown  in lanes 3, 5, and 7 represent transforming derivatives. Lane 1,  mock; lane 2, p185c-neu; lane 3, p185neu; lane 4, CONS.CE→ V; lane  5, CONS.C; lane 6, CONS.AE→ S; lane 7, CONS.AE→ Q.
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Figure 7: Immunoprecipitation/kinase assay of consensus mutants. Lysates from transfected cells, labeled metabolically with [35S]Cys and [35S]Met, were subjected to immunoprecipitation using monoclonal antibody 7.16.4, as described in Materials and Methods. (A) Kinase assay. Immunoprecipitated lysates were subjected to in vitro kinase reactions using γ-[32P]ATP. 32P-labeled proteins were detected by SDS-PAGE and autoradiography. Exposure time was 21 h. (B) Expression. To demonstrate equivalent levels of protein expression for different mutants, identical aliquots of immunoprecipitated lysates as used in A were analyzed by SDS-PAGE followed by fluorography to detect 35S-labeled proteins. Exposure time was 3 d. The samples shown in lanes 2, 4, and 6 represent nontransforming clones, while the samples shown in lanes 3, 5, and 7 represent transforming derivatives. Lane 1, mock; lane 2, p185c-neu; lane 3, p185neu; lane 4, CONS.CE→ V; lane 5, CONS.C; lane 6, CONS.AE→ S; lane 7, CONS.AE→ Q.

Mentions: To confirm kinase activation of the mutants described here, selected mutants were examined for receptor activation using an immunoprecipitation/kinase assay of transfected Cos-1 cells. As shown in Fig. 7 A, p185neu exhibited approximately threefold greater autophosphorylation than p185c-neu (lanes 3 and 2, respectively). The mutants CONS.CE→ V and CONS.C were examined, as they represent an interesting pair of closely related mutants. The first of these is inactive in transformation assays, whereas the latter mutant is active. In the immunoprecipitation/kinase assay, the biologically active mutant CONS.C exhibited a similar increase in autophosphorylation compared with the inactive receptor CONS.CE→ V (lanes 5 and 4, respectively). We also examined another pair of closely related mutants, CONS.AE→ S and CONS.AE→ Q, where once again the first mutant is inactive in transformation assays, but the latter mutant is active. Once again, in the immunoprecipitation/kinase assay, the biologically active mutant CONS.AE→ Q exhibited an increase in autophosphorylation compared with the inactive mutant CONS.AE→ S (lanes 7 and 6, respectively). In this experiment, similar levels of protein expression were achieved for p185c-neu, p185neu, and the various mutants examined, as demonstrated by 35S-metabolically labeled/immunoprecipitated proteins from the same lysates (Fig. 7 B). Thus, consistent with prior experimental results from other laboratories (Bargmann and Weinberg, 1988a; Stern et al., 1988; Weiner et al., 1989a,b; Cao et al., 1992), biologically active derivatives constructed in this work exhibited increased levels of kinase activity, as determined by receptor autophosphorylation in immunoprecipitation/kinase assays.


Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

Chen LI, Webster MK, Meyer AN, Donoghue DJ - J. Cell Biol. (1997)

Immunoprecipitation/kinase assay of consensus mutants. Lysates from transfected cells, labeled metabolically with  [35S]Cys and [35S]Met, were subjected to immunoprecipitation using monoclonal antibody 7.16.4, as described in Materials and  Methods. (A) Kinase assay. Immunoprecipitated lysates were  subjected to in vitro kinase reactions using γ-[32P]ATP. 32P-labeled  proteins were detected by SDS-PAGE and autoradiography. Exposure time was 21 h. (B) Expression. To demonstrate equivalent  levels of protein expression for different mutants, identical aliquots of immunoprecipitated lysates as used in A were analyzed  by SDS-PAGE followed by fluorography to detect 35S-labeled  proteins. Exposure time was 3 d. The samples shown in lanes 2, 4,  and 6 represent nontransforming clones, while the samples shown  in lanes 3, 5, and 7 represent transforming derivatives. Lane 1,  mock; lane 2, p185c-neu; lane 3, p185neu; lane 4, CONS.CE→ V; lane  5, CONS.C; lane 6, CONS.AE→ S; lane 7, CONS.AE→ Q.
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Figure 7: Immunoprecipitation/kinase assay of consensus mutants. Lysates from transfected cells, labeled metabolically with [35S]Cys and [35S]Met, were subjected to immunoprecipitation using monoclonal antibody 7.16.4, as described in Materials and Methods. (A) Kinase assay. Immunoprecipitated lysates were subjected to in vitro kinase reactions using γ-[32P]ATP. 32P-labeled proteins were detected by SDS-PAGE and autoradiography. Exposure time was 21 h. (B) Expression. To demonstrate equivalent levels of protein expression for different mutants, identical aliquots of immunoprecipitated lysates as used in A were analyzed by SDS-PAGE followed by fluorography to detect 35S-labeled proteins. Exposure time was 3 d. The samples shown in lanes 2, 4, and 6 represent nontransforming clones, while the samples shown in lanes 3, 5, and 7 represent transforming derivatives. Lane 1, mock; lane 2, p185c-neu; lane 3, p185neu; lane 4, CONS.CE→ V; lane 5, CONS.C; lane 6, CONS.AE→ S; lane 7, CONS.AE→ Q.
Mentions: To confirm kinase activation of the mutants described here, selected mutants were examined for receptor activation using an immunoprecipitation/kinase assay of transfected Cos-1 cells. As shown in Fig. 7 A, p185neu exhibited approximately threefold greater autophosphorylation than p185c-neu (lanes 3 and 2, respectively). The mutants CONS.CE→ V and CONS.C were examined, as they represent an interesting pair of closely related mutants. The first of these is inactive in transformation assays, whereas the latter mutant is active. In the immunoprecipitation/kinase assay, the biologically active mutant CONS.C exhibited a similar increase in autophosphorylation compared with the inactive receptor CONS.CE→ V (lanes 5 and 4, respectively). We also examined another pair of closely related mutants, CONS.AE→ S and CONS.AE→ Q, where once again the first mutant is inactive in transformation assays, but the latter mutant is active. Once again, in the immunoprecipitation/kinase assay, the biologically active mutant CONS.AE→ Q exhibited an increase in autophosphorylation compared with the inactive mutant CONS.AE→ S (lanes 7 and 6, respectively). In this experiment, similar levels of protein expression were achieved for p185c-neu, p185neu, and the various mutants examined, as demonstrated by 35S-metabolically labeled/immunoprecipitated proteins from the same lysates (Fig. 7 B). Thus, consistent with prior experimental results from other laboratories (Bargmann and Weinberg, 1988a; Stern et al., 1988; Weiner et al., 1989a,b; Cao et al., 1992), biologically active derivatives constructed in this work exhibited increased levels of kinase activity, as determined by receptor autophosphorylation in immunoprecipitation/kinase assays.

Bottom Line: The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu.Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation.These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0367, USA.

ABSTRACT
The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

Show MeSH
Related in: MedlinePlus