ALSCRIPT (2) figure showing alignment of H-Ryk with other RTKs. The alignment of IRK and FGFR tyrosine kinase was performed by the STAMP structural alignment package (61), and the locations of alpha helices (blue cylinders) and beta strands (magenta arrows) for these two kinases, as assigned by DSSP (34) are shown at the bottom. Alignment of IRK with the others was performed using Clustal W (74) and merged with the STAMP alignment. Numbering above the alignment corresponds to H-Ryk. Boxed regions indicate approximate location of the 11 kinase subdomains described by Hanks et al. (22, 23), which are labelled below the aligned sequences. Residues are colored red if they show total conservation across the kinases in the alignment, yellow if they show conservation of hydrophobic character (73), green for polar character, and blue for small character. Residues in H-Ryk described in the text are indicated by circles above the H-Ryk sequence. The National Center for Biotechnology Information protein accession numbers for H-Ryk, H-Cck4, M-Mep1, H-Ror1, H-ErbB3, IR, and FGFR are 1710811, 2136061, 1911183, 346351, 119534, 124529, and 120046, respectively; the protein databank codes for IR and FGFR are 1irk and 1fgi, respectively (4).

In vitro kinase assay of the TrkA and TrkA:Ryk chimeric receptors. Cells were incubated in DMEM supplemented with 0.1% FCS for 24 h prior to immunoprecipitation (I.P.) with MAb 5C3. Half of the sample was used in the in vitro kinase assay (A), while the other half was used to determine the relative amounts of the receptors by Western immunoblotting (I.B.) (B and C). Lanes 1 and 2 in panels A and B represent the TrkA receptor, lanes 3 and 4 in panels A and C represent the TrkA:Ryk chimeric receptor, and lanes 5 and 6 in panels A and C represent the immune complex from the NIH 3T3 parental cell line. Lanes 1, 3, and 5 and lanes 2, 4, and 6 represent before and after 30 min of stimulation with NGF, respectively. The exogenous tyrosine kinase assay was carried out in the presence of 10 mM synthetic Src peptide (D) and 0.2 mg of poly(Glu⁸⁰Tyr²⁰) substrate per ml (E). The assay was carried out in the presence of 10 mM MgCl₂ ions (TrkA and TrkA:Ryk) or 10 mM MgCl₂ and 2 mM Mn²⁺ in the case of the TrkA:Ryk* chimeric receptor. The relative specific activity was determined by calculating the nanomoles of ATP transferred per minute of the reaction by 1.00 mg of total protein.

Activation of the catalytically inactive TrkA:Ryk chimera stimulates the MAPK pathway. The TrkA:Ryk chimera-expressing cell line was made quiescent by incubation in DMEM supplemented with 1% FCS for 24 h followed by another 24 h of incubation in DMEM containing 0.1% FCS prior to stimulation with 100 ng of NGF per ml. The lysates were immunoprecipitated (I.P.) with the Erk1- or Rsk3-specific antibodies and subjected to either immunoblotting (I.B.) with the antiphosphotyrosine antibody (A) or the S6 peptide kinase assay (B). (C) Tyrosine phosphorylation mediated by the TrkA:Ryk K334A chimeric receptor. The TrkA:Ryk K334A chimera-expressing cell line was serum starved for 16 h prior to stimulation with NGF for the time periods indicated. Immunoblotting with 4G10 indicates significantly lower levels of coimmunoprecipitating pp75 compared to the wild-type TrkA:Ryk chimera (prolonged exposure, 30 min). The bottom panel indicates the relative levels of TrkA:Ryk K334A chimera immunoprecipitated. The dominant negative K334A mutant abolishes Erk (D) and p90 Rsk3 (G) activation. Total cellular proteins were resolved by SDS-PAGE and immunoblotted with phospho-Erk (D) and Erk (E). This analysis revealed the absence of Erk activation. (F) Total cellular lysates were equalized for protein content prior to immunoprecipitation with the p90 Rsk3 antibody. Half of the immune complex was used for immunoblot analysis, while the other half was used in the S6 peptide kinase assay (G). The S6 peptide kinase assay demonstrates that the TrkA:Ryk K334A chimera abolishes p90 Rsk3 activation.

Stimulation of the TrkA:Ryk Q307G (A) and TrkA:Ryk Q307G/N454F/A455G (B) chimera-expressing cell lines in response to NGF. Serum-starved cells (DMEM, 0.1% FCS, 16 h) were stimulated with NGF (100 ng/ml). After cell lysis, the cell lysates immunoprecipitated with TrkA MAb 5C3. Immunoprecipitates were analyzed by Western immunoblotting (I.B.) with the antiphosphotyrosine antibody (top panel) and blotted with the H-Ryk C-terminal antibody (bottom panel). (C) Phosphorylation of the synthetic poly(Glu⁸⁰Tyr²⁰) substrate by the TrkA:Ryk Q307 and Q307G/N454F/A455G chimeric mutants. The graph represents the mean of two independent experiments. (D and E) Activation of the Erk MAPK pathway by the TrkA:Ryk Q307 (D) and Q307G/N454F/A455G (E) chimeric mutants in response to NGF stimulation for the time period indicated.

Homology modelling of the H-Ryk catalytic domain. Alignment of sequences of H-Ryk and IRK and manual modelling of three nonconservative mutations within H-Ryk were done as described in Materials and Methods. Corresponding residues of IRK (A) and H-Ryk (B) are labelled. The model is magnified to display the cleft between the two lobes.

Characterization of the TrkA:Ryk chimera-expressing stable cell lines. (A) Schematic diagram of the chimeric fusion constructs consisting of the TrkA extracellular domain (nucleotides 1 to 1444) and the H-Ryk transmembrane (T.M.), juxtamembrane (J.M.), kinase (K.D.), and C-terminal (nucleotides 647 to 2300) domains. The TrkA:Ryk chimeric mutants are labelled according to amino acid change and location; thus, mutant Q307G corresponds to a glutamine-to-glycine change at amino acid 307. (B) Detection of chimeric receptors in NIH 3T3 transfectants. Equal amounts of cell extracts from the NIH 3T3 transfectants were immunoprecipitated (I.P.) with the anti-C-terminal H-Ryk antibody 15.2 and then resolved on an SDS–7.5% polyacrylamide gel under nonreducing conditions. The chimeric receptor proteins were detected by immunoblotting (I.B.) with anti-N-terminal TrkA MAb 5C3. The observed molecular mass (125 kDa) is slightly higher than that observed under reducing conditions. Lanes 1 to 9 represent TrkA:Ryk wild type, TrkA:Ryk Q307G, TrkA:Ryk F332A, TrkA:Ryk K4343R, TrkA:Ryk N454F, TrkA:Ryk A455G, TrkA:Ryk N454F/A455G, TrkA:Ryk Q307G/N454F/A455G chimeras, and the parental NIH 3T3 cell line respectively.

(A) Analysis of total cell proteins for NGF-induced changes in tyrosine phosphorylation of cells expressing the wild-type TrkA:Ryk chimera. The TrkA:Ryk chimera-expressing cell line was cultured in DMEM supplemented with 0.1% for 16 h and then stimulated with NGF for the time periods indicated. The cells were lysed with 2× SDS sample buffer, and an aliquot of the lysate was resolved by SDS-PAGE. Tyrosine-phosphorylated proteins in the total cell lysate were visualized by immunoblotting (I.B.) with the antiphosphotyrosine antibody 4G10. The major phosphorylated bands are indicated by arrows. (B and C) Tyrosine phosphorylation mediated by the TrkA:Ryk chimeric receptor and the TrkA receptor (D). Cells were incubated for 16 h in DMEM containing 0.1% FCS prior to stimulation with NGF (100 ng/ml) for the time period indicated. After stimulation, the cell extracts were equalized for protein content, immunoprecipitated with TrkA MAb 5C3, and immunoblotted with antiphosphotyrosine antibody 4G10. The arrows indicate the coimmunoprecipitated phosphotyrosine-containing 75-kDa protein. The blots were stripped and blotted with either the H-Ryk anti-C terminal antibody (B and C, bottom panel) or the TrkA antibody (D, bottom panel). (E) Immunoprecipitation analysis of the wild-type TrkA:Ryk chimeric cell line under denaturing conditions. The chimera-expressing cells were made quiescent by incubation for 16 h in DMEM–0.1% FCS for 16 h. The cells were lysed in denaturing buffer (boiling SDS buffer) prior to immunoprecipitation (I.P.). Immunoblotting analysis with 4G10 (top panel) and H-Ryk 15.2 (bottom panel) shows absence of pp75 and presence of the TrkA:Ryk chimeric receptor, respectively.

Analysis of the kinetics of tyrosine phosphorylation of the TrkA:Ryk activation domain chimeric mutants in response to NGF. TrkA:Ryk N454F (A), TrkA:Ryk A455G (B), and TrkA:Ryk N454F/A455G (C) chimera-expressing cell lines were made quiescent by incubation in DMEM medium supplemented with 0.1% FCS for 16 h and then stimulated with 100 ng of NGF per ml for the time periods indicated. The tyrosine-phosphorylated chimeric and associated proteins are indicated with arrows (top panel). The blots were stripped and immunoblotted (I.B.) with H-Ryk polyclonal antibody 15.2 (bottom panel). (D) Phosphorylation of exogenous substrate by the activation domain mutants. The synthetic substrate polyGlu⁸⁰Tyr²⁰ was phosphorylated with labelled ATP. Incorporation of ATP into the substrate was determined by a filter paper assay. The graph represents the mean of two independent experiments. (E to G) Activation of the Erk MAPK pathway by the TrkA:Ryk N454F (E), A455G (F), and N454F/A455G (G) chimeric receptors, respectively.

The TrkA:Ryk F332A (A) and TrkA:Ryk K434R (B) chimeric receptors are catalytically inactive. NIH 3T3 cell lines expressing the various chimeras were made quiescent by incubating them in DMEM supplemented with 0.1% FCS for 16 h. Immunoprecipitates were analyzed for tyrosine phosphorylation by immunoblotting (I.B.) with the antiphosphotyrosine antibody (top panel). The coimmunoprecipitated p75 protein is indicated with an arrow. The blot was stripped and probed with the H-Ryk C-terminal antibody (bottom panel). (C) Substrate phosphorylation assay of the TrkA:Ryk F332A and TrkA:Ryk K434R chimeric receptors. The graph represents the mean of two independent experiments.