Evolutionary Divergence in the Catalytic Activity of the CAM-1, ROR1 and ROR2 Kinase Domains

<div><p>Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the <i>C</i>. <i>elegans</i> homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.</p></div

Quantitation of cellular ROR1 phosphorylation.

<p>Data from three technical replicate peptide samples from the ROR1 activation loop are analyzed to determine the quantitation limit for phosphotyrosine autophosphorylation of ROR1 overexpressed in and isolated from Wnt5a-treated HEK293 cells.</p

ROR shows limited residual tyrosine autophosphorylation <i>in vitro.</i>

<p>a) ROR, b) CAM-1, c) MuSK and d) Quantitation of signal intensity per pmol of kinase from the above immunoblots. Error bars represent standard error of the mean. ROR proteins were expressed in insect cells and isolated to >95% purity. Phosphotyrosine was detected by anti-phosphotyrosine, after incubation in the presence or absence of magnesium and ATP. The amount of protein loaded is indicated below each lane in nanograms. These data are representative of two or more independent protein expression and isolation experiments. Quantitation data of experimental replicates can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102695#pone.0102695.s002" target="_blank">Figure S2</a>.</p

Comparison of kinetic parameters for MuSK, CAM-1, ROR1 and ROR2 tyrosine phosphorylation activity.

<p>CAM-1 and ROR data were obtained from a [γ−³²P]ATP-based phosphocellulose binding assay. Data are from three technical replicates, ± standard error. ND, Not determined. ^aMuSK data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102695#pone.0102695-Till1" target="_blank">[24]</a>.</p

Domain structures of ROR and MuSK receptors, kinase mutations in ROR and structural analysis of the ROR2 kinase domain.

<p>a) Domain structures of the ROR, CAM-1 and MuSK receptors showing boundaries of constructs used in experiments. b) Alignment of consensus sequence for regions of kinase domain critical for enzyme activity. Variance from consensus kinase sequence found in ROR is highlighted red; consensus residues are highlighted green. Hs; <i>Homo sapiens</i>, Mm; <i>Mus musculus</i>, Gg; <i>Gallus gallus</i>, Dr: <i>Danio rerio</i>, Xl; <i>Xenopus laevis</i>, Xs; <i>Xenopus</i> (Silurana) <i>tropicalis</i>, Dm; <i>Drosophila melanogaster</i>, Ce; <i>Caenorhabditis elegans</i>, Ac; <i>Aplysia californica</i> (sea slug), Nv; <i>Nematostella vectensis</i> (sea anemone), Sp; <i>Strongylocentrotus purpuratus</i> (sea urchin). c) Alignment of human ROR2 kinase domain (PDB ID: 3ZZW pink) with rat MuSK kinase domain (PDB ID: 1LUF yellow, RMSD C-terminal lobe only, 0.4Å, all atom 0.73 Å). ROR2 Tyrosine 555 is shown in the canonical adenine binding site in the apo structure. d) Alignment of human ROR2 kinase domain (pink) with apo IGF-R (PDB ID: 1P4O cyan) and phosphorylated, activated IGF-R (PDB ID: 1K3A, sky blue, RMSD to ROR2 C-terminal lobe only, 0.8Å, all atom 1.5Å). Tyrosines in the ROR2 activation loop are numbered. The non-hydrolyzable ATP analogue AMP-PCP is shown in dark blue. Human ROR2 kinase domain and human IGF-R kinase domain share approximately 40% sequence identity.</p

ROR1 activation loop is not phosphorylated upon Wnt5a stimulation of cells.

<p>Quantitative mass spectrometry reveals little or no phosphorylation of the ROR1 activation loop following Wnt5a treatment. Stable isotope labeled synthetic peptides corresponding to the kinase activation loop of ROR1 were used to determine the extent of phosphorylation of ROR1 following Wnt5a treatment of 293 cells. a) Representative chromatograms of isotopically labeled apo-peptide (Blue) and native isolated apo-peptide (Red). b) Representative chromatograms of isotopically labeled phosphopeptides (Blue) and endogenous isolated peptides for each of the three tyrosines in the activation loop (Red). A, Integrated peak area. Dotted lines indicate expected peak times in the native chromatogram. Y-axis scale is reduced in b) to illustrate the limits of detection and quantitation.</p

ROR ICD is not required for suppression of canonical Wnt signaling. ROR1 and ROR2 inhibit Wnt3a-stimulated transcription of a luciferase reporter from a Lef/Tcf promoter and this activity is enhanced by Wnt5a ligand.

<p>The ROR1 or ROR2 ECD alone can suppress canonical Wnt signaling when anchored to the plasma membrane. The luciferase reporter data presented in this figure are representative of at least three independent experiments and are normalized to untreated vector transfected cells. Error bars represent standard error.</p