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

Abstract

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 C. elegans 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.

Targeting Wnt signaling pseudokinases in hematological cancers

“…The intracellular domains (ICDs) of RORs include a tyrosine kinase‐like domain followed by two serine/threonine‐rich domains flanking a proline‐rich domain, and a short C‐terminus end (Figure ). RORs are considered pseudokinases due to mutation in the canonical motifs found in bona fide kinases and the lack of detectable kinase activity in vitro using purified recombinant proteins . Mice mutated for both ROR1 and ROR2 phenocopy Wnt5a mouse mutants, indicating that ROR receptors bind to and transduce Wnt5a signaling …”

“…RORs are considered pseudokinases due to mutation in the canonical motifs found in bona fide kinases and the lack of detectable kinase activity in vitro using purified recombinant proteins. 6,23 Mice mutated for both ROR1 and ROR2 phenocopy Wnt5a mouse mutants, indicating that ROR receptors bind to and transduce Wnt5a signaling. 20,21 ROR1 expression in healthy hematological tissue is restricted to the intermediate stage of B-lymphocyte precursors, called hematogones (CD10 + , CD19 + , dim CD45 + , CD34 − , and TdT − ).…”

Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1

“…The cw82 (this study) and xd13 alleles (Song et al 2010) are missense mutations in the kinase domain, changing conserved glycines to charged amino acids. A recent study shows that CAM-1, unlike vertebrate Rors, possesses kinase activity in vitro (Bainbridge et al 2014), suggesting that the cw82 and xd13 alleles disrupt kinase activity. The finding that these kinase missense mutations have stronger effects on ALM polarity than the Class 2 mutations that are predicted to eliminate large parts of the intracellular domain is paradoxical.…”

“…However, a recent study using highly purified Ror2 shows that the protein lacks kinase activity in vitro (Bainbridge et al 2014). Ror2 is best characterized as a positive regulator of a non-canonical Wnt signaling pathway, functioning in mouse embryonic fibroblast (MEF) migration (Nishita et al 2006), in mouse hair cell orientation (Yamamoto et al 2008) and in Xenopus convergent extension (Hikasa et al 2002; Schambony and Wedlich 2007).…”

Receptor tyrosine kinase-like orphan receptor 1: a novel target for cancer immunotherapy

“…In addition, Bainbridge et al have recently reported that ROR1 and ROR2 kinase domains have no catalytic activity. They believe that ROR1 kinase activity is due to its contamination with co-factors or other kinases [32]. Murphy et al studied a nucleotide binding property of different pseudokinases using a thermal-shift assay.…”