The p.R140H mutation occurred right after the ATP-binding P-loop (i.e. Walker A motif), conserved in all kinases. To determine if the mutation alters CLP1 kinase activity we tested recombinant wild type (wt) and mutant GST-tagged human CLP1 protein against a poly(A) RNA oligonucleotide natural kinase substrate in an established assay (Ramirez et al., 2008). Recombinant mutant p.R140H CLP1 was stable to purification (Figure S2A) but displayed defective kinase activity, reduced by more than half of wt levels (Figure 2C). We conclude that CLP1 kinase activity was functionally impaired as a result of the p.R140H mutation.
