Evolution Of Ck1δ/ε Kinase Activity On SR Motif

Orthologous proteins are generally considered to be functionally conserved and most functional variations are reported to happen after gene duplication. Although functional variations between orthologs are recently studied, little is known how the variation in protein sequence contributes to the functional divergence of orhtolgous proteins. Moreover, the use of well-designed laboratory experiments for testing this hypothesis is lacking.Here, we focused on the functional evolution of one branch of casein kinase1(CK1) family. CK1family members are serine and threonin specific kinases, whose functions are considered to be highly similar. The CK18/s(DBT, their sole Drosophila ortholog) subfamily participates in circadian clock. Given the high conservation of circadian system in different species and hints for different effects of mammalian CK18/s and Drosophila DBT on circadian rhythm, we examined the functional evolution for DBT and CK1δ/ε through establishing different model animals and experimental verification of their function in circadian system.By studying the reciprocal interaction of CK1δ/ε and DBT with their substrates hPER2and Drosophila PER, we found that DBT is unable to distinguish the consecutive S/TXXS/T (Serine/Threonin Rich, SR) motif of hPER2which is recoganized by CK15and CK1ε. Moreover, our results support the functional analogy between CK1δ and CK1ε, suggesting that the functional divergence happened before gene duplication. Using orthologous gene recombination, we identified a protein fragment located in the previously unidentified functional domain which contains7amino-acid substitutions between DBT and human CK1Χ is responsible for their functional difference on SR motif. The functional difference between DBT and CK18and the functional importance of the identified responsible fragment were further supported by in vitro kinase assay. These results suggest that even subtle sequence differences between orthologs would underlie evolutionary variation and that functional difference can be generated by reconstructing the domains with weak functional importance. Phylogenetic analysis showed that the responsible sequence is highly homologous in vertebrates CK1δ and CK1ε and some invertebrate chordates which contain single CK1δ/ε orhtologs, but diverged in most arthropods, nematodes and cnidarians CK1δ/ε orthologs, indicating that the functional variation may have happened before the diversification of CK18and CK1ε.Moreover, consistent with the functional evolution of CK1δ/ε, we observed a striking emergence and conservation of CK1δ/ε targeted SR motif in vertebrates as well as putative increase of conserved SR motif in vertebrate proteins, suggesting that co-evolution between kinase and substrate is needed to build new level network without disruption of organism integrity.