The highest observed E-value for that group was taken as the cutoff for confident assignment

ain-Interacting Kinases, CDC-Like Kinases, DualSpecificity Tyrosine Regulated Kinases, and a paralogous superfamily of kinases including LF4, the mammalian RCK kinases, and the fungal IME2 kinases. CMGC kinases coordinate a wide range of cellular functions in different species. CDKs are the major coordinators of cell division in both mitosis and meiosis. MAPKs are crucial for many cellular decisions, including proliferation, differentiation, and stress responses. The Ime2 kinase is crucial for meiosis in S. cerevisiae, while its orthologs in other Ascomycetes control distinct processes including mating, differentiation, and response to light. The Ime2 paralogs in mammals control diverse processes including spermatogenesis and control of retinal cilia-length, as well as intestinal cell biology, control of cell proliferation, organogenesis, and cellular differentiation . Within the evolutionary history of CMGC kinases, gene duplications followed by diversification resulted in multiple paralogous kinases with distinct specificities that coordinate diverse biological functions. For example, the specificities of Cdk1 and Ime2 are mostly non-overlapping. In addition to acquiring distinct modes of regulation, it is likely that the divergence of the biological functions of this kinase family is, in part, due to evolution of their primary specificities. Therefore, understanding the mechanisms that drive specificity change and the consequences of these changes is crucial to rationalize the AIC316 structures of modern phosphoregulatory networks. The shared evolutionary history of CMGC kinases, combined with their diverse specificities, make them an ideal gene family for studying the evolution of kinase specificity. In this study, we determined the primary substrate specificity of eight extant kinases from the IME2/RCK/LF4 group of kinases and found variation in the amino acid that is preferred immediately C-terminal to the phosphoacceptor. To determine the mechanisms by which these specificities evolved, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826300 we used maximum likelihood phylogenetic models to reconstruct sequences for all ancestors of the CMGC kinases. We then resurrected seven ancestral kinases in the lineage starting with AncCMGI, which is the last common ancestor of the CDK, CDKL, MAPK, GSK, CLK, and IME2/RCK/LF4 kinases, up until the modern LF4, RCK, and IME2 kinases. Biochemical characterization of these resurrected kinases allowed us to trace the evolution of primary specificity in this lineage. In addition, we determined a key residue that modulates primary specificity at the +1 position. By mutating this residue in modern IME2 we showed that, at least in some circumstances, the cell can readily tolerate changes that expand kinase specificity. Results The Ime2/RCK/LF4 kinase family has variable +1 specificity To understand how kinase specificity changes over a long evolutionary timescale, we determined the phosphorylation site specificities of eight kinases from the superfamily of kinase paralogs that includes fungal Ime2, the mammalian RCK kinases, and the LF4 kinases in algae and protists. This superfamily controls diverse biological processes, and we hypothesized that differences in primary specificity may underlie some of this functional divergence. In addition, Howard et al. eLife 2014;3:e04126. DOI: 10.7554/eLife.04126 3 of 22 Research Article Biochemistry Genomics and Evolutionary Biology previous work has shown that S. cerevisiae Ime2 and mouse ICK differ in their +1 specificiti