Tracing the origin and evolution of pseudokinases across the tree of life

Abstract

Protein phosphorylation by eukaryotic protein kinases (ePKs) is a fundamental mechanism of cell signaling in all organisms. In model vertebrates, ~10% of ePKs are classified as pseudokinases, which have amino acid changes within the catalytic machinery of the kinase domain that distinguish them from their canonical kinase counterparts. However, pseudokinases still regulate various signaling pathways, usually doing so in the absence of their own catalytic output. To investigate the prevalence, evolutionary relationships, and biological diversity of these pseudoenzymes, we performed a comprehensive analysis of putative pseudokinase sequences in available eukaryotic, bacterial, and archaeal proteomes. We found that pseudokinases are present across all domains of life, and we classified nearly 30,000 eukaryotic, 1500 bacterial, and 20 archaeal pseudokinase sequences into 86 pseudokinase families, including ~30 families that were previously unknown. We uncovered a rich variety of pseudokinases with notable expansions not only in animals but also in plants, fungi, and bacteria, where pseudokinases have previously received cursory attention. These expansions are accompanied by domain shuffling, which suggests roles for pseudokinases in plant innate immunity, plant-fungal interactions, and bacterial signaling. Mechanistically, the ancestral kinase fold has diverged in many distinct ways through the enrichment of unique sequence motifs to generate new families of pseudokinases in which the kinase domain is repurposed for noncanonical nucleotide binding or to stabilize unique, inactive kinase conformations. We further provide a collection of annotated pseudokinase sequences in the Protein Kinase Ontology (ProKinO) as a new mineable resource for the signaling community.

A Structurally-Validated Multiple Sequence Alignment of 497 Human Protein Kinase Domains

“…We first removed 55 pseudokinases from the alignment. Following Kwon et al 23 , these proteins were identified by substitutions in one or more of the three conserved residues crucial for catalysis -DFG-Asp, HRD-Asp and β3-Lys. We then identified the highly conserved residue positions across kinases by the columns which are occupied by the same residue in at least 400 kinases in our MSA (90% of sequences).…”

“…for our MSA. We have also compared the quality of our alignment with the previously published alignments by Manning et al5 , Möbitz16 , Kwon et al23 , a hidden Markov model (HMM) derived from our MSA, and the initial ClustalOmega alignment. The average TPR for our MSA is 0.97, which is significantly better than the Möbitz (0.88), Kwon (0.90), and Manning (0.80) MSAs.…”

A resource for exploring the understudied human kinome for research and therapeutic opportunities

“…The human kinome 5 includes ~50 pseudokinases that lack one or more residues generally required for catalytic activity. These residues include the ATP -binding lysine (K) within the VAIK motif, the catalytic D within the HRD motif and the magnesium binding D within the DFG motif 19 . Many pseudokinases function in signal transduction despite the absence of key catalytic residues.…”

A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

“…We previously showed that FN3Ks belong to a large super-family of protein kinase-like (PKL) enzymes that include eukaryotic protein kinases, small molecule kinases, and atypical kinases (21,22). FN3Ks are more closely related to small molecule kinases, such as aminoglycoside kinase (APH) and choline kinases, than ePKs and more distantly related to atypical pseudokinases such as Fam20C and SelO (23)(24)(25). Through quantitative comparisons of the evolutionary constraints acting on diverse PKL-fold enzymes, we previously demonstrated that ePKs share sequence and structural similarity with small molecule kinases in the N-terminal ATP binding lobe, but diverge significantly in the C-terminal substrate binding lobe (21,22,26).…”