![]() ![]() (including Greg Plowman, Gerard Manning, and Sucha Sudarsanam) to characterize the full ePK complements of model eukaryotes from genomic sequence data. Tony Hunter continued to work with bioinformaticians at SUGEN, Inc. īy 1995, with the advent of genome-sequencing projects, the task of cataloging and classifying the members of the ePK superfamily had grown to become too distracting from my funded research and I discontinued my efforts in this area. The 390 ePK domain alignment was made publicly available through the Protein Kinase Resource website. By early 1994, the ePK domain alignment had grown to contain 390 sequences including 205 non-orthologous vertebrate ePKs, and a fourth major ePK group (CMGC, comprising the CDK, MAPK, GSK, and CLK families) had been added through phylogenetic analysis. Over the next 7 years I continued to add new sequences to the alignment as they became available and to construct phylogenetic trees as a means of classifying the burgeoning ePK superfamily. These groupings indicated that ePK domain phylogeny reflects substrate specificity and/or mode of regulation and could therefore serve as a useful classification tool. The phylogenetic tree revealed major clusters including the tyrosine kinases (the TK group), cyclic nucleotide- and calcium-phospholipid-dependent kinases (the AGC group including the PKA, PKG, and PKC families) and calmodulin-dependent kinases (the CAMK group). More detailed discussions of ePK subdomains and conserved residues in relation to crystal structures and catalytic function can be found in. Particularly important for catalytic function are the invariant lysine in subdomain II and the invariant aspartate in subdomain VII that function to anchor and orient ATP, and the invariant aspartate in subdomain VIB which is the likely catalytic base in the phosphotransfer reaction. Binding of Mg-ATP is largely the function of the amino-terminal lobe and hinge region, while peptide-substrate binding is mediated by the carboxy-terminal lobe. Crystal structures show that ePK domains adopt a common fold consisting of amino-terminal and carboxy-terminal lobes connected by a hinge region. The positions of amino-acid residues and motifs highly conserved throughout the ePK superfamily are indicated above the subdomains, using the single-letter amino-acid code with x as any amino acid. The 12 conserved subdomains are indicated by Roman numerals. The alignment (produced manually at the word-processor) defined the boundaries of the eukaryotic protein kinase (ePK) catalytic domain, revealed conserved subdomains that were never interrupted by amino-acid insertions, and identified highly conserved individual amino acids and motifs (Figure 1). In collaboration with the Salk's resident protein kinase guru Tony Hunter and biocomputing specialist Anne Marie Quinn, we aligned the homologous catalytic-domain amino-acid sequences of 65 distinct protein kinases from diverse eukaryotes (including 45 nonorthologous vertebrate enzymes) and constructed a phylogenetic tree to visualize their overall relationships. This interest stemmed from my having identified several novel human protein kinases using a homology-based cDNA cloning strategy and wanting to determine their relationships to other known protein kinases. About sixteen years ago, while working at the Salk Institute, my colleagues and I undertook a comparative analysis of all the available sequences of protein kinase catalytic domains. ![]()
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