The completion of the first draft of the human genome sequence presented an opportunity to determine the full complement of human protein kinases. The first analysis came from a group led by Mitch Kostich at Schering-Plough Research Institute (SPRI) 9. This group mined public GenBank records (available before December, 2001) for ePK sequences by performing BLAST searches using known ePK domains as queries. The resulting hits were consolidated, and efforts were made to remove non-human sequences, pseudogenes, and poor-quality sequences that could represent duplicate hits. The SPRI investigators chose to err on the side of inclusion rather than exclusion, however, and many cases of 'single hit' sequences were retained. Their effort culminated in a collection of 510 potentially unique human ePKs. A color-coded alignment that accompanied their article 9 nicely illustrates the ePK domain sequence conservation.

The SUGEN group, led by Gerard Manning and Sucha Sudarsanam, carried out a more comprehensive effort to describe and classify all human ePKs 10. They employed a dataset that included, in addition to the public databases, genomic reads from Celera that are not publicly available, non-public expressed sequence tags (ESTs) from Incyte and SUGEN, and they searched using a hidden Markov model of the ePK domain that allowed detection of very divergent family members. The sequence data were further searched for members of the various known aPK families. Using stringent criteria to eliminate false positives (including verification of novel sequences by cDNA cloning) they compiled a list of 478 human members of the ePK superfamily and another 40 aPKs, bringing their human kinome total to 518 (approximately 1.7% of all predicted human genes). They also identified 106 ePK or aPK pseudogenes.

A comparison of the SPRI-510 and SUGEN-518 lists reveals 474 protein kinases in common (see the additional file). Of the 44 SUGEN-specific kinases, 32 are aPKs; the other 8 aPKs identified by SUGEN, from the ABC1 and RIO families, were included in the SPRI list as a result of their having weak ePK domain similarity. Of the remaining 12 SUGEN-specific ePKs, five (TAK1, MLKL, NEK5, SgK307, and TBCK) were not available in the public data used in the SPRI analysis; another five (SgK196, SgK223, SgK424, SgK493, and Slob) have rather divergent ePK domains that lack many of the highly conserved residues and are unlikely to have catalytic activity, so it is easy to see how these might have been excluded by visual inspection; and the final two are SgK110 and NEK10. SgK110 was actually detected by the SPRI search, but it was erroneously merged with a related sequence AC008735_EPK1 (SgK069) on the same genomic contig; and it is unclear why the SPRI group missed NEK10. Most, if not all, of the 36 SPRI-specific ePKs represent over-inclusion errors (Table 1): 14 correspond to sequences determined to be pseudogenes by the SUGEN group; 19 are based on single sequences that are (or appear to be) either poor-quality duplicates of other ePKs or interspecies contaminants; and the remaining three are duplicates arising by virtue of non-overlapping partial sequences.

Thus the SUGEN compilation of 478 human ePK superfamily genes represents the accurate count based on current sequence data. If one subtracts those that lack key conserved residues, we are left with 428 human ePKs with known or likely kinase function (Table 2), 99% of which were included in the SPRI list; 365 of these fall within the seven major ePK groups: TK, 84 in total; CAMK, 66; AGC, 61; CMGC, 61; STE, 45; TKL, 37; and CK1, 11. The remaining 63 are in the 'Other' category, falling outside the main ePK group branches. Krupa and Srinivasan 11 have also recently searched the public human genome data with a focus on identifying functional protein kinases; their efforts resulted in a list of 448 distinct human ePK sequences, but around 90 of these appear to represent duplicate entries, and no novel protein kinases were identified that were not present in the SUGEN compilation.

Knowing the full complement of ePK family members and functional ePKs encoded by eukaryotic genomes will have great impact upon many areas of scientific investigation. As mentioned above, an obvious benefit relates to understanding of how signal transduction pathways evolved during the course of eukaryotic evolution. Both SUGEN 10 and Krupa and Srinivasan 11 extended their analyses to describe other domains present in the various human ePKs which are likely to function in directing the enzymes to relevant substrates or modulating kinase activities. Further analysis of the ePK domain sequences uniquely conserved within the major groups and families, together with comparisons of ePK domain crystal structures, should ultimately allow a full understanding of how different classes of peptide substrate are recognized. For example, Figure 2 shows consensus sequences for the catalytic loop region in subdomain VIB (which includes the invariant aspartate thought to function as the catalytic base) and the activation loop region in subdomain VIII (which includes the highly conserved glutamine in the 'APE' motif) - two regions that have been recognized as being primarily involved in peptide-substrate recognition 1213. A number of group-specific differences are apparent (highlighted in Figure 2) that correlate with unique peptide-recognition tendencies for the ePKs that fall within a given group 14. Beyond sequence analysis, the kinome data will allow for the development of comprehensive tools (such as full-length cDNAs, microarrays, antibodies, and fusion protein and RNAi constructs) that will greatly aid laboratory investigations aimed at understanding cell signaling through analysis of kinase function. As an example of such proteomic approaches to the study of protein kinases, nearly all yeast protein kinases have been expressed in bacteria and analyzed for their ability to phosphorylate an array of protein or peptide substrates using protein-chip technology 15. Finally, the human kinome data will have benefits in the understanding and treatment of human diseases. The ePK genes that map within disease loci are attractive etiological candidates, and knowledge of the full repertoire of human protein kinases will greatly aid in the development of drugs that target specific protein kinases or protein kinase families whose function contributes to disease-associated cellular defects.