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Reversible protein phosphorylation plays a central role in regulating basic functions of all eukaryotes such as DNA replication, cell cycle control, gene transcription, protein translation, and energy metabolism. Protein phosphorylation is also required for more advanced functions in higher eukaryotes such as cell, organ, and limb differentiation, cell survival, synaptic transmission, cell-substratum and cell-cell communication, and to mediate complex interactions with the external environment. Because aberrant protein phosphorylation is commonly the cause of cancer and other human diseases, a comprehensive knowledge of the key enzymes that regulate these functions can provide the basis for novel therapeutic intervention strategies.

The genomic revolution promises to provide a new paradigm for drug discovery, allowing one to selectively target the molecular basis of human disease. The completion of the Caenorhabditis elegans genome sequence gives us an opportunity to decipher the molecular nature of its signal transduction machinery. Several global analyses of proteins and protein domains present in C. elegans have been presented elsewhere (1-4), revealing that protein kinases comprise the second largest family of protein domains in worms. The three most frequently occurring protein domains found in worms are seven transmembrane chemoreceptors (650 domains, 3.5% of genome), protein kinases (496 domains, 2.6% of genome), and zinc finger C4 domains, including nuclear hormone receptors (275 domains, 1.4% of genome). A more in-depth analysis has been performed on the 535 worm proteins containing zinc-binding domains, including the C4, C2H2, and C3HC4 ring finger types (3), and on the 83 worm homeobox transcription factors (4). Here, we present a comparative analysis of the enzymes and adaptor molecules that are the key components of the protein phosphorylation signaling network present in C. elegans.

Identification and Classification of C. elegans Protein Kinases. To identify worm protein kinases, we first used an HMMER 2.1.1 (http://hmmer.wustl.edu/) profile search against the 19,099 predicted worm proteins, the finished and unfinished C. elegans genomic sequence, and the worm chromosome assemblies. The nucleic acid databases were first translated in all six frames, and ORFs longer than 30 amino acids were parsed into a relational database. We generated a hidden Markov model based on 70 representative yeast and human protein kinases whose catalytic domains share <50% sequence identity with each other (5). Using a similar strategy, additional profiles were generated for other protein kinase-like domains (phosphoinositide kinases, atypical A6 kinases, diacylglycerol kinases, aminoglycoside resistance kinases, and microbial kinases), protein phosphatases, and domains capable of specifically binding to phosphotyrosine (P.Tyr) or phosphoserine/threonine residues (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW domains). Scripts were written for reassembly of contiguous exons identified from genomic sequence to generate the predicted catalytic domain sequence of each kinase. Pairwise BLAST 2.0 (ftp://ncbi.nlm.nih.gov/blast/executables/) analysis was performed to identify redundant entries, and putative protein kinases with low profile scores were manually inspected to determine whether they should be included in subsequent analyses.

View larger version (135K): [in this window] [in a new window]   Fig. 1.   Hyperbolic tree representation of C. elegans protein kinases. Major protein kinase groups are labeled in different colors. A JAVA tool for viewing this dendrogram can be found at www.kinase.com.

Yeast- and Fungal-Specific Kinases. The first complete eukaryote sequence, that of the budding yeast Saccharomyces cerevisiae, was reported in 1996 (6). Shortly thereafter, we presented a comprehensive analysis and classification of yeast protein kinases (7). Now, with the availability of a second eukaryotic genome, C. elegans, we can perform a similar analysis and make more informed generalizations on which of these protein kinases are unique to yeast or fungi, and also on which protein kinases evolved during the emergence of multicellular organisms and are therefore not represented in yeast or fungi.

Worm-Specific Protein Kinases. Which protein kinases are specific to worms? Protein kinases that are absent from yeast yet present in worms are likely to be involved in the complex signal transduction pathways that are required for the existence of multicellular organisms. These might include protein kinases involved in cell-substratum and cell-cell adhesion, transmembrane signaling in response to humoral factors, protein kinases involved in cell survival or programmed cell death, and protein kinases whose signals regulate metazoan-specific transcription factors, particularly those containing Zn-finger domains.

Conserved Metazoan Protein Kinase Signaling Transduction Pathways. Worms provide an elegant model system for studying signal transduction. This transparent animal is comprised of 959 somatic cells plus 131 cells destined for programmed cell death. The C. elegans hermaphrodite contains 302 neurons and 81 muscle cells and has a brain, reproductive system, and digestive tract (ref. 14; http://dauerdigs.biosci.missouri.edu/Dauer-World/Wormintro.html). It provides a complex yet tractable system for studying development, metabolism, aging, and behavioral responses to a number of stimuli. Regulation of many of these processes is carried out through signal transduction pathways that are also present in humans. Not surprisingly, all of the major protein kinase groups found in worms are also conserved in humans (15). The number of protein kinases classified into each major group from yeast and worms, along with a current estimate from humans, is provided in Table 1. These numbers represent a current analysis, but new protein kinases are being discovered every month as the worm genome sequencing project continues. Some of these entries may also represent pseudogenes containing frameshifts that result in incomplete translation into a full kinase catalytic domain.

AGC Group. The AGC group of worm protein kinases contains representatives of many of the known types of cyclic nucleotide-dependent, NDR or DBF2, and ribosomal S6 kinase families. Worms also contain members of the cGMP-dependent kinase (PKG), RSK, and G-protein coupled receptor kinase families that are absent from budding yeast. Two of the S6 kinase members have dual catalytic domains similar to vertebrate RSK enzymes, where the N-terminal domain clusters into the AGC group and the C-terminal kinase domain is most related to the CaMK group. Worms have four members of the AKT family, two being close orthologues of mammalian AKT1/PKB/RAC, and two related to the AKT upstream kinase, PDK1. AKT is a mammalian protooncoprotein regulated by phosphatidylinositol 3-kinase (PI3-K), which appears to function as a cell survival signal to protect cells from apoptosis (16). Insulin receptor, RAS, PI3-K, and PDK1 all act as upstream activators of AKT whereas the lipid phosphatase PTEN functions as a negative regulator of the PI3-K/AKT pathway (17). Downstream targets for AKT-mediated cell survival include the proapoptotic factors BAD and Caspase9 and transcription factors in the forkhead family, such as DAF-16 in the worm. AKT is also an essential mediator in insulin signaling, in part because of its use of GSK-3 as another downstream target. Each of these components of the AKT/PI3-K pathway is conserved in worms, providing a powerful system for genetic dissection of a major cell survival signal.

CaMK Group. In the CaMK group, the most abundant representatives include Ca²⁺/calmodulin-regulated and AMP-dependent protein kinases and EMK-related kinases. Worms also contain members of the death-associated protein kinase, mitogen-activated protein kinase (MAPK)-associated protein kinase, myosin light chain kinase, and phosphorylase kinase families that are absent from budding yeast. All of these protein kinase families have likely evolved as a result of the demands of multicellularity and the emergence of complex organ systems. For example, even though yeast have myosin homologues, they lack myosin light chain kinases. These protein kinases have presumably evolved to regulate myosin during muscle contraction. A worm contig still under construction appears to contain a phosphorylase kinase catalytic  subunit orthologue, consistent with the presence of two orthologues of the noncatalytic phosphorylase kinase  subunits, which facilitate calmodulin-binding and are required for activation of the mammalian holoenzyme.

CMGC Group. In the CMGC group of serine/threonine kinases, all of the main subfamilies are conserved between yeast, worms, and mammals, including cyclin-dependent kinase (CDK), MAPK, GSK-3, and CLK. An exception is the RCK family, which is absent from yeast but has two members in worms and at least seven in humans. The worm RCK kinases are most similar to mammalian MAK, or male germ cell-associated kinase, which has been implicated in spermatogenic meiosis and in signal transduction pathways for sight and smell. Worms have 14 CDKs (compared with 5 CDKs in yeast) including orthologues of CDC2, CDK3, CDK5, CDK7, and CDK8, and contain 34 cyclins, compared with 23 in budding yeast (Table 1), including one cyclin H orthologue, which we predict will interact with worm CDK-7 to generate a functional cyclin-activated kinase.

STE Group: MAPK Pathways. The STE family refers to the three classes of protein kinases that lie sequentially upstream of the MAPKs. In worms, this group includes 10 STE7 (MEK or MAPKK) kinases, 2 STE11 (MEKK or MAPKKK) kinases, and 12 STE20 (MEKKK) kinases. Based on the number of MAPK and STE-family kinases in C. elegans, we predict worms will contain at least 8-10 MAPK pathways. In humans, several protein kinase families that bear only distant homology with the STE11 family also operate at the level of MAPKKKs, including RAF, MLK, TAK1, and COT. Except for COT, worms also have orthologues of each of these kinases. Because crosstalk takes place between protein kinases functioning at different levels of the MAPK cascade, the large number of STE family kinases could translate into an enormous potential for upstream signal specificity and diversity.

Protein-Tyrosine Kinase Group: Receptor Protein-Tyrosine Kinases (RTKs). The largest group of protein kinases in worms are the protein-tyrosine kinases (PTKs), with 92 members and 5 fragments. We predict this will also remain the largest group of protein kinases in higher eukaryotes, including humans, where the current count is 100. These numbers are impressive when one considers that this family is absent from budding yeast. Yeast, however, do have a "budding" tyrosine phosphorylation signaling system, with several dual-specificity kinases (CLK-like, Ste7/MEK family, Swe1, Spk1/Rad53, Mps1), an atypical A6 PTK, 3 protein-tyrosine phosphatases, 16 dual-specificity and low molecular weight phosphatases, and 6 "infant" P.Tyr-binding proteins comprising an apparently nonfunctional SH2 domain protein and 5 phosphatase-like STYX domains. Budding yeast lack PTB domains, and none of the six potential P.Tyr-binding domains have been functionally verified.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the human and C. elegans receptor protein-tyrosine kinase families. Catalytic domains are shown in yellow. The names of the human RTKs are in black, and the names of the worm RTKs are in red.

Protein-Tyrosine Kinase Group: CTKs. Most of the 52 CTKs in worms belong to the single SH2-containing FER family. Of the remaining 10 CTKs, there are 2 orthologues of the SH3-containing ACK, and 1 each of FYN (SRC family CTK), FRK, CSK, ABL, and FAK, plus 3 unclassified CTKs. In vertebrates, CSK negatively regulates FYN-family kinases by phosphorylation of a C-terminal tyrosine facilitating a conformational change through an intramolecular SH2-P.Tyr interaction (25). We predict a similar functional interaction between worm FYN and CSK. Co-evolution of this regulatory pair suggests even early metazoans required a means to dampen signaling through CTKs. Notably absent in worms are protein kinases related to the ZAP70 and JAK CTKs, whose primary role in mammals is in signaling through the T cell and cytokine receptors, both of which represent more specialized pathways not present in worms. Humans have eight SRC-family kinases whereas worms have only one. This redundancy has confounded efforts to dissect out the precise role of these CTKs in human biology, often requiring "triple knockouts" to demonstrate a deficiency. The simplicity of non-FER-like CTKs in worms may be helpful in placing these CTKs within specific signaling cascades.

Protein-Tyrosine Kinases: Adaptor and Docking Molecules. Ligand activation of RTKs results in tyrosine phosphorylation of both the receptor itself (autophosphorylation) and of downstream substrates. These phosphorylated tyrosines then function as attachment sites for proteins containing SH2 and other P.Tyr-binding domains. We have identified 74 proteins containing a total of 77 SH2 domains in worms. The majority of these SH2 domains are in CTKs, two are present in a SHP2-related PTP, and the remainder are predicted to represent orthologues of a variety of adaptor molecules, including phospholipase C, CBL, CIS4/SOCS5, CRK, NCK, SEM-5/GRB2, SHC, tensin, STAT, and VAV. Worms also contain at least 16 PTB domains, which in some cases have been found to interact specifically with tyrosine phosphorylated proteins. Worm PTB-containing proteins include orthologues of SHC, which also contains an SH2 domain, neuronal transmembrane protein X11, and an insulin receptor substrate (IRS) family member. The mammalian X11 PTB domain does not to bind to P.Tyr, so we anticipate only a few of these worm domains will function as P.Tyr-binding domains. Additional potential phosphoprotein-binding domains identified in worms include three 14-3-3 domains, 22 WW domains, and 11 FHA domains.

Other Protein Kinases. Approximately 15% of the worm protein kinases do not fall into one of the six major groups but include smaller families with representatives in higher eukaryotes, including CHK1, DYRK, MLK, TAK1, PIM, RAF, STKR, and the mitotic kinases (BUB1, AURORA, PLK, and NIMA/NEK). Recent genetic and biochemical data place TAK1 (transforming growth factor -associated kinase) on a MAPK-like pathway at the level of a MAPKKK acting upstream of the MAPK-family member NLK. The worm orthologues of TAK1 and NLK regulate Wnt-mediated cell polarization during embryogenesis (21). Biochemical data also demonstrate that this MAPK-like pathway negatively regulates Wnt signaling because NLK phosphorylates the TCF/LEF HMG transcription factors, thereby inhibiting Wnt-regulated binding of the -catenin-TCF complex to DNA. Both of these pathways are conserved between mammals and worms. The likely orthologous human/worm pairs on the TAK1 MAPK-like pathway include TAK1/MOM-4, NLK/LIT-1, and TCF4/POP-1. Upstream regulators may include TGF1/DBL-1, TGF type I receptor/SMA-6, TGF type II receptor/DAF-4 (worms have three receptor serine kinases). Additional components of the Wnt-signaling pathway, such as cadherin, the adenomatous polyposis coli tumor suppressor gene (APC), disheveled, and GSK-3 kinase are also present in worms, suggesting that there may be a primordial connection between polarized control of cell division/migration and cellular transformation in vertebrates (26).

Microbial-Like Kinases: Origin of Protein Kinases? The availability of the sequence of the first complete metazoan genome, combined with the sequence of budding yeast and several prokaryotic and Archaea genomes, provides an excellent opportunity to reassess current theories on the evolutionary origin of protein kinases. Pkn1 is a bacterial protein kinase-like sequence first described in the Gram-negative bacteria Myxococcus xanthus, which functions in growth and differentiation and in the ability of this prokaryote to form a fruiting body in response to nutrient starvation. Pkn-related proteins are present in other prokaryotes, including Streptomyces, Bacillus, Mycobacterium, Pseudomonas, Chlamydia, and Synechocystis, where they are involved in virulence, secondary metabolism, sporulation, and complex growth cycles (27). However, there are no Pkn homologues in bacteria with less complex life cycles, such as Escherichia coli, and Haemophilus influenzae, or in any Archaea, suggesting they may have been acquired by horizontal transmission from an early eukaryote, and are unlikely to represent the ancestral founders to protein kinases.

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Atypical Protein Kinases and Protein Kinase-Like Domains. Worms contain 26 kinase-like domains present in receptor guanylyl cyclases (there are 10 additional soluble guanylyl cyclases), and at least 7 diacylglycerol kinases, 7 choline/ethanolamine kinases, and 30 YLK1-related antibiotic resistance kinases. Each of these families contain short motifs that were recognized by our profile searches with low scoring E-values, but a priori would not be expected to function as protein kinases. Instead, the similarity could simply reflect the modular nature of protein evolution and the primal role of ATP binding in diverse phosphotransfer enzymes. However, two recent papers on a bacterial homologue of the YLK1 family suggests that the aminoglycoside phosphotransferases (APHs) are structurally and functionally related to protein kinases (28, 29). There are over 40 APHs identified from bacteria that are resistant to aminoglycosides such as kanamycin, gentamycin, or amikacin. The crystal structure of one well characterized APH reveals that it shares >40% structural identity with the two-lobed structure of the catalytic domain of cAMP-dependent protein kinase (PKA), including an N-terminal lobe composed of a five-stranded antiparallel  sheet and the core of the C-terminal lobe, including several invariant segments found in all protein kinases (29). APHs lack the GxGxxG normally present in the loop between  strands 1 and 2 but contain 7 of the 12 strictly conserved residues present in most protein kinases, including the HGDxxxN signature sequence in kinase subdomain VIB (29). Furthermore, Daigle et al. (30) have demonstrated that this APH also exhibits protein-serine/threonine kinase activity, suggesting that the worm YLK1-related molecules may indeed be functional protein kinases.

Protein Phosphatases. Because of their important role in signal transduction, it is not surprising that the activity of protein kinases must be tightly regulated. This is accomplished through autoinhibition, autophosphorylation, transphosphorylation, dimerization, and cellular localization. Equally important is the role of protein phosphatases, which act to remove these regulatory phosphates from the protein kinase and its substrates. Because our analysis reveals worms to have a mature P.Tyr-signaling network, especially when compared with the yeast genome, we surveyed the worm genome for protein-tyrosine phosphatases.

Conclusions. What does the worm genome sequence tell us about mammalian signal transduction? First, it has provided an ideal model to highlight the bioinformatics challenges that lie ahead with the human genome effort and allows us to test our analysis tools and database organization. Second, it lets us refine our expectations as to the diversity and absolute number of unique protein kinases that we can expect to find in the human genome. Based on our count of 493 (411 conventional and 82 PK-like proteins) worm kinases, minus the 197 kinases that appear to be worm-specific expansions of certain families such as the CK1, FER, and KIN-15 families, multiplied by the 4-fold greater number of genes in humans compared with worms, we predict the human genome to contain 1,100 protein kinases (PTKs and serine/threonine kinases). A similar extrapolation predicts 300 human protein phosphatases (PTPs, dual-specificity phosphatases, and serine-threonine phosphatases). Because our current count of human protein kinases and phosphatases stands at 600 and 130, respectively, we still have about half the work ahead of us. However, recent claims predict the human genome may contain as many as 140,000 genes, compared with previous estimates of 80,000. Such calculations would result in a significant increase in our predictions of the total number of human protein kinases and phosphatases.