Department of Ecology and Evolution, University of Chicago
Correspondence: E-mail: whli{at}uchicago.edu.
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Abstract |
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Key Words: tyrosine kinase gene family gene duplication gene loss
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Introduction |
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Tyrosine kinases form a monophyletic group within the large protein kinase superfamily (Hanks and Hunter 1995), which includes also plant receptor-like kinases (RLK), Pelle kinases, and Raf kinases (Shiu and Bleecker 2001). In this study, this relationship is used to distinguish the tyrosine kinase family from other kinase families capable of phosphorylating tyrosine, such as MAP kinase kinase and casein kinase II. Members of the tyrosine kinase family contain a canonical kinase domain that is capable of phosphorylating tyrosines on substrate proteins. Outside of the kinase domain, these proteins possess a diverse array of sequence motifs responsible for interaction with other components in signal transduction pathways (Hubbard and Till 2000). On the basis of these sequence motifs and sequence similarity, human tyrosine kinases have been classified into multiple subfamilies (Robinson, Wu, and Lin 2000; Blume-Jensen and Hunter 2001; Manning et al. 2002). Interestingly, tyrosine kinases have so far been isolated only from animals, including sponge and hydra (Gamulin et al. 1997; Suga et al. 1999; Steele, Stover, and Sakaguchi 1999) and from the choanoflagellate Monosiga brevicollus, a unicellular relative of metazoans (King and Carroll 2001). Saccharomyces cerevisiae (budding yeast) is devoid of this gene family all together (Hunter and Plowman 1997), and it has been reported that receptor tyrosine kinases are absent from the Arabidopsis genome (Arabidopsis Genome Initiative 2000). Therefore, it is generally believed that tyrosine kinases are specific to metazoans.
Similar to many other multigene families, the tyrosine kinase family underwent differential expansion during the course of metazoan evolution. Several Caenorhabditis elegansspecific tyrosine kinases were found to have undergone expansions (Plowman et al. 1999; Popovici et al. 1999). In a comparison of all kinases from budding yeast, Drosophila melanogaster (fruit fly), and C. elegans (Manning et al. 2002), it was noted that the tyrosine kinase subfamilies in fruit fly and C. elegans were of different sizes. In addition, the orthologous relationships for quite a few tyrosine kinases could not be readily established. Based on surveys of GenBank sequences and targeted sequencing of selected tyrosine kinase subfamilies, it was proposed that two major episodes of duplications have occurred in this gene family (Iwabe, Kuma, and Miyata 1996; Suga et al. 1997; Suga et al. 1999). The first seems to have occurred before the split between poriferans and the other metazoans. The second occurred around the time of the split between the cyclostomes, such as lamprey, and the gnathostomes, including jawed fishes and tetrapods. However, these assertions were based on limited numbers of tyrosine kinases identified using degenerate primers, raising the question of whether the pattern observed is representative of this gene family across different taxa.
In this study, we sought to address several key questions in tyrosine kinase evolution. In eukaryotes, only in budding yeast has the absence of tyrosine kinases been rigorously examined on the basis of whole-genome analyses. Therefore, we searched for tyrosine kinases in 28 eukaryotic genomes including seven metazoans, four fungi, one microsporidian, one green alga, two flowering plants, and nine unicellular eukaryotes to examine the notion that tyrosine kinases are metazoan-specific. This search was further extended to GenBank polypeptide and expressed sequence tag (EST) databases. Previous studies on the expansion of tyrosine kinases did not use completely or nearly completely sequenced genomes. We constructed a phylogeny for all tyrosine kinases found in the genomes analyzed to determine the extents of family expansion in different evolutionary lineages. We also examined the domain organizations of tyrosine kinases in an attempt to determine the timing of their establishment and to identify domain recruitment events. Finally, gene duplications and losses are two counteracting mechanisms in controlling gene family size. To understand the relative contribution and timing of these events in different tyrosine kinase subfamilies, we determined the numbers of duplications and losses of tyrosine kinases among the organisms analyzed.
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Methods |
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Obtaining Tyrosine Kinases from GenBank Polypeptide and EST Databases
The kinase domain sequences of the tyrosine kinase set obtained above were used to BLAST against the GenBank polypeptide and EST sequence release 132 with the cutoff threshold of 1. These candidate kinase sequences were used to search against the combined kinase set from Arabidopsis, human, and all four fungi using BLAST. Sequences with a known human tyrosine kinase as the top match were regarded as tyrosine kinase candidates and those candidates from non-metazoans were verified further by determining their affiliation with kinase representatives. Seven human and three mouse sequences in these GenBank-derived sequences were not found in the Ensembl sequence release used. These sequences were incorporated into the tyrosine kinase set described in the previous section. For a list of tyrosine kinases from organisms other than the genomes analyzed, see Supplement B of the Supplementary Material online.
Classification of Tyrosine Kinase Subfamilies and Inference of Duplications and Losses
A phylogeny of the kinase domain protein sequences of all tyrosine kinases identified from the genomes listed in table 1 was constructed as described earlier but rooted with human cyclindependent kinase 3 (CDK3, NP_001249). The phylogeny was then compared to the published classification scheme (Blume-Jensen and Hunter 2001), and all subfamilies except JAK had more than 40% support, whereas the majority has more than 80% support (for the full phylogeny of tyrosine kinases, see Supplement C of the Supplementary Material online). Therefore, a cutoff of 40% bootstrap support was used for defining subfamilies. The subfamilies were further joined or divided based on the presence or inferred presence of any C. elegans tyrosine kinase. For each clade with two basal bifurcating branches, those with more than 40% bootstrap support and possessed of at least one C. elegans tyrosine kinase in one branch but not the other were defined as subfamilies. Any clade with higher than 40% support but without a C. elegans tyrosine kinase was also regarded as a subfamily only if its sister group is a subfamily with a C. elegans tyrosine kinase. The other clades with more than two sequences and more than 40% bootstrap support were also classified as subfamilies. The rest were defined as singletons.
The kinase domain protein sequences from each of the 43 subfamilies were aligned and manually inspected according to the kinase subdomain signatures (Hanks and Hunter 1995). The phylogeny for each subfamily was generated as described above, but with 1,000 bootstrap replicates. The rooted phylogenies were superimposed on species trees to infer gene duplications and losses using the program GeneTree (Page 1998).
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Results |
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Differential Lineage Expansion in Tyrosine Kinase Subfamilies
The size of the tyrosine kinase family in animal genomes ranges from 30 in arthropods to 115 in Tagifugu. This family can be divided into subfamilies with a wide range of domain organizations and functions (Robinson, Wu, and Lin 2000; Blume-Jensen and Hunter 2001). In this study, we used the kinase domain sequences to construct a phylogeny to define 43 subfamilies for further comparative analysis as outlined in Methods (fig. 2; for the full phylogeny, see Supplement C of the Supplementary Material online). Under the assumption that C. elegans is the earliest diverging taxa among the metazoans examined, 19 subfamilies were defined with at least one C. elegans tyrosine kinase. These subfamilies are likely to have been present in the common ancestor of these metazoans. An additional 11 subfamilies were defined because they are sister groups to the 19 C. elegans containing subfamilies (fig. 2A, arrowheads). Many of these subfamilies were also likely present in the common ancestor, and the absence of C. elegans sequences might be due to gene loss. These inferences suggest that the number of tyrosine kinase genes in the common ancestor of the metazoans examined was at least 19 and could be up to 30 or more. Thirteen additional subfamilies were defined, although they did not have any C. elegans sequence and they were not a sister group to any subfamily with C. elegans sequences (fig. 2A, arrows). In addition, 19 singletons do not fall into the subfamilies defined (for their identity, see Supplement A of the Supplementary Material online).
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Establishment of Domain Configurations in Tyrosine Kinases
In addition to gene duplication, one mechanism to generate molecular novelties is the recruitment of additional protein domains through domain shuffling. Multiple domains are found in tyrosine kinases. Therefore, an intriguing question is whether domain addition in the tyrosine kinase family occurred frequently after the divergence of the animals analyzed. Tyrosine kinases with known domains outside the kinase regions were compared among organisms to uncover the pattern of conservation or divergence in domain organization.
Interestingly, the domain organization and composition of subfamily members are in general similar among organisms (fig. 3). Among the 13 subfamilies shared among C. elegans, arthropod (fruit fly or mosquito), and chordates (at least one of the four species), the domain organizations are essentially the same in 11 subfamilies with minor differences such as the number of repeated motifs. In the subfamilies shared among the four chordates, most subfamily members have the same organization. In particular, no difference is found between mouse and human or between fruit fly and mosquito. Nevertheless, differences in domain organization are found in some subfamilies. For example, in the HGFR subfamily, all chordate members have large, homologous extracellular domains, which were not found in the C. elegans ortholog. Because the N-terminal region of the C. elegans HGFR member did not have sequence resembling the chordate HGFR extracellular domain, this absence may indicate a domain loss in the C. elegans lineage or a domain recruitment before the divergence of chordates. Taken together, the domain configurations in most subfamilies were established before the divergence of the metazoan genomes analyzed. Few clear changes in domain configurations were identified, and conservation of organization seems to be the rule.
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Two alternative species trees were used. The first assumed a closer relationship between deuterostomes and arthropods (fig. 4A), and the second assumed that nematodes and arthropods belong to Ecdysozoa (Aguinaldo et al. 1997; fig. 4B). Both topologies were evaluated but the details of only the first are shown in figure 4C. The numbers of duplications and losses were scored on each branch of the trees. Interestingly, there are more inferred duplications in branch 6, right after the split of the vertebrate lineage from the Ciona lineage, than in any other branches (fig. 4A). For most subfamilies, the numbers of duplications on branch 6 are higher than on most other branches (fig. 4C). This finding indicates that the tyrosine kinase family underwent an episodic expansion in the lineage leading to the common ancestor of Tagifugu, mouse, and human after its split from the Ciona lineage. In addition, the number of duplications in branch 9 is also large compared to the sister lineages that lead to either mouse or human (branches 7, 10, 11), consistent with the proposed genome duplication in the ray-fin fish lineage. Very few or no duplications were inferred in the arthropod lineage (branches 2, 3, 4) and in the lineages leading to either mouse or human (branches 7, 10, 11). In the two alternative topologies, most branches have similar numbers or the same number of inferred duplications. The only large difference is seen in branch 0, which represents the common ancestor of the species analyzed. More duplication events were inferred when assuming the presence of Ecdysozoa in the species tree.
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Duplications and Losses After the Split Between Ciona and the Other Chordates
Because different tyrosine kinase subfamilies play substantially different roles in animals, some may be retained at a higher rate than the others. To determine the retention rates, we analyzed the relationships between duplication and loss in tyrosine kinase subfamilies. Because the frequency of duplication was highest in the lineage leading to the vertebrates after its separation from the Ciona lineage (branch 6; fig. 4A and fig. 4C), the number of duplications was compared to the number of losses in the chordate lineages excluding Ciona.
Nearly all subfamilies have undergone more gene duplications than losses, as indicated by their deviations from a one-to-one relationship between duplication and loss (fig. 5). The deviation is most pronounced in the EPHR subfamily. Interestingly, the relationships between the numbers of duplications and losses fit a linear model well (fig. 5, solid line), suggesting that, despite the differences in duplication rate among subfamilies, the rates of net gene gain are quite similar. The slope indicates one gene loss per 2.16 duplication events. Therefore, about one out of every two tyrosine kinase duplicates has been retained during the period examined.
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Discussion |
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Among the protein kinase superfamily, tyrosine kinase is related to Raf kinases and the receptor-like kinase/Pelle family that defines the "receptor kinase group" (Shiu and Bleecker 2001). Because tyrosine kinases are able to phosphorylate tyrosine, this gene family may have been derived from another kinase family that exhibits similar specificity such as MAP kinase kinases (Dhanasekaran and Premkumar Reddy 1998), casein kinase II (Litchfield 2003), and Dictyostelium dual-specificity kinases. Among these kinase families, only the Dictyostelium dual-specificity kinases are closely related to the receptor kinase group (data not shown). Interestingly, Raf kinases and nearly all RLK/Pelle family members tested to date are serine/threonine kinases. The kinase domains in guanylyl cyclases are also closely related to tyrosine kinases (fig. 2A, RETGC) but autophosphorylating on serine residues only (Aparicio and Applebury 1996). Therefore, the ability to phosphorylate tyrosine in the tyrosine kinase family might have evolved several times independently in the protein kinase superfamily, and the tyrosine kinase family may be descendents of Dictyostelium dual-specificity kinase-like genes.
Conservation of Domain Organization at the Subfamily Level
The domain organizations are similar among members of each tyrosine kinase subfamily (see Supplement C of the Supplementary Material online), providing additional support for our classification of subfamilies. As expected, this conservation is most pronounced between more closely related organisms, such as mouse and human. Interestingly, the conservation of domain organization between distantly related organisms such as C. elegans and human is still quite high. These findings indicate that some of the additions of domains to tyrosine kinases occurred before the divergence among the organisms studied 600 to 1,000 MYA (Ayala and Rzhetsky 1998; Hedges 2002). Based on the analysis of a limited number of tyrosine kinase subfamilies and other gene families in metazoans, Iwabe, Kuma, and Miyata (1996) suggested that duplications leading to different subfamilies with distinct domain organizations occurred before the protostome-deuterostome split. Our findings based on the whole tyrosine kinase family from multiple genomes are mostly in line with this notion. Nevertheless, there are some interesting exceptions. In addition to the HGFR subfamily, the vertebrate AXL members contain a large extracellular domain, whereas the C. elegans counterpart F11E6.8 is devoid of the extracellular part. The immunoglobulin domains in vertebrate MUSKs and RORs are not found in their arthropod relatives.
Given the proposed increase of molecular complexity expressed in the form of domain recruitment in the human lineage (Lander et al. 2001; Venter et al. 2001), what we found was a relative stasis in domain configuration that lasted for hundreds of millions of years. This observation suggests that most domain additions may be selected against due to interference with normal developmental programs. Indeed, tyrosine kinases have been implicated in several malignancies because of their fusion to other proteins, as occurs in chronic myelogenous leukemia (Groffen et al. 1984; Shtivelman et al. 1985), anaplastic large-cell lymphoma (Morris et al. 1994), and congenital fibrosarcoma (Knezevich et al. 1998). These examples suggest that most, if not all, tyrosine kinase fusions significantly decrease the fitness of the individuals. This strong negative effect may contribute substantially to the lack of changes in domain organization among the metazoans examined.
Similarity in domain organization alone may not be sufficient for establishing homology between genes. In previous studies, three C. elegans sequences were designated as VEGFR orthologs because they contained similar numbers of immunoglobulin repeats (Plowman et al. 1999; Popovici et al. 1999). Our study indicates that their kinase sequences are more closely related to kinases in the CeKIN15 subfamily instead of VEGFR. This raises the possibility that the similar extracellular domains in VEGFRs and the C. elegans sequences may have been recruited independently by different tyrosine kinases. In addition, the C. elegans FER subfamily was designated as mammalian FES/FER relatives (Plowman et al. 1999). However, our phylogeny of tyrosine kinases does not lend support to this designation. Further analysis is needed to resolve these conflicting results.
One limitation of our analysis is that certain sequences were not clustered with other sequences because of their high sequence divergencei.e., the singletons that did not fall into any subfamilies. The extracellular domains of C. elegans singletons C16D9.2 and C25F6.4 are similar to those of ROS and DDR, respectively. In addition, a mosquito singleton contains a WSC domain and a fibronectin 3 domain in its extracellular region, representing a novel domain configuration not found in other organisms except fruit fly. It is not known if sequence divergence, domain shuffling, and/or gene conversion had confounded the inference of relationships between singletons and subfamilies. Nonetheless, most tyrosine kinases can be readily placed in subfamilies, and these singletons represent interesting exceptions that require further examination.
Expansion of the Tyrosine Kinase Family
The family of tyrosine kinases would have been small in the common ancestor of metazoans and the choanoflagellates, but it had expanded to approximately 30 members before the divergence of the metazoans examined. The expansion continued after the divergence of the metazoans examined, especially in C. elegans and vertebrates.
Interestingly, the subfamily sizes are generally similar among C. elegans, fruit fly, and mosquito with few exceptions. Most subfamilies shared among chordates are larger in Tagifugu, mouse, and human than in Ciona, which diverged from the three other chordates analyzed 550 MYA (Dehal et al. 2002). Furthermore, an episodic increase of duplications was found in a number of subfamilies in the ancestral lineage of Tagifugu, mouse, and human after its split from the Ciona lineage. Similar patterns of duplications have been reported in several tyrosine kinase subfamilies (Suga et al. 1997). This pattern of duplication is also found in other gene families and its timing coincides with the large-scale duplication in early vertebrate evolution (McLysaght, Hokamp, and Wolfe 2002; Gu, Wang, and Gu 2002). The increase of gene duplications during this time period is regarded as evidence for a whole-genome duplication by some investigators, but this is hotly debated (Wolfe 2001, Hokamp, McLysaght, and Wolfe 2003, Hughes and Friedman 2003). Another explanation for the observed increase in duplication events is that multiple, independent segmental duplications of chromsomes occurred within a relatively short time frame. In any case, our study does not support or refute either possibility. In mouse and human, we also found that one-third of tyrosine kinases are located within tandem clusters (Shiu and Li, unpublished data). It is likely that the combined action of tandem duplication and large-scale duplication contributed to the expansion.
One intriguing finding is that no duplication event was inferred in human and mouse after the split of their common ancestor from the Tagifugu lineage. Few duplications were inferred in fruit fly and mosquito after their split from all the other metazoans examined. In addition, all human genes have mouse orthologs, and all but three fruit fly tyrosine kinases have mosquito counterparts. In both cases, the one-to-one relationships are rather remarkable considering the fact that reciprocal best matches covered only 80% of the genes in mouse and human (Mouse Genome Sequencing Consortium 2002) and only 45% of the genes in fruit fly and mosquito (Zdobnov et al. 2002). These findings indicate gain or loss events that abolish a one-to-one relationship occurred rarely during the period of 90 Myr since the divergence between mouse and human and
260 Myr in the case of fruit fly and mosquito. Because large-scale duplications seem to be the major mechanism for the expansion of the tyrosine kinase family, the low number of gains in these lineages may simply reflect a low rate of gene duplication. On the other hand, the low number of losses may be due to the deleterious consequences of loss-of-function mutations in tyrosine kinases (Robertson, Tynan, and Donoghue 2000). Interestingly, among the 74 tyrosine kinase knockouts generated in mice, five of them do not have obvious phenotypes, which were knockouts of BLK (Texido et al. 2000), SRM (Kohmura et al. 1994), BMX (Rajantie et al. 2001), CTK/Matk (Hamaguchi et al. 1996), and EphA6 (Shimoyama et al. 2002).
One rather unexpected finding is the linear correlation between duplications and losses. Because genes in different subfamilies have different functions, one might expect that some subfamily members have been retained more than the others. Surprisingly, the number of duplications and losses in different subfamilies is strongly correlated with a duplication-to-loss ratio of 2.16. That is, approximately every other duplicated tyrosine kinase was retained. This finding indicates the presence of a general trend for the retention of tyrosine kinases from different subfamilies. However, there is likely substantial variation in the net gain rate among different vertebrates. The common ancestor of Tagifugu and mammals might have approximately 90 genes. If a whole-genome duplication occurred in the ray-finned fish after its divergence from lobe-finned fish, as proposed (Amores et al. 1998; Gates et al. 1999; Postlethwait et al. 2000), we expect to see 135 tyrosine kinases in Tagifugu, assuming a gene-loss rate of 50%. Instead, there are only 105. In a comparative analysis between zebrafish and Tagifugu, it was shown that Tagifugu has lost many duplicates retained in zebrafish (Taylor et al. 2003). This may partially explain the smaller than expected number of tyrosine kinase genes in Tagifugu. In addition, we cannot rule out the possibility that a fraction of tyrosine kinases is not present in the current release of the Tagifugu genome.
Pattens of Gains and Implications on Functional Divergence
The expansion of the tyrosine kinase family in animals implies that these tyrosine kinases are somehow beneficial and might have been retained by some mechanisms. One possible mechanism is expression divergence in time or tissues (Ferris and Whitt 1979; Force et al. 1999). For example, the two PDGFR isoforms have distinct expression patterns (Ataliotis and Mercola 1997). Moreover, although their kinase domains are interchangeable under certain conditions (Klinghoffer et al. 2001), the two PDGFR isoforms have different ligand affinities. Thus, another retention mechanism may involve the evolution of divergent ligand-binding capacities. Intuitively, these isoforms bind to the same ligands but with different signal outputs, providing additional regulatory mechanisms in the signaling network, and they are therefore retained. This model is not restricted to the extracellular ligand-binding domain because a change in activity modulation may be selected for.
Another possible mechanism for retention may be gene dosage effect. The AXL subfamily members, AXL, MER, and Tyro3, are involved in spermatogenesis and the mice that are devoid of all three genes have various defects including infertility (Lu et al. 1999). However, various double mutant combinations have only limited phenotypes and are fertile, and individual gene knockouts are phenotypically the same as the wild type. In light of the mutant phenotypes and differences in severity, it is possible that the increased number of AXL members contributed to the increase in fecundity and were selected for.
With the wealth of sequence information and our current understanding of tyrosine kinase functions, we conducted this analysis to uncover the history and mechanisms of tyrosine kinase family expansion. We found that this gene family is not animal-specific and the expansion was preceded and followed by periods of stasis. Its presence in Chlamydomonas, Phytophthora, and Entamoeba genomes represents the only known examples outside of the animal kingdom, including choanoflagellates. We speculate that whole genome duplications were the major means for the expansion of this gene family. The domain architectures are conserved among members of most subfamilies, suggesting that most, if not all, domain acquisition events occurred prior to the divergence of the metazoans examined. We also found that gene gains are often followed by gene losses. Given the important roles of tyrosine kinases in essentially all stages of metazoan life, it is likely that tyrosine kinase duplicates were retained for increasing the capacity of cell-cell communication and intracellular signal transduction. Nevertheless, many gene losses have occurred. It is difficult to explain why certain duplicates were retained or lost simply based on the generalized functions of each subfamily. Further molecular genetic or biochemical analyses of members within a subfamily in multiple organisms may shed light on the relative contributions of various retention mechanisms in the tyrosine kinase family.
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Acknowledgements |
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Footnotes |
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