Coevolution of the Domains of Cytoplasmic Tyrosine Kinases

Martin Nars and Mauno Vihinen

*Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland;
{dagger}Institute of Medical Technology, University of Tampere, Tampere, Finland;
{ddagger}Tampere University Hospital, Tampere, Finland


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Many signaling molecules are multidomain proteins that have other domains in addition to the catalytic kinase domain. Protein tyrosine kinases almost without exception contain Src homology 2 (SH2) and/or SH3 domains that can interact with other signaling proteins. Here, we studied evolution of the tyrosine kinases containing SH2 and/or SH3 and kinase domains. The three domains seem to have duplicated together, since the phylogenetic analysis using parsimony gave almost identical evolutionary trees for the separate domains and the multidomain complexes. The congruence analysis of the sequences for the separate domains also suggested that the domains have coevolved. There are several reasons for the domains to appear in a cluster. Kinases are regulated in many ways, and the presence of SH2 and SH3 domains at proper positions is crucial. Because all three domains can recognize different parts of ligands and substrates, their evolution has been interconnected. The reasons for the clustering and coevolution of the three domains in protein tyrosine kinases (PTKs) are discussed.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Cellular signaling molecules generally consist of several domains, all having their distinct functions and specificities. Protein tyrosine kinases (PTKs) form a large group of enzymes which contain other modules in addition to the catalytic kinase domain. The most common additional domains are the SH2 (Src homology 2) and SH3 modules (for review, see Cohen, Ren, and Baltimore 1995Citation ; Pawson 1995Citation ). SH2 domains bind to phosphorylated tyrosine (pY) residues in peptides and proteins, while left-handed polyproline type II (PP-II) helices in proteins and peptides interact with SH3 domains.

Cytoplasmic PTKs have clearly different organization, structure, and functions from integral membrane-bound receptor tyrosine kinases (Hardie and Hanks 1996Citation ). The two prominent families of protein kinases, based on substrate specificity, are the tyrosine and serine/threonine kinases (PSKs). In addition to different substrates, the latter group differs from PTKs especially in the nature of additional domains.

PTKs are regulated in many ways. Phosphorylation of a tyrosine(s) in the so-called activation loop is essential for optimal catalytic activity. Interactions between the domains play a critical role and facilitate several regulatory modes (Vihinen and Smith 1998Citation ). Phosphorylation of certain residues is important in enhancing or preventing interactions. Many PTKs are normally inactive but will be activated by autophosphorylation or by special proteins when needed. Several PTKs seem to be regulated by intracellular interactions between the domains (Cantley et al. 1991Citation ; Roussel et al. 1991Citation ). The three-dimensional structures of the Src family members Hck (Sicheri, Moarefi, and Kuriyan 1997Citation ) and c-Src (Williams et al. 1997Citation ; Xu, Harrison, and Eck 1997Citation ; Xu et al. 1999Citation ) has revealed that the SH2 domain binds to the regulatory C-terminal tyrosine and the SH3 domain interacts with the linker connecting the SH2 and kinase domains. The catalytic site and substrate-binding region are accessible, but the enzyme is in inactive conformation due to the organization of the two kinase lobes. The upper lobe is twisted relative to the lower lobe in the inactive open form. In the active form, ATP and Mg2+ ions are bound between the two lobes of the kinase (Knighton et al. 1991Citation ; Hubbard 1997Citation ).

The regulatory mechanisms require certain structural features, and therefore it was of interest to address the evolution of PTKs, especially to study the evolution of different domains and their combinations. It has been shown that several of the domains can be involved simultaneously in recognition and binding to substrate or ligand(s). Therefore, to maintain the function of the SH2, SH3, and kinase domain cluster, it is tempting to assume that they have evolved together. PTKs have almost without an exception the same domain organization, where the SH3 domain is followed by the SH2 domain, and the kinase domain is in the C-terminus. Since most of the known PTKs contain both SH2 and SH3 domains, we investigated how the three domains have evolved over time. Although sequences for numerous signaling molecules are available, an extensive analysis of the evolution of the domains in protein kinases has not been performed. We examined representatives from each of the cytoplasmic tyrosine kinase families and performed evolutionary analysis of the separate domains and their combinations in order to study their evolution. Only Fak and Jak family kinases of the major cytoplasmic PTKs were excluded from the analyses, since they do not contain SH2 or SH3 domains. It was found that the SH2, SH3, and kinase domains have coevolved and multiplied simultaneously in gene duplications. Also, the order of the domains, SH3-SH2-kinase, has remained unchanged.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Sequences and Alignments
The sequences for signaling molecules were collected from sequence databases. One sequence was chosen to represent each kinase type (table 1 ). Mammalian sequences (human or rodent) were used when available; otherwise, avian sequences were used. To include all of the cytoplasmic PTKs, it was necessary to also include some nonhuman sequences.


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Table 1 The Sequences for the Domains of the Analyzed Kinases

 
The amino acid sequences were aligned using the program Pileup in the GCG program package (Devereux, Haeberli, and Smithies 1984Citation ). The final adjustment of sequences was made based on the multiple-sequence alignment and knowledge from three-dimensional structures of the domains, as well as hallmark residues (in the kinase domains; Hanks and Hunter 1995Citation ; Neet and Hunter 1996Citation ). The sequences were trimmed to be of equal lengths. The three dimensional structures were taken from Protein Data Bank (table 2 ) (Abola et al. 1997Citation ).


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Table 2 Protein Data Bank Entries of Signalling Molecules

 
Phylogenetic Analyses
The aligned sequences were subject to the maximum-parsimony analysis using PAUP* (Swofford 1998Citation ) and the PROTPARS program of PHYLIP package (Felsenstein 1997Citation ). The algorithms insist that amino acid changes are consistent with the genetic code, and therefore they consider only those nucleotide substitutions that result in amino acid substitutions. Thus, the programs recognize whether more than one change is required at the nucleotide level. The kinase domain of Irk was used as an outgroup in the kinase domain calculations, and the domains of Grb2 were used as an outgroup in the SH2 and SH3 domain phylogeny. Phylogenetics with PAUP* was estimated with weights equal for all characters. The robustness of the nodes in parsimony analyses was tested with bootstrap (Felsenstein 1985Citation ) and jackknife (Farris et al. 1996Citation ) values. Bootstrap was performed with both PHYLIP and PAUP*, and jackknife was performed with PAUP*. Confidence limits were tested by bootstrapping the trees 100 times in PHYLIP analysis and 1,000 times in PAUP* studies.

The congruences of the different domains of PTKs were estimated with the incongruence length difference (ILD) test (Farris et al. 1994, 1995Citation ) in PAUP*. The sequences were partitioned domainwise with PAUP* for incongruence tests.

Conservation of Sequence and Function
The program MultiDisp (unpublished data) was used to study the conservation and numbers of possible character sets at each position. Large sequence alignments were used for comparison. The SH3 domain alignment was taken from Protein Kinase Resource at http://bioinfo.weizman.ac.il/Kinases/pkr/3D/xray/sh3/sh3walk_thru.html, and those for SH2 and kinase domains were taken from Pfam (Bateman et al. 2000). The sequences studied here were excluded from the large alignments along with their homologs. The MultiDisp program visualizes multiple-sequence alignment by indicating with character height the conservation of amino acids. In addition, the type of residue is shown with color coding.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
The genetic relationships between PTKs were analyzed through the comparison of their SH2 and/or SH3 and kinase domains. Phylogenetic trees were determined for each domain separately, as well as for the whole SH3-SH2-kinase sequence for those entries containing all three domains. The flanking regions surrounding these domains were not included in the analysis because they do not share similarity.

Protein Kinase Families
Kinases have been divided into families based on their sequence similarities in the kinase domain (Hanks and Quinn 1991Citation ; Hanks and Hunter 1995Citation ; Neet and Hunter 1996Citation ). In all of the PTKs, either the SH2 or the SH3 domain is immediately N-terminal to the kinase domain (fig. 1 ), except for Ack, a PTK in which the SH3 domain is C-terminal to the kinase domain.



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Fig. 1.—Domain organization of the studied tyrosine kinase families

 
The largest family of cytoplasmic PTKs is the Src family, some members of which were originally found as viral transforming oncogenes. Src family kinases are negatively regulated by phosphorylation of a tyrosine in the C-terminal tail. They are myristylated and membrane-bound. The Src family kinases are regulated by the Csk (C-terminal Src kinase) family PTKs, which phosphorylate the regulatory tyrosine. The members of this family are the smallest known PTKs and, in addition to the SH3, SH2, and kinase domains, have only a few residues.

The Abl is a large PTK of molecular weight ~150 kDa which also contains DNA and actin-binding domains. It is ubiquitous in both cytoplasm and cellular compartments, as well as in the nucleus. It is involved in cytoskeletal events, and it seems to have a transcription regulatory function in the nucleus. The genes for both family members, Abl and Arg, are alternatively initiated and spliced, yielding two transcripts.

The Fes family members do not have an SH3 domain. These kinases also appear both in the cytoplasm and in the nucleus. The Syk family members do not have any SH3 domain, but they contain two SH2 domains. This family plays an essential role in the activation and development of lymphocytes. The two SH2 domains bind to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequences in the stimulated antigen receptors of B and T cells. Tec family members contain two additional domains in the amino terminus: the pleckstrin homology (PH) and Tec homology (TH) domains. Tec family kinases are primarily expressed in hematopoietic cells, except for Bmx. Txk has the closest relatives in the Tec family, although it is missing the signature PH domain and the Btk motif of the TH domain.

Recently, some new PTKs not belonging to any of the above-mentioned families have been identified. Brk is a breast tumor kinase, and Frk is found in tumor cells. The kinase domain of Srm shares similarity with both the Src and the Tec families. Ack (activated Cdc42Hs-associated kinase) does not have any SH2 domain but contains an SH3 domain.

Multiple-Sequence Alignments
Sequences are known from several hundred protein kinases. Only one sequence, from a human if available, representing each kinase family was included in the multiple-sequence analysis. Figure 2 shows sequence alignments of the SH3, SH2, and kinase domains. In the final adjustment of the alignments, information from three-dimensional structures of the domains was used to match the sequences optimally. Linkers between the domains were discarded, and insertions appearing only due to the outgroup sequences were truncated. There were, altogether, 26 proteins with the SH2 domain, 22 with the SH3 domain, and 26 with the kinase domain (table 1 ). Of these proteins, 21 contained all three modules. Also shown in the sequence alignment are the residues forming the secondary-structural elements identified in the three-dimensional structures. The residues shown to be involved in ligand binding in some of the proteins were noticed to be conserved throughout the sequences (fig. 2 ). The lowest sequence identities in the domain alignments varied from 18% to 35%, indicating substantial variation.



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Fig. 2.—Sequence alignment of the analyzed PTKs. The secondary-structural elements are named above the sequence: {alpha}-helices are in red, and ß-strands are in blue according to the information in the Protein Data Bank entries. A, SH3 domain alignment. Residues binding to ligands are in boldface and underlined (Feng et al. 1994Citation ; Musacchio, Saraste, and Wilmanns 1994Citation ; Morton et al. 1996Citation ; Wittekind et al. 1997Citation ). B, SH2 domain alignment with secondary structures and consensus sequence (bottom). Invariant residues are indicated with capital letters, and conserved residues in lowercase letters. Amino acids binding to pY in ligand are in bold and underlined, and residues recognizing amino acids +1 and +3 are underlined (Eck, Shoelson, and Harrison 1993Citation ; Waksman et al. 1993Citation ; Hatada et al. 1995Citation ; Rahuel et al. 1996Citation ; Mulhern et al. 1997Citation ). C, Protein kinase domain alignment. Invariant residues are indicated at the bottom of each panel

 
The Fes family, Syk, and Zap70 do not contain the SH3 domain, although they have SH2 domains. Bmx, which is a Tec family member, does not contain an SH3 domain. Sequences for SH3 domains are relatively conserved, with two invariant residues and a large number of conserved positions (fig. 2A ). The first invariant residue is a leucine that ends the ßA strand, and the other residue, glycine, is in the ßD strand, which is next to a conserved ligand-binding residue. There are very few insertions and deletions in the alignment. The gaps are located on both sides of the ßC and ßD strands, i.e., in the loops on the protein surface. The conserved residues are involved mainly in ligand binding.

In the SH2 domain alignment, the alterations to polypeptide chain length are outside the conserved secondary-structural elements. The domain has six invariant residues and a large number of conservative substitutions (fig. 2B ). The invariant residue ßD4 (numbering according to Waksman et al. 1993Citation ) is responsible for the recognition of the phosphotyrosine ligand. Also, the other ligand-binding residues are very conserved. The most variable parts, both in sequence and in polypeptide chain length, are between ß-strands C and D and between strands D and E. All of the secondary structures are located in the corresponding positions in the determined SH2 and SH3 domain structures. The C-terminal SH2 domain of Zap70 was used because it contains the intact pY peptide-binding site (Hatada et al. 1995Citation ).

Protein kinases have several conserved or invariant hallmark residues in the catalytic domain (Hanks and Hunter 1995Citation ; Neet and Hunter 1996Citation ) that are scattered all along the length of the domain. Thirty-five residues were invariant in the sequence alignment of kinase domains (fig. 2C ). These residues have a number of functions, including ATP and substrate binding, formation of the glycine-rich loop and the catalytic site, and providing several structurally important interactions. The major positions of insertions and deletions are in the loops between ß2 and ß3, in the activation loop, and in the kinase insert region.

Molecular Phylogeny of PTKs and Their Domains
The tree topologies of the PTK domains obtained by the maximum-parsimony analysis are depicted in figure 3A D. Each domain was analyzed separately and in the multidomain complexes. The adaptor protein Grb2, which contains two SH3 domains and one SH2 domain, was used as an outgroup in the phylogenetic analysis of both the SH3 and the SH2 domains, and the kinase domain of the insulin receptor kinase (Irk) was used as an outgroup for the kinase domain phylogenies. The Grb2 is an adaptor protein without enzymatic activity, and the Irk is a receptor PTK, while all of the analyzed kinases were cytoplasmic.



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Fig. 3.—Evolutionary trees of (A) SH3 domains, (B) SH2 domains, (C) kinase domains, and (D) SH3-SH2-kinase multidomain complexes. The single most-parsimonious trees found from 1,000 replicate runs of the PAUP* program are shown. The numbers indicate percentage of optimal trees in which the given branch appeared; above are values from bootstrap analysis, and below are values from the jackknife test.

 


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Fig. 3 (Continued)

 
Parsimony analysis was performed with both the PAUP* and the PHYLIP programs. The results from the PAUP* study are shown, because the phylogenetic trees obtained with PHYLIP were essentially similar with slight changes in bootstrap values and positions of the deeply branched nodes. The conclusions drawn with both of the methods were the same in each case.

The SH3 domain tree is displayed in figure 3A. To further verify the observed branching patterns, bootstrap analysis was performed. Bootstrap and jackknife probabilities were generally large, indicating that many of the features in the phylogeny are reliable. Results from three node robustness studies, bootstrap with PAUP* and PHYLIP and jack-knifing with PAUP*, were evaluated (fig. 3A ). The results are in concordance with the classification of PTKs made solely based on the kinase domain information (Hanks and Quinn 1991Citation ), suggesting that the domains have evolved together. The Src family is clearly divided into two halves. Also, the Tec and Abl families are distinct. The SH3 domain tree has the lowest support in the analyzed domains because of the lower number of informative characters.

The phylogenetic tree for SH2 domains in figure 3B is very similar to that in figure 3A with respect to the positions of common sequences. The PTKs can be divided into the same general families (the Csk, Abl, Tec, and Src families) as in the SH3 phylogeny. In addition, there are enzymes from the Fes family, as well as Syk and Zap70. The Src family falls into the two known subfamilies, SrcA and SrcB (Neet and Hunter 1996Citation ). Brk and Srm form separate branches, whereas Frk forms the deepest branch to the Src family group.

The phylogeny based on the 26 kinase domain sequences (fig. 3C ) indicates a clearer distinction between the Src subfamilies. Also, the other families have strongly supported branching. The Syk, Tec, Src, Srm/Brk, Abl, Fes, and Csk families each form a branch. Ack is connected to the Syk branch, and Frk is connected to the Src branch. The bootstrap probabilities are very high for the tree due to the larger number of information-containing residues than in the SH3 and SH2 domain trees. The classification and phylogenetics of kinase domains (Hanks and Quinn 1991Citation ) agree with the tree in figure 3C.

SH2, SH3, and Kinase Domains Have Coevolved in PTKs
The phylogenies were also determined for the sequences containing all the three studied domains. The bootstrap and jackknife probabilities were high for this tree. In the SrcB subfamily, the deepest branch was formed by the Blk. The trees for each domain (fig. 3AC ) and for the SH3-SH2-kinase complex (fig. 3D ) were consistent. The SH3 domain phylogeny indicated the division of the kinases into the families, but in the other trees the probabilities were higher due to longer sequences containing more information.

The coevolution of the PTK domains was further studied by performing the ILD test (Farris et al. 1994, 1995Citation ) in PAUP*. Sequences for each of the three domains were compared with the other two. The statistical analysis of the ILD test did not show significant incongruence among the partitions. For the SH3 domain versus the SH2 domain, P = 0.13; for the SH3 domain versus the kinase domain, P = 0.17; and for the SH2 domain versus the kinase domain, P has an even larger value, 0.3. All of the domain pairs had insignificant P values, indicative of common evolutionary history. Thus, the ILD test also supports the observation of the coevolution in the three domains. ILD tests have previously been used to analyze the evolution glutamate receptors (Chiu et al. 1999Citation ) and nuclear receptors (Thornton and De Salle 2000).

Phylogeny has previously been estimated for kinase domains of 11 receptor PTKs containing immunoglobulin-like domains (Rousset et al. 1995Citation ). Also, these kinases could be classified to subgroups based on sequence identities and number of Ig-like domains, which, with the data presented here, suggest kinase domains to be tightly linked to the other domains, such as the SH2, SH3, and Ig-like domains present in PTKs. This implies that generally the complete or almost-complete genes for PTKs (excluding, e.g., the unique domains in the Src family) have duplicated at some time.

In another study, the evolutionary trees were determined for SH3-SH2-kinase complexes of PTKs from many sources, but the phylogenetics of the separate domains was not studied (Hughes 1996Citation ). Several new PTKs have been published since then. In this study, we concentrated on sequences from one organism (humans); sequences from other phyla were used only if the human counterpart was not available. In this way, it was possible to assess whether the domains had been linked during evolution. The trees were consistent with those in figure 3D for the entries present in both studies.

The consistency of phylogenies for each domain and the SH3-SH2-kinase complex suggests that the PTKs have had all three domains together during evolution and domain swapping or that additions have not occurred within these domains. The observation is also in line with the regulatory mechanism in the Src family. The different domains can function in a concerted way which does not allow replacements of individual domains without functional consequences. Outside the three domains, there is a great variation between and within the PTK families. The majority of the PTKs contain both the SH2 and the SH3 domains. Those missing one of the three domains usually form their own branches in the different trees, like Syk and Zap70, and Fes and Fer.

Domain swapping would most likely have produced proteins with defective ligand binding and substrate recognition and/or intramolecular regulation—most likely both. In Abl, malignant transformation or viral transduction can be caused by chromosomal rearrangements (Kurzrock, Gutterman, and Talpaz 1988Citation ; Rosenberg and Witte 1988Citation ). The mechanism involving intramolecular binding of the SH2 and SH3 domains to the kinase domain tail and linker, respectively (Sicheri, Moarefi, and Kuriyan 1997Citation ; Williams et al. 1997Citation ; Xu, Harrison, and Eck 1997Citation ), has been suggested to be widely adopted by PTKs. This mechanism does not allow major changes to the interacting domains. Abl is negatively regulated by its SH3 domain. Mutations in the linker between the SH2 and kinase domains prevent the SH3 domain binding and activate the kinase (Barilá and Superti-Furga 1998Citation ). The same effect was obtained by mutating ligand-binding residue in the SH3 domain (Barilá and Superti-Furga 1998Citation ). Binding of the SH2 and SH3 domains to their recognition sequences increases the specificity of the kinase domain (Kanemitsu et al. 1997Citation ; Thomas et al. 1998Citation ). Also, the tandem SH2 domains increase kinase domain substrate specificity (Ottinger, Botfield, and Shoelson 1998Citation ). Involvement of the SH2 and SH3 domains in kinase domain regulation and substrate binding have presumably forced the domains of PTKs to coevolve, because alterations like domain swapping and even single amino acid substitutions can lead to impaired function.

Structure-Function Correlations
The three-dimensional structure has been determined for a number of SH2, SH3, and kinase domains. The secondary structures are highly conserved. To further investigate the function, evolution, and structure of the domains, the sequences and conservation of residues were studied. The SH3 domains contained two, the SH2 domains 6, and the kinase domains altogether 35 invariant residues. In addition, a large proportion of the amino acids were conserved. Visualization of the conservation within the SH3 domains is shown in figure 4 . Similar analysis was also performed for the SH2 and kinase domains.



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Fig. 4.—Multiple-sequence alignment of the SH3 domains in figure 2A (top) and a large set of SH3 domains (bottom) excluding those above visualized with MultiDisp. The character height indicates its proportion in all the sequences. The characters are colored based on their properties: polar negative in red, polar positive in blue, neutral and polar in gold, aliphatic in green, aromatic in purple, cysteine in yellow, proline and glycine in magenta, and amide residues in black. Gaps in the alignment are indicated with rectangles.

 
The most conserved residues appear generally within the secondary-structural elements. The SH3 domain is a binding module that recognizes PP-II structures. The ligand is bound mainly by conserved aromatic residues (figs. 2A and 4 ). In these positions, substitutions are very conservative.

More variable character states can be seen on the surface loops. This feature is especially prominent for the so-called n-Src and distal loops of SH3 domains. In the SH2 and kinase domains, more variable loops are also present with higher variation in the lengths of the loops. In conclusion, the character states and conservation, the lengths of loops, and the functions of the amino acids correlate at most of the positions.

PTKs that phosphorylate tyrosines usually contain SH2 domains, whereas no PSKs are known with the added SH2 domain. The substrate binding and regulation of PTKs is also dependent on other domains in addition to the kinase region. Mutations in kinases lead to many diseases (Stenberg, Riikonen, and Vihinen 1999, 2000Citation ), including cancers, immunodeficiencies, endocrine disorders, and cardiovascular diseases. Kinases have such finely tuned reaction and regulation mechanisms that even small alterations can be fatal. Therefore, the domains have remained and evolved together because, e.g., domain swapping might have caused fatal disease.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 literature cited
 
Financial support from European Union BIO4-CT98-0142, the Finnish Academy, the Sigrid Juselius Foundation, and the Tampere University Hospital Medical Research Fund is gratefully acknowledged.


    Footnotes
 
Antony Dean, Reviewing Editor

1 Keywords: evolution phylogenetics SH2 SH3 protein kinase tyrosine kinase structural conservation Back

2 Address for correspondence and reprints: Mauno Vihinen, Institute of Medical Technology, FIN-33014 University of Tampere, Finland. E-mail: mauno.vihinen{at}uta.fi Back


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Accepted for publication September 26, 2000.