Analysis of the MAPK pathways, in silico and in vitro

LEABHARLANN CHOLAISTE NA TRIONOIDE, BAILE ATHA CLIATH

TRINITY COLLEGE LIBRARY DUBLIN

OUscoil Atha Cliath

The University of Dublin

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Analysis of the MARK pathways,

in silico and

in vitro.

Thesis subm itted to the University of Dublin

for the degree of Doctor of Philosophy

By

Daniel Richard Caffrey

Department of Biochemistry

Trinity College Dublin

Declaration

This thesis is submitted by the undersigned to the University o f Dubiin for

the examination of Doctorate in Philosophy. The work herein is entirely my

own and has not been submitted as an exercise for a degree to any other

university. The library of Trinity College has my permission to lend or copy

this thesis upon request.

Summary

1

Acknowledgments

iii

Abbreviations

iv

List of Figures

V

List of Tables

vii

Chapter 1

1

Introduction

1

A Bioinformatic approach

1

Protein evolution and Sequence analysis

1

Neutral and adaptive protein evolution

2

Phylogenetic trees

4

Predicting functional sites in proteins

4

THE MAPK pathvi^ays

8

O bjective of thesis

11

Chapter 2

15

Introduction

15

Material and Methods

17

Sequences used in analysis

17

Multiple sequence alignments.

18

Phylogenetic tree reconstruction.

18

Bootstrapping

21

Additional information

21

Results

22

Term inology and evolutionary interpretation

22

Evolution of the MAPK family

22

Evolution of the MEK family

36

Evolution of the MEKK family

48

Evolution of the STE20/PAK family

60

Discussion

72

Yeast hyper-osm olarity and mammalian stress pathway evolution

72

Novel substrate specificities tend to evolve over long periods

75

An evolutionary model for the yeast pherom one and the mammalian ERK pathway

76

The yeast hypo-osm olarity pathway and the mammalian oxidative stress pathway

78

The yeast sporulation pathway

79

Conclusions

80

Introduction

82

Materials and methods

87

Multiple alignm ent and Prediction of ancestral sequences

87

Calculation of Burst After Duplication (BAD) scores

88

Calculation of subfam ily scores (SS) which ignore ancestral nodes

92

Analysis of BADT and SST predictions versus experimental evidence

93

Solvent accessibility and residue contacts

94

Results

96

Comparison of p38 and ERK

96

Comparison of p38 and JNK

111

Relationships between BAD / BADT scores and structure

124

Sites identified in MKK3/6, MKK4, and MKK7

126

Discussion

138

C h a p t e r 4

143

Introduction

143

Materials and Methods

146

Plasmids

146

Plasmid purification for Cloning

146

DNA Ligation and transform ation into competent cells

147

PCR conditions for Chimera construction

148

Chimera construction

151

Plasmid purification for transfection

154

Culture and transfection OF 293RI cells

155

Bradford assay

157

Immuno-precipitation protein kinase assay

157

Western blot procedure for detection of substrate phosphorylation

159

Western blot procedure for detection of flag-tagged protein expression

160

Results

161

Chimeric proteins require upstream activation in order to activate downstream targets

166

Activation of chim eric MAPKs by upstream MKKs.

173

Inhibition of MAPK signal by over-expression of JIP.

184

Discussion

186

C h a p t e r 5

iss

Final conclusions and com m ents

198

C h a p t e r 6

201

References

201

Appendix II

List of sup p liers

207

Summary

This thesis takes a combined computational and experimental approach to

study the MARK pathways. These proteins were chosen as they are highly

conserved in both sequence and function across all eukaryotes.

An evolutionary analysis demonstrated that the MARK pathways in

animals arose largely, by a co-ordinated set of gene duplications. This involved

the JNK and p38 pathways at the MARK and MKK levels. These pathways are

primarily involved in stress and immune responses. The gene duplications

occurred after the divergence of animals from fungi. However, a more ancient

cross talk involving STE11 has been retained in both animals and fungi.

A novel prediction method was developed in an attempt to delineate the

residues that were conferring pathway specificity between JNK and p38. The

method compared predicted ancestral residues at the gene duplication node with

the first animal speciation node, to identify sites of interest. The method was also

applied to predicting residues conferring functional differences between p38 and

ERK. This allowed a partial assessment of the method, as several experiments

analysing p38 and ERK specificity had been previously published. It was found

that the method improved differentiation between subfamily specific sites and

other sites, compared with an almost identical method that did not use ancestral

information. There were other predicted sites that represented either false

positives or uncharacterised sites. The prediction method was also applied to the

MKK components of the p38 and JNK pathways. It was found that all predictions,

also distinct results. This suggested that a similar region of these proteins were

involved in protein-protein interactions.

The p38 and JNK predictions were used to design p38/JNK chimeric

proteins. Some of these chimeras failed to express in cells and their role could

not be assessed. However, a key residue was identified as being important for

both upstream and downstream specificity in the JNK pathway. Introduction of

additional residues often disturbed this specificity, suggesting that they may have

been indirectly involved but not essential to subfamily specificity. The results

suggested that the prediction method was useful but also likely to give many

Acknowledgements

This work would not have been realised without the kind help and guidance of

Abbreviations

ERK

Extracellular Regulated Kinase

JNK

c-Jun-N-terminal Kjnase

MEK

MAPK/ERK Kinase

MEKK

MEK Kinase

MKK

Mitogen Activated Protein Kinase Kinase

MARK

Mitogen Activated Protein Kinase

PAK

p21 Activated Kinase

SAPK

Stress Activated Protein Kinase

IL1

Interleukin

1

.

DMSO

Dimethyl sulphoxide

DTT

Dithiothreitol

EDTA

Etylene-diamine-tetra-acetic acid

PCS

Foetal calf serum

List of Figures

Figure 1.1

A proposed model of residue types involved in protein-protein interactions.

7

Figure 1.2

A schematic diagram of the MARK pathways for S.

cerevisiae.

12

Figure 1.3

A schematic diagram of the MARK pathways for animals.

13

Figure 1.4

The three dimensional structures for p38 (A), JNK (B), and ERK (C).

14

Figure 2.1

Alignm ent of the MARK family.

34

Figure 2.2

Rhylogenetic tree for the MARK family.

35

Figure 2.3

Alignm ent for the MEK family.

46

Figure 2.4

Rhylogenetic tree for the t\/IEK family.

47

Figure 2.5

.Alignment for the MEKK family.

58

Figure 2,6

Phylogenetic tree for the MEKK family.

59

Figure 2.7

Alignment for the Ste20-PAK family.

70

Figure 2.8

Rhylogenetic tree for the STE20-RAK family.

71

Figure 3.1

Schematic diagram of the stress Induced MAR kinase pathways in

animals and yeast at the MARK and MKK levels.

85

Figure 3.2

Rhylogenetic trees for the JN K/p38 pathway.

86

Figure 3.3

Example of ancestral residues with multiple probabilities for a site

91

Figure 3.4

Alignm ent of sequences used in comparison between ERK and p38.

106

Figure 3.5

Prediction of sites conferring functional differences between ERK

and p38.

107

Figure 3.6

Prediction of sites conferring functional differences between ERK

and p38 as adapted from [29],

108

Figure 3.7

Alignm ent of JNK, p38, and ERK with structural features.

109

Figure 3.8

Alignm ent of sequences used in comparison between JNK and p38.

119

Figure 3.9:

Prediction of regions that have evolved functional differences in JNK

since its duplication from the Ik ' ancestor.

120

Figure 3.10

Prediction of regions that have evolved functional differences in p38

since its duplication from the 1k"ancestor.

121

Figure 3.11

Prediction of regions conferring functional differences between JNK

122

133

134

135

136

137

142

145

165

169

170

176

177

178

179

180

181

182

183

185

191

192

193

194

195

196

and p38.

Alignm ent of sequences used in comparison between MKKs.

Prediction of regions that have evolved functional differences in MKK7

since its duplication from the 2k"ancestor.

Prediction of regions that have evolved functional differences in MKK4

since its duplication from the 2k” ancestor.

Prediction of regions that have evolved functional differences in

MKK3/6 since its duplication from the 2k' ancestor.

Prediction of regions conferring functional differences between

MKK7, MKK4, and tVlKK3/6.

Carbon alpha trace of the p38 structure.

Schematic diagrams of the MAPK chimeras.

Aligned sequences for chimeric (C1-C9) and wild type proteins as

inferred from DNA sequence.

Substrate specificity of p38, JNK and JNK/p38 chimeras.

Residues differing between p38 and JNK/p38 chimeras.

Activation of p38 and JNK by various MKKs.

Activation of p38, JNK, and

p38/JNK chim eras by MKK3.

Activation of p38, JNK, and

p38/JNK chimeras by MKKS.

Activation of p38, JNK, and

p38/JNK chimeras by MKK4.

Activation of p38, JNK, and

p38/JNK chimeras by MKK7.

Activation of C5 by various MKKs.

Activation of C2 by various MKKs.

Activation of C4 by various MKKs.

The effect of JBD on p38, JNK, and JN K/p38 chim era activity.

Schem atic summ ary of experimental results.

List of Tables

Table 3.1

Sequences used in analysis

Table 3.2

Relationships between evolutionary change in physicochemical

properties and structural function for selected residues across

ERK (top) and p38 (bottom).

Table 3.3

Relationships between evolutionary change in physicochemical

properties and structural function for selected residues across

JNK (top) and p38 (bottom).

Table 3.4

The relationship between BADT values and exposed/buried for

JNK and p38.

Table 4.1

Primers used in study.

Table 4.2

The PCR conditions for chimera construction.

Table 4.3

Residues that differ between chimera 9 and chimera 2.

Table 4.4

Residues that differ between chimera 2 and chimera 4.

95

110

123

125

149

150

171

172

Chapter 1

Introduction

A Bioinformatic approach

One of the main challenges facing biologists, is to extract meaningful

knowledge from the wealth of information that is generated from various genomic

and proteomic projects. For example, it is relatively easy to sequence a genome,

but more difficult to identify the genes from the rest of the DNA [1], It is also

difficult to predict the function of a protein encoded by a gene unless it has

sequence similarity with another protein that has already been biochemically

characterised. It is likely that the most successful post genomic science will

involve a team of biologists and computational biologists, where a computer

based prediction will be made based on a biologists prior knowledge. This

prediction will in turn be tested by the biologist and reported back so as

eventually a reliable predictive model will exist for biologists to exploit. Similarly,

this project attempts to take a combined computational and experimental

approach.

Protein evolution and Sequence analysis

The majority of techniques applied in the following chapters are founded

on our knowledge of protein evolution and sequence analysis. The first matrices

for amino acid replacements were derived in the 1970’s [2]. PAM (Percentage

Accepted Mutations) matrices are based on the assumption that all sites in a

protein evolve independently and are essentially a matrix of weights that is

derived from how often different amino acids replace other amino acids as

observed in related proteins. In other words, matrices allow for a quantitative

measure of evolutionary distances between proteins and tell us how likely it is

that a particular residue will be replaced by another. Several other matrices have

been constructed since the pioneering work of Margaret Dayhoff. Weights have

been constructed on the basis of chemical and structural features of the different

amino acids [3 , 4]. The BLOSUM matrices [5] were constructed from alignments

of different percentage identities, such that a BLOSUM 45 (derived from

alignment of 45% identity) is more tolerant of non-conservative substitutions than

a BLOSUM 80 (derived from alignment of 80% identity). Almost all sequence

analysis programs use a substitution matrix, and their role is paramount.

The sequence families that were originally identified by Dayhoff relied

heavily on manual editing and were not automated. It was only in 1987, that

progressive multiple alignment schemes were devised [6-8] and a year later

before such programs became available for the microcomputer [9, 10]. All of

these methods use a substitution matrix to align their sequences. Since then,

multiple alignments have been used to various applications. For example, to

search for distantly related proteins [11], to predict secondary structure [12], to

predict protein 3D structure, (reviewed in [13 ]), to reconstruct phylogenetic trees

[14], and to predict contact maps [15].

Neutral and adaptive protein evolution

reconstructed from multiple alignments. Tree reconstruction is founded on some

fundamental theories of evolution that were developed in the 1960’s. As

sequence data for haemoglobins and cytochrome c became available, it was

observed that the rate of amino acid substitution was

approximately the

same

across different mammals [16-18]. This approximately constant rate of evolution

is referred to as the molecular clock. It is an important observation, as these

constant rates facilitate reconstruction of phylogenetic relationships between

different species and gene families. This in part led to the neutral mutation

hypothesis [19]. It argues that the majority of molecular changes in evolution are

the result of neutral mutations or random genetic drift rather than natural

selection. Of course, there are striking exceptions, where Darwinian selection is

the dominant form of evolution, e.g. the binding sites of the MHC molecules [20].

Positive selection or convergent evolution can cause the incorrect reconstruction

of a tree, e.g. the lysozyme family [21, 22]. In the case of the lysozymes, they

function as a bacteriolytic enzyme in both ruminents (e.g. cows) and colobine

monkeys (e.g. langur). This required that the proteins adapt to the harsh

environment of the stomach. Thus, lysozyme had undergone similar selective

changes in both ruminents and colobine monkeys such that the colobine

sequences cluster with ruminents rather than primates in the wrong tree.

However, it is likely that the mutation rates for most proteins are approximately

constant with only slight periodic changes in rates that will usually be reflected in

the branch length of the reconstructed tree. These changes in rates may reflect a

relaxation in constraint, particularly in a duplicated gene that is free to evolve as

the other copy remains conserved so that it can perform its function.

Phylogenetic trees

There are several methodologies for constructing trees, such as maximum

parsimony methods e.g. [23], maximum likelihood methods e.g. [24], and

distance based methods e.g. [14]. The latter method is used to reconstruct the

trees in this project, and generally performs well when the distances are

estimated accurately. However, accurate estimation of distances can be difficult

when the rate varies greatly among sites [25]. The primary application of trees in

this work is to distinguish between paralogues (arose by gene duplcation) and

orthologues (arose by speciation events). It is assumed that in general,

paralogues will have slightly different functions and that orthologues will have the

same function. It also allows investigation of the possibility that interacting

proteins arise by co-ordinated gene duplication events (as reviewed in [26 ]).

Predicting functional sites in proteins

This project also uses multiple alignment to predict residues conferring

functional differences between related proteins. Other groups have looked at

similar problems. In general, the underlying assumption is that a functionally

important site will be conserved through out evolution. The Valencia group

extended their idea of correlated mutations [15] to the problem of predicting sites

involved in protein-protein interactions [27]. Correlated mutations are essentially

sites that co-evolve due to their functional interaction. For example, a mutation in

one site will cause a compensatory mutation in another site that interacts with it.

The same group also developed a method for identifying tree determinant

residues [28]. These are essentially residues that are conserved within a

subfamily but differ from other subfamilies. Livingstone and Barton took a similar

approach, although their methodology for measuring differences between

subfamilies was more vague and was implemented in AMAS [29]. A similar

method using profiles and entropy as a measure of conservation has also been

described [30]. Armon

et al used phylogenetic information (evenly distributed

taxonomic sampling) to get a more informed measure of conservation for

predicting functional sites [31]. An alternative approach was taken by Gallet

et al

who analysed hydrophobicity distribution along linear stretches of sequences to

predict interacting sites [32]. Jones ef a/claim 66% accuracy in predicting the

"surface patch" of the protein that forms the protein-protein interface. [33].

Lichtarge ef a/developed a method that attempts to predict binding sitesby

mapping alignment information to a known three-dimensional structure [34].

Despite the encouraging results that these different methods yielded, it is clear

that predicting sites involved in protein-protein interactions is not trivial. A recent

analysis of 621 protein-protein interfaces by Glaser ef a/suggests that the

residue composition does not differ significantly from other regions of the protein

[35], They also looked at residue-residue contact preferences and their

observations were similar to previous studies. The most common residue pairing

was between 2 hydrophobic residues. They found that arginine and tryptophan

were the most common hydrophobic-charged pairing. The interaction of two

oppositely charged residues (salt-bridge) are also quite common, and previous

studies suggest that these account for 13% of residue interactions [36], Contacts

between two positive residues were more common than contacts between two

negative residues. There is an average of 18 Water molecules found between the

protein-protein interface, these often form hydrogen bonds between the protein-

protein interface, thus increasing the complexity of the protein-protein interaction

[37]. Taken together, these studies suggest that accurate prediction of functional

or binding sites in proteins will be a difficult task. A proposed model of the

different types of residues involved in protein-protein interaction is shown in

Figure 1.1

Conserved

for family

Conserved

for

subfamily

Correlated/

compensator

residues

Variable

Figure

A proposed of model of residue types involved in protein - protein interactions.

The most critical residues are likely to be conserved across the fam ily (triangles) or conserved

across a subfam ily (squares). Other interacting sites will be variable across a fam ily but their

interacting proteins will have compensatory mutations (diamond/wedge) to maintain the

interaction. It is likely that variable residues are less important for interactions. It is possible that

conserved residues interact with variable residues but this is probably quite rare. It is not clear if

indirect interactions through water molecules are more prominent in conserved or variable

residues.

THE MARK pathways

The Mitogen Activated Protein Kinase (MARK) signalling pathways were

chosen as the proteins to model for a number of reasons. The signalling

pathways, the specificity of their protein-protein interactions, and biochemistry

are reasonably well characterised and are summarised in Figures 1.2 and 1.3.

Sequences are available from a large and diverse number of eukaryotes, but

have remained relatively conserved. This means that they can be aligned to

provide a rich source of evolutionary information that can be potentially exploited.

The high degree of similarity is reflected in the three dimensional structures that

are illustrated in Figure 1.4. Despite the availability of structures for the MAPKs,

the multiple alignment is the primary source of information used in the analysis.

This is due to the simple fact that the number sequences exceeds the number of

structures. Indeed, this will be the case for any protein family, thus the need to

develop tools that can extract important information form such alignments.

A typical MARK pathway (MEKK->MEK->MARK) consists of a

serine/threonine MARK that is activated by its upstream dual specificity MARK /

ERK Kinase (MEK), which is activated by its upstream serine / threonine MEK

Kinase (MEKK). The MARK modules appear to be fundamental in relaying a

diverse set of signals into the cell. Depending on the cell type or organism a

MARK pathway can be activated by a diverse range of extra-cellular signals and

the downstream effect is also context dependent. Extracellular signals such as

pheromones, mitogens, osmotic stress, UV stress, and pro-inflammatory

number of activators. Likewise, the best known effects include mitosis and

programmed cell death, but it is clear that many genes are increased and

decreased in expression and the physiological effects will be cell and context

dependent. Some of the characterised examples are outlined below.

There are five distinct pathways within the budding yeast

S.cerevisiae.

In

mammals, there are several more MAP kinases. These cascades are

summarised in Figure 1.2 and Figure 1.3 and reviewed in [38-41]. The resulting

activation of the MAPKs allows them to bind and phosphorylate a wide variety of

substrates. Their translocation to the nucleus and subsequent activation of

transcription factors is probably documented best [42-44]. Other substrates of

MAPKs include the cytoplasmic phosphlipase A2 [45], and the EGF receptor [46],

The yeast pheromone and filamentation MAPK pathways are almost

identical with the exception of their preference at the MAPK level for FUS3 and

KSS1 respectively [47, 48]. Kinases belonging to these pathways share closest

sequence similarity with the animal classical (mitogenic) pathway, but the

physiological activators and responses are not necessarily similar [42]. For

example, mitogens and growth factors activate the classical pathway that leads

to multiple differentiation and developmental processes, while the pheromone

activated cascade causes cell arrest, morphogenic changes, and transcription of

genes necessary for mating. In yeast, the serine/threonine kinase STE20 has

been shown to act upstream of the MEKK STE11 [49]. It was proposed that

CDC42 is upstream of this pathway [50, 51]. An alternative view is that STE20

may be activated in the pheromone pathway by the Gpy protein complex via the

adapter protein STE5 and that CDC42 binding to STE20 is only essential for

pseudohyphal growth and cytokinesis [49]. In animals, the STE20 relatives are

called P21-Activated protein Kinases (PAKs) and were shown to interact with

Rac and human CDC42 to varying extents [52-54], The PAKs are likely to

activate MEKK1/2/3 (similarity to STE11). However, subsequent activation of the

classical pathway in this context is unclear as it appears that MEKK1 then

activates the p38 and JNK pathways [52, 55-60]. In the classical pathway, the

small G-protein RAS recruits a RAF kinase to the cell membrane, thus resulting

in its activation [61-64]. The activated RAF acts as a MEKK in the classical

pathway but sequence similarity with MEKK1/2/3/4 in not significant.

Interestingly, PAK3 has been shown to activate RAF [65].

Like the yeast hyper-osmolarity pathway, the animal JNK and p38

pathways are also activated by high salt concentrations [66]. It is unclear if the

upstream osmo-sensing components are ancestrally shared. In addition, the p38

and JNK pathways are activated by LPS, UV light, and cytokines [66]. The yeast

hyper-osmolarity pathway leads to activation of transcription factors that initiate

transcription of glycerol phosphate dehydrogenase, while the known targets of

JNK and p38 are necessary for mitotic arrest, expression of cytokines, and

apoptosis [42, 44].

The yeast hypo-osmolarity pathway is activated by PKC1, which lies

downstream of Rho1 [67]. PKC may by-pass BCK1 in the activation of this

pathway [68]. It is not known what genes are expressed as a result of this

pathway being activated. There are no clear orthologues identified in animals to

date and activation of the mammalian MARK pathways by PKC isoforms may

simply reflect its promiscuity

in vitro [69, 70].

Other potential activators of animal MARK pathways include: Mixed

lineage kinases (MLK3/SPRK/PTK-1) [71, 72], (DLK/MUK/LZK/ZPK) [73-75],

(MST/MLK2) [76], Ste20-like oxidant stress response kinase-1 (S0K-1/YSK-1)

[77, 78], Tpl-2 [79], TGPp activate kinase (TAK1) [80], and Apoptosis signal-

regulating kinase 1 (ASK1) [81]. Meanwhile, more recently discovered MAPK

components such as the MEK5-ERK5 pathway appear to be involved in

response to hyper-osmolarity, oxidative stress and EGF [82, 83]. Similarly, ERK3

and ERK4 are relatively uncharacterised [84, 85].

Objective of thesis

This project attempts to understand the evolution of the MARK pathways.

The evolutionary model is put in the context of kinase and pathway function.

Sites that confer functional differences between kinase subfamilies are then

predicted by exploiting the evolutionary model. The predictions are then tested

by standard experimental procedures.

S cerevisiae

Filam entous

Pheromone

H yper-osm olari^

H ypo-osm olarity

Sporulation

Level

i

M E K K

i

MAPK

1

1

1

X ^ S T E 1 1 _ SSK22/2

C ^ S T E ? PBS2

i

* C ^ r u s 3 ^ H O G l

RHOl,

PKC

BCKl

4

SPSl

T

Figure

1.2:

A schematic diagram of the MAPK pathways for S.

cerevisiae.

Kinases that are studied in this thesis are surrounded by ovals, other kinases are

in triangles, small G proteins are in pentagons, and arrows denote

phosphorylation. Many other activators of the MAPK modules have been omitted

for simplicity. Reviews or individual citations within the text should be consulted

for a more comprehensive description of the pathways, as experimental evidence

for activation of substrates is often ambiguous.

Mammals

C la s s ic a l J N K P38 O x id a tiv e G icw tli factors Sbfiss siiitTuh andpio-irifUimBtorycytokuies EGF, oxidative stress

RAS

Level

PAK.

KKKK

RAF

MEKK

MEKK1/2J

ASKl

MEKK4

MEK.

M EK1/2V V M K K V VMKK^

I

MAPK

Figure 1.3:

A schematic diagram of the i\/IAPK pathways for animals.

Kinases

that are studied in this thesis are surrounded by ovals, other kinases are in

triangles, small G proteins are in pentagons, and arrows denote phosphorylation.

Many other activators of the MAPK modules have been omitted for simplicity.

Reviews or individual citations within the text should be consulted for a more

comprehensive description of the pathways, as experimental evidence for

activation of substrates is often ambiguous.

Chapter 2

Introduction

The first objective of the thesis was to establish the evolutionary

relationship between the kinases at different levels. This would allow the

distinction between orthologues and paralogues, that is, proteins that have arisen

by speciation events and those that have arisen by gene duplication respectively.

The reasoning behind this is that orthologues (often but not always) have similar

function, while paralogues often allow for novel or slightly different function to be

acquired.

The second objective was to establish if gene duplication patterns at

different levels in the cascade correlated with pathway and substrate specificity.

There are many examples of two interacting proteins being duplicated so that

there are two sets of interacting proteins, as reviewed in [26].

Thirdly, there are several so-called isoforms in the MAPK pathways that

have subtle differences in function. It was hoped that evolutionary analysis might

provide insight into their functional differences.

Briefly, a select number of kinases from each level and pathway were

searched against a copy of the non-redundant protein database to identify

candidate homologues. The kinases from each level were aligned using clustalw.

Alignments were then edited manually to ensure a correct and optimal alignment

was used for construction of evolutionary trees. Upon tree construction,

sequences that appeared to be evolving abnormally (long-branch attraction) were

omitted to allow reconstruction of a more stable and accurate tree. A bootstrap

analysis was performed as an assessment of the reliability of the tree

construction.

Material and Methods

Sequences used in analysis

The catalytic domains of 35 kinases belonging to the PAK-MEKK-MEK-

MAPK pathways were searched against the Non-redundant Protein database at

NCBI on November 1®'1998 using the NETBLAST program [86]. The search

data-set consisted of

Dictyostelium discoideum: ERK1 (SP P42525);

Saccharomyces cerevisiae: H0G1 (SP P32465), FUSS (SP P16892), KKQ1 (SP

P36005), KSS1 (SP P14681), SLT2 (SP Q00772), SMK1 (SP P41808), MKK1

(SP P32490), MKK2 (SP P32491), PBS2 (SP P08018), STE7 (SP P06784),

SSK2 (SP P53599), SSK22 (SP P25390), CLA4 (SP P48562), K 0L3 (SP

Q12469), SPS1 (SP P08458), STE20 (SP Q03497);

Schizosaccharomyces

pombe : BYR2 (SP P28829):Canc//da

albicans: STE20 (SP Q92212);

Homo

sapiens: ERK5 (SP Q13164), MEK2 (SP P36507), MKK3 (SP P46734), MEKK3

(SP Q99759), ASK1 (GB D84476), PAK1 (GB U51120), PAK3 (GB U25975) ;

xenopus laevis: p38 (SP P47812), MEK2 (SP Q07192);

Caenorhabditis elegans

SUR1 (SP P39745);

Mus muscuiis

(SP P47809), MEKK1 (SP P53349),

MEKK2 (SP Q61083), MEKK3 (SP Q61084), MEKK4a (GB U85607);

Rattus

norvegicus: PAK2 (SP Q62829). All proteins with P values of 1e-65, 1e-50, 1e-

40, 1e-50 or less (a raw score of approximately 250++) for MAPK, MEK, MEKK,

or PAK searches respectively were fetched locally from the

EMBI_/SWISSPROT/PIR databases at the Irish National Centre for

Bioinformatics.

Multiple sequence alignments.

Residues that were N and C terminal to the kinase catalytic domain could

not be aligned very well and were consequently removed as this would distort

tree construction. The catalytic domain for each kinase was identified using

previously described criteria as a guide [87], The catalytic domain of each protein

was aligned to a structural alignment that was held fixed using the

CLUSTALW1.7 package [88]. The structural alignment was based on the crystal

structures of the catalytic domains for Rat ERK2, Human Insulin receptor, Human

CDK2, Mouse p38, Human SRC, Mouse cAMP, Rabbit Phosphorylase B Kinase,

S Pombe casein Kinase, Rat Casein Kinase (SP P27703 [89]; SP P06213 [90];

SP P06213 [91]: SP P24941 [92]; SP P47811 [93]; SP P12931 [94], SP P05132

[95], SP P00518 [96]; SP P40233 [97]).

Phylogenetic tree reconstruction.

A neighbour-joining tree was drawn for the entire dataset using the

CLUSTALW tree drawing option [14]. The tree consisted of four major groupings

that coincided with the MAPK family, the MEK family, the MEKK family, and the

STE20-PAK family. Alignments for MAPK, MEK, MEKK, and STE20-PAK families

were extracted from the large alignment. To reduce errors in the tree

construction, the alignments were edited by hand before neighbour-joining trees

were constructed using the bootstrap option in CLUSTALW. All trees were

constructed with gaps excluded and corrected for multiple substitutions. Human

CDK2 (SP P24941) was used to root the MAPK and the MEK tree. The MEKK

and STE20-PAK trees were rooted with yeast STE20 (SP Q03497) and STE11

(SP P23561) respectively. Similar, redundant, outlying, unalignable, or fast

evolving sequences were removed and the trees were re-constructed using the

same options described above. The final trees were derived from the following

sequences that can be accessed at the Genbank™/EMBL (GB), Swissprot (SW),

and PIR (PIR) databases: SMK1_SC (SP P41808), MKC1_CA (SP P43068),

MPS1_MG (GB AF020316), SPM1_SP (SP Q92398), SLT2_SC (SP Q00772),

ERK5_HS (SP Q13164), SUR1_CE (SP P39745), ERKA_DM (SP P40417),

ERK1_HS (SP P27361), ERK2_HS (SP P28482), FUS3_SC (SP P16892),

KSS1_SC (SP PI 4681), SPK1_SP (SP P27638), ERK1_CA (SP P28869),

MAPK_PNC (GB AF043941), FSMAPK_NH (GB U52963), PMK1_MG (GB

U70134), ERK1_DD (SP P42525), MPK6_AT (SP Q39026), MAPK_PS (SP

Q06060), NTF4_NT (SP Q40532), MPK_AS (PIR S56638), MPK3_AT (SP

Q39023), MAPK1_PC (GB Y12785), NTF6_NT (SP Q40531), MPK5_AT

(Q39025), MPK4_AT (SP Q39024), MMK2_MS (SP Q40353), MPK7_AT (SP

Q39027), MPK1_AT (SP Q39021), MPK2_AT (SP Q39022), STY1_SP (SP

Q09892), H0G1_CA (SP Q92207), H0G1_SC (SP P32485), H0G2_ZR (GB

AB012088), H0G1_ZR (GB AB012146), B0218_CE (GB U58752), F42G8.3_CE

(GB AF038618), P38G_HS (SP P53778), P38D_HS (GB Y10488), P38_DM (GB

U86867), P38_CC (SP Q90336), P38_XL (SP P47812), P38A_HS (SP Q16539),

P38B_HS (SP Q15759), P38B2_HS (GB AF031135), B0478_CE (GB U57054),

JNK_DM (GB U73196), JNK2_HS (SP P45984), JNK1_HS (SP P45983),

JNK3_RN (SP P49187), MEK1_ZM (GB U83625), MEK1_LE (GB AJ000728),

MEK1_AT ( GB AF000977), MKK1_SC (SP P32490), MKK2_SC (SP P32491),

K08A8_CE (GB U38377), HEP_DM (SP Q23977), YR62_CE (SP Q20347),

VZC3741_GE (GB Z95122), MEK2_XL (SP Q07192), MKK4_HS (SP P45985),

MKK4_MM (SP P47809), R03G5_CE (GB U51994), MKK3_MM (SP 009110)

MKK3_HS (SP P46734), MKK6_HS (SP P52564), MKK6_MM (SP P70236),

PBS2_SC (SP P08018), WIS1_SP (SP P33886), MEK5 ANIMAL MEK5B_HS

(GB U71087), MEK5_RN (SP Q62862), MEK2_CE (PIR A56466), DSOR_DM

(SP Q24324), MEK2_RN (SP P36506), MEK2_HS (SP Q63932), MEK1_XL (SP

Q05116), MEK1_HS (SP Q02750), BYR1_SP (SP PI 0506), FUZ7_UM (GB

U07801), STE7_SC (SP P06784), STE7_CA (SP P46599), CDC7_SP (SP

P41892), CDC15_SC (SP P27636), MEKK4A_MM (GB U85607), MTK1_HS (GB

AF002715), SSK22_SC (SP P25390), SSK2_SC (SP P53599), WAK1_SP (GB

Y11989), WIN1+_SP (GB AJ223190), F9A6_CE (GB U41994), ASK1_HS (GB

D84476), MEKK5_DM (PIR JC4673), ATMEKK1_AT (GB D50468), MAP3KG_AT

(GB D50468), PMK1_SP (SP Q10407), BCK1_KL (GB AJ005079), BCK1_SC

(SP Q01389), BYR2_SP (SP P28829), NRC1_NCGB (AF034090), STE11_SC

(SP P23561), MEKK2_MM (SP Q61083), MEKK3_MM (SP Q61084),

MEKK3_HS (SP Q99759), MEKK1_HS (GB AF042838), MEKK1_MM (SP

Q62925), NPK1_NT ( PIR A48084), ANP1_AT (GB AB000796), NRK1_SC (SP

P38692), STE20_DD (GB U51923), DDPAK_DD (GB Y10158), MIHCK_AC (GB

U67056), SHK2_SP (SP Q10056), CLA4_CA (GB U87996), CLA4_SC (SP

P48562), SHK1_SP (GB L41552), PAK1_SP (SP P50527), STE20_SC (SP

Q03497), STE20_CA (SP Q92212), DPAK_DM (GB U49446), PAKLIKE_CE (GB

D83215), CEPAK_CE (GB U63744), PAK1_XL (GB AF000239), PAK1_RN (SP

P35465), PAK1_HS (SP Q13153), PAK3_HS (SP Q13177), PAK3_RN (SP

Q64303), PAK2_MM (SP Q61036), PAK2_RN (SP Q64303), LOK_MM (GB

D89728), KIAA020_HS (GB D86959), SLK_MM (GB AF039574), ZC_CE (GB

U55363), SIK1_AT (GB U96613), PRKSD_SD (GB Y13101), KRS1_RN (GB

U60206), KRS2_HS (GB U60207), SPAC9_SP (GB Z98763), SEVERIN_DD (GB

AF059534), T19A5_CE (GB U53153), S0K1_HS (GB X99325), YSK1_HS (GB

D63780), MST3_HS (GB AF024636), SPS1_SC (SP P08458), and SPAC_SP

(GB Z99259).

Bootstrapping

Bootstrapping is a standard statistical technique used to assess the

confidence in an inferred branching order. The bootstrap percentage corresponds

to the level of statistical confidence in the branching order, assuming that amino

acid positions are independent. This may be violated when correlated changes

occur or when there are alignment errors. In general, a bootstrap of 70% is likely

to be correct at the 95% level [98]

Additional inform ation

All sequences included and excluded from the analysis, the alignments,

their respective trees, plus additional information can be found at

http://acer.gen.tcd.ie/~dcaffrey/mapk/

Results

Terminology and evolutionary interpretation

Sequences are defined as orthologous, when the duplication arose via

speciation. Thus, a yeast kinase and a mammalian kinase are orthologous if they

were encoded by the same gene in their common ancestor, and non-orthologous

if they were encoded by two separate genes in their common ancestor. In

inferring relationships, it was generally assumed that mammalian kinases did not

have yeast orthologues that were deleted, in order to keep the interpretation

simple. In order to help interpret the trees, we arbitrarily chose a kinase that

would lie outside of the main cluster. This outgroup provides a root for the

sequences of interest, which indicates the branching order within the group since

the divergence from the last common ancestor.

Evolution of the MARK family

The MARK alignment (Figure 2.1) consists of 52 sequences along with

human CDK2. The CDK2 sequence was included to indicate the root of the

MARK tree that is shown in Figure 2.2. The branch length represents the inferred

number of amino acid replacements over evolutionary time. The rate of evolution

is more or less constant across the different lineages. Exceptions such as KKQ1

do not appear to represent alignment error, so that this acceleration may reflect

reduced functional constraints or else adaptation to a novel function.. The tree

falls into two broad groups, one including H0G1 FUNGI group, the animal groups

for p38 and JNK (F42G8 ANIMAL, P38DG ANIMAL, P38AB ANMAL, JNK

ANIMAL); the other containing everything else.

The H0G1 FUNGI group clusters with the F42G8 ANIMAL, P38DG

ANIMAL, P38AB ANIMAL, and JNK ANIMAL groups (70% bootstrap). Since the

JNK and p38 clusters form a very distinct group from the H0G1 FUNGI group

(96% bootstrap), this suggests that a H0G1 like progenitor duplicated to give the

present day JNK and p38 found in animals. For the single budding yeast H0G1

gene there are at least seven human relatives. While the greatest sequence

identity is with the p38 isoforms, this is simply because the JNK genes have

undergone a more rapid evolution. It is clear that this accelerated evolution in

JNK occurred after the divergence from yeast but before the

C. elegans

and

mammalian split. This accelerated evolution in JNK most likely represents a

selection for novel function, given our biochemical knowledge of JNK in each

organism.

The absence of any animal sequences along with the SMK1 FUNGI and

the SLT2 FUNGI cluster suggest that SMK1 duplicated from SLT2 since the

animal/fungal split, although this grouping is weakly supported. Similarly, it

appears that KKQ1 and SLT2 duplicated after the animal/fungal split. ERK5

ANIMAL does not cluster strongly with any of the yeast members. This suggests

that either the tree topology is incorrect so that its yeast orthologue is one of the

ungrouped yeast kinases (such as SLT2 FUNGI) or a deletion of the yeast

orthologue occurred. The duplication of KSS1 and FUS3 appears to have

occurred after the animal/fungal split (92% bootstrap), but before the speciation

of many fungal groups. Thus, it is unlikely that there is a mannmalian orthologue

for both KSS1 and FUSS of the filamentous and pheromone pathways

respectively. Instead, the ERK1 ANIMAL group is orthologous to both KSS1 and

FUSS.

B0478_CE ... ... ... YQNLRLIG- - SGAQG1 VC SAF DT...

JNK_DM ... ....YINLRPIG--SGAQGIVCAAYDT--...

JNKl.HS --- --- YQNLKPIG--SGAQGIVCAAYDA...

JNK^_RN -... YQNLKPIG--SGAQGIVCAAYDA-...

JNK2_HS -...- - YQOLKPIG- -SGAQGIVCAAFDT...

B0218_HS -.... -... ... YINLTPIG-

-TGAYGTVCAAECT---F42G8 . 3_CE -... YNSLKPLG--EGAYGWCTAEYE-...

P3 8G_HS -... ... YRDLQPVG- -SGAYGAVCSAVDG-...

P3 8D_HS --- --- --- YVSPTHVG--SGAYGSVCSAIDK ....

P3 8_XL -.... --- --- YQNLTPVG- SGAYGSVCSSFDT ...

-P38A_HS -.... YQNLSPVG-

-SGAYGSVCAAFDT---P38_CC -...

YQNLSPVG--SGAYGTVCSAYDE---P3 8B_HS LQGLRPVG - - SGAYGSVCSAYDA...

P38_I»! -... -...

YQGLQPVG--SGAYGQVSKAWR---H0G2_ZR -... -... YTDLNPVG-

-MGAFGLVCSATDT---HOGl.ZR YTDLNPVG-

-MGAFGLVCSATDT---H0G1_SC --- ---

---YNDLNPVG--MGAFGLVCSATDT---H0G1_CA ... ... ...YTELNPVG- -MGAFGLVCSAVDR- -

-STYl.SP -... -YSDLQPIG-

-MGAFGLVCSAKDQ---ERK5_HS -... ...YEI lETIG- -NGAYGWSSARRR...

ERK1_HS -... YTQLQYIG- - EGAYGMVSSAYDH -...

ERK2_HS -YTNLSYIG- -EGAYGMVCSAYDN...

ERKA_DM -...

YIKLAYIG--EGAYGMWSADDT---SUR1_CE -... -... YVNLSYIG- -EGAYGMVASALDT ...

FSMAPK_NH -... - -YDIQDWG- -EGAYGWCSAIHK...

PMK1_MG -... -... Y DIQ D W G - - EGAYGWCSAIHK...

MAPK_PNC YEILDV1G--EGAYGIVCSAIHK...

ERK1_CA -... - - YQILEIVG- -EGAYGIVCSAIHK...

KSS:_SC ... YKLVDLIG--EGAYGTVCSAIHK...

SPKl.SP -...-YEMINLIG-'QGAYGWCAALHK...

FUS3_SC ... FQLKSLLG- -EGAYGWCSATHK ....

ERKi_DD YSIVKCIG--HGAYGWCSAKDN...

MAPK_PS RPPIMPIG--KGAYGIVCSAHNS...

NTF4_NT

-KPPIMPIG--KGAYGIVCSALNS---MPK6_AT KPPIMPIG--KGAYGIVCSAMNS... ...

MPK3.AT -... RPPIIPIG--RGAYGIVCSVLDT...

MAPKi_PC RPPIMPIGRGAYGIVCSIMNT...

-MPK^AS -...

QPPIMPIG--RGAYGIVCSVMNF---MPK4_AT -... - - VPPLRPIG-

-RGAYGIVCAATNS---MMK2_MS ... VPPIRSVG-

-RGAYGIVCAAVNA---MPK5_AT

VPPIRPIG--RGAYGFVCPAVDS---NTF6_NT

IPPIQPVG--RGAYGMVCCATNS---MPK1_AT

YMP1KPIG--RGAYGWCSSVNS---MPK2_AT

YMPIKPIG--RGAYGWCSSVNR.-MPK7_AT

--YVPIKPIG--RGAYGWCSSINR---MPS 1_MG -... -...-... YTVTKELG - - QGAYGIVCAAVNN ...

-SPM1_SP ... ...

...FKWKELG--QGAYGIVCAARNVAS---MKC1_CA -... FKIVKELG--HGAYGIVCSAKYDNGSKKVPDS

SLT2_SC ... ...FQLIKEIG--HGAYGIVCSARFAEA...

KKQ1_SC ... FHLTGKIG--RGSHSLICSSTYTES...

SMK1_SC -... -.... - YEIIQFLG- - KGAYGTVCSVKFKGR...

P38B2_HS ... LQGLRPVG--SGAYGSVCSAYDAR...

CDK2_HS MENFQKVEKIG--EGTYGWYKARNK...

-B0478_CE FONVTHAKRAYRELKLMSLVN-JNK_DM FQNVTHAKRAYREFKLMKLVN-JNK1_HS FQNQTHAKRAYRELVLMKCVN-JNK3..RN -FQNOTHAKRAYRELVLMKCVN-JNK2_HS FQNOTllAKRAYRELVLLKCVN-B0218_HS FQSIIHARRTYRELRLLRCMC-F42G8.3_CE FQSTIHAKRTYRELKLLRTLQ--P38G_HS FQSELFAKLAYRELRLLKHMR--P38D_HS FQSEIFAKRAYRELLLLKHMQ--P38_XL FQSIIHAKRTYRELRLLKHMK--P38A_HS FQSIIHAKRTYRELRLLKHMK-P3 8_CC FQSIIKAKRTYRELRLLKHMK-P38B_HS FQSLIHARRTYRELRLLKHLK-P38_DM FQSAVHAKRTYRELRLLKHMA--H0G2_ZR FSTAVLAKRTYRELKLLKHLR--H0G1_ZR FSTAVLAKRTYRELKLLKHLR--H0G1_SC FSTAVLAKRTYRELKLLKHLR--H0G1_CA FSTSVLAKRTYRELKLLKHLK-STY1_SP FSTPVLAKRTYRELKLLKHLR-• ERK5_HS AFDWTNAKRTLRELKILKHFK--ERK1_HS FEHQTYCQRTLREIQILLRFR--ERK2_HS FEHQTYCQRTLREIKILLRFR--ERKA_DM -FEHQTYCORTLREITILTRFK--SUR1_CE FEHQTFCQRTLREIKILNRFK-• FSMAPK_NH FDHSMFCLRTLREMKLLRYFN-• PMK1_MG FDHSMFCLRTLREMKLLRYFN--MAPK.PNC FDHSMFCLRTLREMKLLRYFN-• ERK1_CA FERSMLCLRTLRELKLLKHFN--KSS1_SC FSKKLFVTRTIREIKLLRYFHE-SPK1_SP -FNHPVFCLRTLREIKLLRHFR- • FUS3_SC FDKPLFALRTLREIKILKHFK-• ERK1_DD AFDNLKDTKRTLREIHLLRHFK--MAPK_PS AFDNKIDAKRTLREIKLVRHMD--NTF4_NT ---- AFDNKIDAKRTLREIKLLRHMD--MPK6_AT .... AFDNKIDAKRTLREIKLLRHMD--MPK3_AT .... AFDNHMDAKRTLREIKLLRHLD--MAPK1_PC .... AFDNYMDAKRTLREIKLLRHLD--MPK_AS ---- AFDNNMDAKRTLREIKLLRHLD--MPK4_AT AFDNIIDAKRTLREIKLLKHMD--MMK2_MS AFDNRIDAKRTLREIKLLRHMD--MPK5_AT AFDNKVDAKRTLREIKLLRHLE--NTF6_NT AFENRIDAKRTLREIKLLSHMD--MPK1_AT VYENR1DALRTLRELKLLRHLR--MPK2_AT VFENRIDALRTLRELKLLRHLR--MPK7_AT VFENRVDALRTLRELKLLRHVR--MPS1_MG VFSKKILAKRALREIKLLQHFRG-SPMl.SP VFSKSILTKRALREIKLLIHFRN-MKC1_CA IFSKNILCKRALRELKLLQFFRG-SLT2_SC VFSKTLLCKRSLRELKLLRHFRG-KKQ1_SC AFGNKLSCKRTLRELKLLRHLRG-SMK1_SC IFNKEILLKRAIRELKFMNFFKG-P38B2_HS FQSLIHARRTYRELRLLKHLK--CDK2_HS

TETEGVPSTAIREISLLKELN--•HKNIIGILNCFTPQKK... LDEFNDL ■ HKNIIGLLNAFTPORN... LEEFQDV -HKNIIGLLNVFTPQKS... LEEFQDV ■HKNIISLLNVFTPQKT... --LEEFQDV ■HKNIISLLNVFTPQKT-.... --LEEFQDV ■HENIIDLLDVFTPNEN--- VNDIEDV •HDNVLBMIDVFTPDPD... ASSLNNV -HENVIGLLDVFTPDET--... LDDFTDF -HENVIGLLDVFTPASS- -.... -LRNFYDF -HENVIGLLDVFSPAKS... FEEFNDV •HENVIGLLDVFTPARS--... LEEFNDV -HENVIGLLDVFTPATS--... LEEFNDV •HENVIGLLDVFTPATS--... lEDFSEV •HENVIGLLDIFHPHPA---- NGSLENFQQV •HENLICLQDIFLSP--- LED---•HENLICLQDIFLSP HENLICLQDIFLSP... LED---•HENLITLDDIFISP--- LED---HENIISLSDIFISP ... FED--•HDNIIAIKDILRPTVP--- YGEPKSV •HENVIGIRDILRAST LEAMRDV -HEN IIGINDIIRAPT... IEQMKDV •HENIIDIRDILRVDS IDQMRDV •HENIINIQEIIRSET... VDSLKDI HENIISILDIQKPRN... YESFNEV •HENIISILDIQKPRS... YETFNEV •HENIISILDIQQPQD--- FESFSEV HENIISILAIQRPIN...-YESFNEI HEN 11SILDK VR PVS... IDKLNAV HENIISILDILPPPS... YQELEDV HENIITIFNIQRPDS... FENFNBV HENLISIKDILKPNSK... EQFEDV HENWAIRDIVPPPQR.... ....EVFNDV HENIVAIRDIIPPPQR.... EAFNDV HENIVAIRDIIPPPLR... N AFNDV HENIIAIRDWPPPLR... RQFSDV HENVIAITDVIPPPLR... REFTDV HENIVGLRDVIPPSIP... QSFNDV HENVIAVKDIIKPPQR... ENFNDV HENVMS1KDIIRPPQK--- ENFNHV HENVWIKDIIRPPKK...---EDFVDV HENIIKIKDIVRPPDR--- EEFNDV HENVIALKDVMMPIHK... MSFKDV HENWALKDVMMANHK--- RSFKDV HENVIALKDVMLPANR... TSFKDV HRNITCLYDMDIPRPD... NFNET HRNITCIYDLDIINPY--- NFNEV HKNITCLYDLDIIPNP... MTGEFNEI HKNITCLYDMDIVFYP--- DGSINGL HPNIVWLFDTDIVFYP---- N---GALNGV HKNIVNLIDLEIVTSS-... PYDGL HENVIGLLDVFTPATS---lEDFSEV HPNIVKLLDVIHTEN ... KL

B0478_CE JNK_DM JNK1_HS JNK1.RN JNK2_HS B0218_HS F42G8.3_C) P38G_HS P38D_HS P38_XL P38A_HS P38_CC P38B_HS P38_DM HOG2_2R H0G1_ZR H0G1_SC H0G1_CA STY1_SP ERK5_HS ERK1_HS ERK2_HS ERKA_DM SUR1_CE FSMAPK.NH PMK1_MG MAPK_PNC ERK1_CA KSS1_SC SPK1_SP njs3_sc ERK1_DD MAPK_PS NTF4_NT MPK6_AT MPK3_AT MAPK1_PC MPK_AS MPK4.AT MMK2_MS MPK5_AT NTF6_NT MPK1_AT MPK2_AT MPK7_AT MPS1_MG SPMl.SP MKC1_CA SLT2_SC KKQ1_SC SMK1_SC P38B2_HS CDK2_HS

YIVMELMDANLCQVIQMD- -... LDHERLSYLLYQMLCGIR- - -... YLVMELMDANLCQVIQMD... -LDHDRMSYLLYQMLCGIK-.. YIVMELMDANLCOVIQME...LDHERMSYLLYQMLCGIK... YLVMELMDANLCQVIQME...LDHERMSYLLYQMLSAIK-... . YLVMELMDANLCQVIHME... -LDHERMSYLLYQMLCGIK-... YFVSMLMGADLSNILKIQR--- --- LNDDHIQFLVYQILRGLK-... YFVSVLMGSDLQNIMKIQR... LTDEQIQLLIYQVLRGLKYMSHQNFNS YLVMPFMGTDLGKLMKHEK... --LGEDRIQFLVYQMMKGLR---YLVMPFMQTDLQKIMGMEF... -SEEKIQYLVYQMLKGLK... YLVTHLMGADLNNIVKCQK... LTDDHVQFLIYQILRGLK... -YLVTHLMGADLNNIVKCQK... LTDDHVQFLIYQILRGLK--... YLVTHLMGADLNNIVKCQK-...LTDDHVQFLIYQILRGLK... YLVTTLMGADLNNIVKCQAGAHQG---ARLALDEHVQFLVYQLLRGLK---YLVTHLMDADLNNIIRMQH ... LSDDHVQFLVYQILRGLK---YFVTELQGTDLHRLLQTRP--... LEKQFVQYFLYQILRGLK---YFVTELQGTDLHRLLQTRP...LEKQFVQYFLYQILRGLK-.... - - -YFVTELQGTDLHRLLQTRP-... LEKQFVQYFLYQILRGLK-... YFVNELQGTDLHRLLNSRP...LEKQFIQYFTYQIMRGLK---YFVTELLGTDLHRLLTSRP- --- LETQFIQYFLYQILRGLK---YWLDLMESDLHQIIHSSQP- ... LTLEHVRYFLYQLLRGLK---YIVQDLMETDLYKLLKSQQ... LSNDHICYFLYQILRGLK---YIVQDLMETDLYKLLKTQH-... LSNDHICYFLYQILRGLK... YIVQCLMETDLYKLLKTQR... LSNDHICYFLYQILRGLK.... .... YIVQCLMETDLYKLLKTQK--- -LSNDHVCYFLYQILRGLK---YLIQELMETDMHRAIRTQD-... -LSDDHCQYFIYQTLRALK... . YLIQELMETDMHRVIRTQD --- LSYDHCQYFIYQTLRALK.... .... YLIQELMETDMHRVIRTQD... LSDDHCQYFIYQILRALK... YLIQELMETDLHRVIRTQN... LSDDHIQYFIYQTLRALK.... .... YLVEELMETDLQKVINNQNSG---... FSTLSDDHVQYFTYQILRALK... YIVQELMETDLYRVIRSQP--- LSDDHCQYFTYQILRALK.... .... YIIQELMQTDLHRVISTQM... LSDDHIQYFIYQTLRAVK... . YIVSELMDTDLHQIITSPQP... LSDDHCQYFVYQMLRGLK... YIAYELMDTDLHQIIRSNQ...ALSEEHCQYFLYQILRGLK... YIAYELMDTDLHQIIRSNQG...LSEEHCQYFLYQILRGLK... YIAYELMDTDLHQIIRSNQ...ALSEEHCQYFLYQILRGLK... YISTELMDTDLHQIIRSNQS- - -.... LSEEHCQYFLYQLLRGLK... YIATELMDTDLHQIIRSNQG... . . LSEEHCQYFLYQLLRGLK... YIATELMDTDLHHIIRSNQE- -... LSEEHCQYFLYQLLRGLK... YIVYELMDTDLHQIIRSNQP... LTDDHCRFFLYQLLRGLK... YIVSELMDTDLHQIIRSNQP--...MTDDHCRYFVYQLLRGLK... YIVFELMDTDLHQI1RSNQS... LNDDHCQYFLYQI LRGLK... YIVYELMDTDLHQIIRSSQ... ALTDDHCQYFLYQLLRGLK-- -...* YLVYELMDTDLHQIIKSSQR--... -LSNDHCQYFLFQLLRGLK... YLVYELMDTDLHQIIKSSQ...VLSNDHCQYFLFQLLRGLK... YLVYELMDTDLHQIIKSSQS--- LSDDHCKYFLFQLLRGLK... YLYEELMECDLAAHRSGQP... LTDAHFQSFIYQILCGLK...-YIYEELMEADLNAIIKSGQP... LTDAHFQSFIYQILCGLK---YLYEELMECDMHQIIRSGQP- -... ... LSDQHYQSFIYQVLCGLN... YLYEELMECDMHQIIKSGQP--- LTDAHYQSFTYQILCGLK---YLYEELMECDLSQIIRSEQR... ... LEDAHFQSFIYQILCALK---YCYQELIDYDLAKVIHSSVQ... LSEFHIKYFLYQILCGLK---YLVTTLMGADLNNIVKCQA... LSDEHVQFLVYQLLRGLK---YLVFEFLHQDLKKFMDASALTG...

B0478_CE HLHSA-GIIHRDLK--PSNIWR... -... JNK_DM -... HLHSA-GIIHRDLK--PSNIWK... . JNK1_HS -... HLHSA-GIIHRDLK- - P S N I W K ... -... JNK3..RN HLHSA-GI1HRDLK--PSNIWK... ... ... JNK2_HS -...- -HLHSA-GIIHRDLK- -PSNIWK ... .... B0218_HS YIHSA-DIIHRDLK--PSNIAVN-... F42G8.3_CE TIILKKLMHPFQRRNTRFRLYIHSA-GIIHRDLK--PSNIAVNERCEVKVFLSFSQLSFL P38G_HS YIHAA-GIIHRDLK--PGNLAVN---- ---P38D_HS YIHSA-GWHRDLK--PGNLAVN... ... ... P38_XL YIHSA-GIIHRDLK--PSNLAVN... ... P38A_HS --- ---YIHSA-DIIHRDLK--PSNLAVN... P38_CC YIHSA-DIIHRDLK--PSNLAVN... ... -.... P38B_HS YIHSAGIIHRDLKPSNVAVN... -P38_DM ---... -... YIHSA-GVIHRDLK- -PSNIAVN ... H0G2_ZR --- ---YVHSA-GVIHRDLK--PSNILIN-... H0G1_2R --- --- ---YVHSA-GVIHRDLK--PSNILIN ... H0G1_SC --- --- YVHSA-GVI HRDLK--PSNI LIN---H0G1_CA ... ... YIHSA-GVIHRDLK--PSNILIN... STY1_SP ... ... FVHSA-GV1HRDLK--PSNILIN... ERK5_HS --- --- YMHSA-QVIHRDLK--PSNLLVN... ERK1_HS -.... -... YIHSA-NVLHRDLK- - PSNLLIN ---- -... ERK2_HS -... YIHSA-NVLHRDLK--PSNLLLN... ERKA_DM -... -YIHSA-NVLHRDLK- - PSNLLLN ... SUR1_CE -... - -YIHSA-NVLHRDLK- - PSNLLLN... -... FSMAPK_NH -... - - AMHSA-NVLHRDLK- - PSNLLLN ... PMKl.MG AMHSA-NVLHRDLK--PSNLLLN... MAPK_PNC ... AMHSA-DILHRDLK--PSNLLLN... ERK1_CA ... - - AMHSA-NVLHRDLK- - PSNLLLN- -.... ... KSS1_SC ... SIHSA-OVIHRDIK--PSNLLLN... ... SPK1_SP -... - -AMHSA-GWHRDLK- - PSNLLLN... FUS3_SC VLHGS-NVIHRDLK--PSNLLIN... ... ERKl.DD ... ... HIHSA-NVLHRDLK--PSNLLIN... MAPK_PS -.... --YIHSA-NVLHRDLK--PSNLLLN... NTF4_NT ---... YIHSA-NVLHRDLK--PSNLLLN -... MPK6_AT -... YIHSA-NVLHRDLK- - PSNLLLN... MPK3_AT .. ... -YIHSA-NI IHRDLK--PSNLLLN-... MAPK1_PC -...YIHSA-NI IHRDLK--PSNLLLN... MPK_AS ... ... YIHSA-NVIHRDLK- - PSNLLLN...

MPK4_AT -YVHSA-NVLHRDLK--PSNLLLN...

MMK2_MS ... - YVHSA-NVLHRDLK - - PSNLLLN... MPK5_AT ... ... YIHSA-NVLHRDLK--PSNLLLN... ... NTF6_NT ... ... YVHSA-NVLHRDLK--PSNLLLN-- ---- ---MPKl^AT ... ... YIHSA-NILHRDLK- - PGNLLVN... --- ---MPK2_AT ... ... - - - YIHSA-NILHRDLK- - PGNLLVN... MPK7_AT ... YLHSA-NILHRDLK- - PGNLLVN... -...-.... MPS 1_HG -... YIHSA-NVLHRDLK- - PGNLLVN... -... SPM1_SP YIHSA-NVIHRDLK--PGNLLVN---- ---MKC1_CA ... ....FIHSA-DVLHRDLK--PGNLLVN... -... SLT2_SC ... ... YIHSA-DVLHRDLK--PGNLLVN ... ... KKQ1_SC -YIHSA-NVLHCDLK--PKNLLVN... ... SMK1_SC YIHSA-DVIHRDLK--PGNILCT... ... P3 8B2_HS -...YIHSA-GI IHRDLK--PSNVAVN... CDK2_HS ... FCHSH-RVLHRDLK- - PQNLLIN... -... ...

B0478_CE JNK_DM JNK1_HS JNK3_RN JNK2_HS B0218_HS F42G8.3_C1

P3 8G_HS

P38D_HS

P38_XL

P3 8A_HS

P3 6_CC

P38B_HS P3B_DM H0G2_ZR HCX31_ZR H0G1_SC H0G1_CA STY1_SP ERK5_HS ERK1_HS ERK2_HS ERKA_DM SUR1_CE FSMAPK_NH PMK1_MG MAPK_PNC ERK1_CA KSS1_SC SPK1_SP FUS3„SC ERK1_DD KAPK_PS

N T K 4 _ N T

MPK6.AT MPK3.AT MAPKi.PC MPK_AS MPK4_AT MMK2_MS MPK5_AT NTF6_NT MPK1_AT MPK2_AT MPK7_AT MPSl.MG SPMl.SP MKC1_CA SLT2.SC KKQ1_SC SMK1_SC P38B2_HS CDK2_HS

SDCTLKILDFGLAR --TAIE-A--... -... FMMTPYWT

ADCTLKILDFGLAR... -TAGT-T... FMMTPYWT

SDCTLKILDFGLAR...TAGT-S ... -... FMMTPYWT

S DCTLKILDFG L AR... TAGT - S... ...- FMMT P Y W T

SDCTLKILDFGLAR... -TACT-N - -.-... FMMTPYWT

EDCELKILDFGLAR... (JTDS- E... -... MTGYVAT

ILSFFKILDFGLAR--....AQDA- E--- -... MTGYVAT

EDCELKILDFGLAR... QADS-E- -... -.... -... M T G Y W T

EDCELKILDFGLAR...KADA-E--- M T G Y W T

EDCELKILDFGLAR- -... HTDE-E... -... MTGYVAT

EDCELKILDFGLAR...HTDD-E... -... MTGYVAT

EDCELKILDFGLAR...HTDD- E... -... MTGYVAT

EDCELRILDFGLAR---QADE-E--... -... MTGYVAT

EDCELRILDFGLAR- - PTEN-E... -...-... MTGYVAT

ENCDLKICDFGLAR... IQDP-Q- -... -... MTGYVST

ENCDLKICDFGLAR---lODP-Q- -... MTGYVST

ENCDLKICDFGLAR--- IQDP-Q- -... -...MTGYVST

ENCDLKICDFGLAR---LQDP-Q--- -MTGYVST

ENCDLKICDFGLAR---IQDP-Q--- -MTGYVST

ENCELKIGDFGMAR---GLCTSPAEHQY --- F-MTEYVAT

TTCDLKICDFGLAR--....lADPEHDHT... F-LTEYVAT

TTCDLKICDFGLAR--- VADPDHDHTG-... -... F-LTEYVAT

KTCDLKICDFGLAR---lADPEHDHTG... F-LTEYVAT

TTCDLKICDFGLAR---VTDPQTDHTG... ...F - LTEYVAT

ANCDLKVCDFGLAR-...SAASQEDNSG--- F-MTEYVAT

ANCDLKVCDFGLAR-...SAASQENNSG... F-MTEYVAT

ANCDLKVCDFGLAR-... SAVSTEDSSS... -.... F-MTEYVAT

SNCDLKICDFGLAR...SIASQEDNYG-... -... F-MTEYVAT

SNCDLKVCDFGLAR--- CLASSSDSRETLVG... F-MTEYVAT

ANCDLKVADFGLAR...STTAQGGNPG-... F-MTEYVAT

SNCDLKVCDFGLAR---IIDESAADNSEPTG(MSG... MTEYVAT

EDCLLKICDLGLAR...VED.... ATHQG... F-MTEYVAT

ANCDLKICDFGLAR...VTS.... ETD... F-MTEYWT

ANCDLKICDFGLAR...VTS.... ETD... F - M T E Y W T

ANC DLKIC DFGLAR -.... VTS.... ESD... F - MTE Y W T

ANCDLKICDFGLAR...PTS.... END... F-MTEYWT

ANCDLKICDFGLAR... HNT DDE ... -... F-MTEYWT

ANCDLKICDFGLAR...PSS.... ESD---... M-MTEYWT

ANCDLKLGDFGLAR... TKS ETD... -... F-MTEYWT

ANCDLKIGDFGLAR... TTS ETD--... -... F-MTEYWT

SNCDLKITDFGLAR... TTS ETE... - -Y-MTEYWT

ANCDLKICDFGLAR...TTS.... EAD... F-MTEYWT

ANCDLKICDFGLAR... ASNT KGQ... -... F-MTEYWT

ANCDLKICDFGLAR--- TSNT--- KGQ... F-MTEYWT

ANCDLKICDFGLAR... TSQG NEQ... -... F-MTEYWT

ADCELKICDFGLAR... -GFSVDPEENAG-... Y-MTEYVAT

ADCELKICDFGLAR... GCSENPEENPG... F-MTEYVAT

ADCELKICDFGLAR... GFSENPDENAG... -... F-MTEYVAT

ADCQLK ICDFGLAR -....GYSENPVENSQ --- F-LTEYVAT

SDCQLKICNFGLSC... SYSENHKVNDG... -... --F-IKGYITS

LNGCLKICDFGLAR--- GIHAGFFKCHSTVQP--- H-ITNYVAT

EDCELRILDFGLAR--- QADEE... -... MTGYVAT

TEGAIKLADFGLAR.... - - AFGVPVRT... YTH E W T

C-KYYSTAVDIWS--LGCIFAEMVTRR-ALFPGDSEID-B0478_CE QWTRIIEO--- L--- GTP---DRSFLERLQPTV--RN... -.... -... JNK_EW QVmKI lEQ L GTP- - -SPSFMQRLQPTV- - RN... -... JNK1_HS QWNKVIEQ--- L--- GTP---CPEFMKKLQPTV--RT... JNK3 _RN QWNKVIEQ-L- - - OTP- - -CPEFMKKLQPTV- - KN... ... JNK2_HS OWNKVIEQ L GTPSAEFMKKLQPTVRN... .... ... -B0218_HS QLTRIMSV--- T --- GTP--- DEEFLKKISSEE-ARN... ... F4 2G8. 3_CE QLTKIMSV--- V --- GTP---KEEFWSKIQSEE-ARN-.... ... P3 BG_HS QLKEIMKV--- T --- GTP---PAEFVQRLOSDE-AKN... P3 8D_HS QLTQILKV--- T --- GVP---GTEFVQKLNDKA-AKS... P3 8_XL QLKLILRL--- V --- GTP---EPELLQKISSEA-ARN... -.... P3 8A_HS QLKLILRL--- V --- GTP---GAELLKKISSES-ARN... ... P38_CC QLQQIMRL--- T --- GTP- - - PASLISRMPSHE-ART... -...-... P36B_HS QLKRIMEV--- V --- GTP---SPEVLAKISSEH-ART... P38_DM QLNLIMEM--- L--- GTP-- - PAEFLKKISSES-ARS-.... -... H0G2_ZR QFSIITDL--- L--- GSP---PRDVINTICSENTLK... ... H0G1_ZR QFSIITDL--- L--- GSP---PRDVIITICSEDTLK... H0G1_SC QFSIITDL--- L--- GSP---PKDVINTICSENTLK--... H0G1_CA QFSIITEL--- L--- GSP---PADVIDT1CSENTLR--- -STY1_SP QFSIITEL--- L--- GTP---PMEVIETICSKNTLR-- ---ERK5_HS QLQLIMMV--- L--- GTP---SPAVIQAVGAER-VRA... ... ERK1_HS QLNHILGI----L--- GSP---SQEDLNCIINMK-ARN-... ... ... ERK2_HS QLNHILGI--- L--- GSP---SQEDLNCIINLK-ARN... ERKA_DM QLNHILGV--- L--- GSP SRDDLECIINEK-ARJJ-... ... . SUR1_CE QLNLILAV--- V --- GSP---SNADLQCIINDK-ARS... ... FSMAPK.NH QLTLILDV--- L --- GTP---TMEDYYGIKSRR-ARE--... PMK1_MG QLTLILDV--- L --- GTP---TMEDYYGIKSRR-ARE... -.... MAPK_PNC QLMLILDV---- L--- GTP---TMEDYYGIKSRR-ARE... ERK1_CA QLWLIMEV--- L --- GTP-- NMEDYYNIKSKR-ARE... ... KSSl. SC QLWLILEV--- L--- GTP- - -SFEDFNQIKSKR-AKEYIA--- ---SPK1_SP QITLILNI--- L--- GTP---TMDDFSRIKSAR-ARK--- ---FUS3_SC QLLLIFGI--- 1--- GTP- --HSDNDLRCIESP-RAR... -... ERK1_DD QITLIIET--- 1--- GSP---SEEDICNIANEQ-ARQ... MAPK_PS QLRLLMEL--- I--- GTP---SEADLGFLNEN--AKRY... NTF4_NT QLRLLMEL--- I--- GTP---SEAEMEFLNEN--AKRY... MPK6_AT QLRLLMEL--- I--- GTP---SEEELEFLNEN--AKRY... MPK3.AT QMRLLTEL--- L --- GTP---TESDLGFTHNED-AKR... ... MAPK1_PC QMRLLTEL--- L--- GSP---TEADLGFVRNED-AKR... MPK_AS QMRLITEV--- I--- GTP---TDDDLGFIRNED-ARR-- ---MPK4.AT QLRLITEL--- 1--- GSP---DDSSLGFLRSDN-ARR... .... MMK2_MS QLRLVTEL--- I--- GSP-- -DDASLGFLRSEN-ARR... MPK5_AT QLKLITEL--- 1--- GSP---DGASLEFLRSAN-GGK... NTF6_NT QLGLI lAL---- L--- GSP- - -EDSDLGFLRSDN-ARK... MPK1_AT QLKLIVNI---- 1--- GSQ REEDLEFIVNPK-AKR... MPK2_AT QIKLIINI---- L--- GSQ REEDLEFIDNPK-AKR ... .... MPK7_AT QLKLIINV--- V --- GSQ---QESDIRFIDNPK-ARR... MPS1_MG QLNQILHI---- L--- GTP NEETLSRIGSPR-AQE- ---SPMl.SP QLNLILHQ---- L--- GTP DEETLSHISSSR-AQE... MKC1_CA QLNQILMI----L --- GTP-- PESTLQRIGSHR-AQN--- ---SLT2_SC QLNQILQV--- L--- GTP- - - PDETLRRIGSKN-VQD... -... .... KKQ1_SC HLNHILQI--- L--- GTP---PEETLQEIASQK-VYN--- -... SMK1_SC QIFEIIKV L GTP DKDILIKFGTIKAWNLG... -P38B2_HS QLKRIMEV--- V--- GTP---SPEVLAKISSEH-ART.... .... ... CDK2_HS QLFRIFRT---- L--- GTP DEWWPGVTSMP-DYK---

B0478_CE JNK_DM JNK1_HS JNK3_RN JNK2_HS B0218_HS F42G8.3_CE P38G_HS P38D_HS P38_XL P38A_HS P38_CC P38B_HS P38_DM H0G2_ZR H0G1_ZR H0G1_SC H0G1_CA STY1_SP ERK5_HS ERK1_HS ERK2_HS ERKA_DM SUR1_CE FSMAPK_NH PMK1_MG MAPK_PNC ERKl.CA KSS1_SC SPK1_SP FUS3_SC ERK1_DD MAPK_PS NTF4_NT MPK6_AT MPK3_AT MAPK1_PC MPK_AS MPK4_AT MMK2_MS MPK5_AT NTF6_NT MPKl.AT MPK2_AT MPK7_AT MPS1_MG SPMl.SP MKC1_CA SLT2_SC KKQ1_SC SMK1_SC P38B2_HS CDK2_HS

YVENRPRYQATPFEVLFSDNMFPMTADSS RLTGAQARDLLSRMLVI ---YVENRPRYTGYSFDRLFPDGLFPNDNNQNS--RRKASDARNLLSKMLVI ---YVENRPKYAGYSFEKLPPDVLFPADSEHN---KLKASQARDLLSKMLVI ---YVENRPKYAGLTFPKLFPDSLFPADSEHN---KLKASQARDLLSKMLVI •--YVENRPKYPGIKFEELFPDWIFPSESERD---KIKTSQARDLLSKMLVI - - -YIRNLPKMTRRDFKRLFAQAT--- PQAIDLLEKMLHL - - -YIKNRSPIIRODFVTLFPMAS--- PYALELLEMMLIL YMKGLPELEKKDFASILTNAS ... PLAVNLLEKMLVL YIOSLPQTPRKDFTOLFPRAS--- PQAADLLEKMLEL - • - YIQSLPYMPKMNFEDVFLGAN ... PQAVDLLEKMLVL - - - YIQSLTQMPKMNFANVFIGAN---PLAVDLLEKMLVL - - - YINSLPQMPKRNFSEVFIGAN--- PQAVDLLEKMLVL - - - YIOSLPPMPQKDLSSIFRGAN ... PLAIDLLGRMLVL - - - YIQSLPPMKGRSFKNVFKNAN--- PLAIDLLEKMLEL - - -FVTSLPHRDPVPFQERFKTVE--- PDAVOLLRRMLVF - - -FVTSLPHRDPVPFQERFKAVE--- PDAVDLLGRMLVF ---FVTSLPHRDPIPFSERFKTVE...PDAVDLLEKMLVF - - -FVQSLPHRDPIPFSERFASCT- -... HVEPEAIDLLAKLLVF FVQSLPQKEKVPFAEKFKNAD...PDAIDLLEKMLVF - - - YIQSLPPRQPVPWETVYPGAD ... RQALSLLGRMLRF - - -YLQSLPSKTKVAWAKLFPKSD-... SKALDLLDRMLTF - - - YLLSLPHKNKVPWNRLF PNAD... SKALDLLDKMLTF - - - YLESLPFKPNVPWAKLFPNAD ... ALALDLLGKMLTF YLISLPHKPKQPWARLYPGAD...PRALDLLDKMLTF ---YIRSLPFKKKVPFRTLFPKTS ... DLALDLLEKLLAF YIRSLPFKKKVPFRTLFPQDF --- GSRLDLLEKLLAF - - -YIRSLPFKKRVSFASIFPRAN... -.... PLALDLLEKLLAF ---YIRSLPFCKKIPFSELFANTNNNTSTSNTGGRTNINPLALDLLEKLLIF

NLPMRPPLPWETVWS-KTDLN- -... -... PDMIDLLDKMLQF - - -YIKSLPFTPKVSFKALFPQAS- -...- PDAIDLLEKLLTF - -EYIKSLPMYPAAPLEKMFPRVN ... -PKGIDLLQRMLVF -FIRSLNMGNQPKVNFANMFPKAN...-PDAIDLLERMLYF 1RQLPLYRRQSFOEKFPHVH... PEAIDLVEKMLTF IRQLPLYRROSFVEKFPHVN---...PAAIDLVEKMLTF IRQLPPYPRQSITDKFPTVH... PLAIDLIEKMLTF YIRQLPNFPRQPLAKLFSHVN... PMAIDLVDRMLTF ---FILQLPRHPRQPLROLYPQVH--- PLAIDLIDKMLTF - - - YMRHLPQFPRRPFPGQFPKVQ... PAALDLIERMLTF - - - YVRQLPQYPRQNFAARFPNMS... -.... AGAVDLLEKMLVF - - - YVRQLPQYPKQNFSARFPNMS-... -... PGAVDLLEKMLIF YVXELPKFPRQNFSARFPSMN ... STAIDLLEKMLVF - - -YVKHLPRVPRHPFSQKFPDVS---PLALDLAERMLVF YIRSLPYSPGMSLSRLYPCAH--- VLAIDLLQKMLVF ---YIESLPYSPGISFSRLYPGAN... VLAIDLLQKILVL FIKSLPYSRGTHLSNLYPQAN---...PLAIDLLQRMLVF YVRNLPFMAKKPFPTLFPNAN ... PDALDLLDRMLAF YVRSLPKQRPIPFETNFPKAN -... PLALDLLAKLLAF YVRSLPITRKASYEELFPDAN... PLALDLLERMLTL YIHQLGFIPKVPFVNLYPNAN... -... SQALDLLEQMLAF YIFQFGNIPGRSFESILPGAN---PEALELLKKMLEF KNSNNPVYKKIPWSNIFPFAS--- HEAINLIESLLHW ---YIQSLPPMPQKDLSSIFRGAN-...PLAIDLLGRMLVL PSFPKWARQDFSKWPPLD... EDGRSLLSQMLHY

B0478_CE DPERRIS-JNK^DM DPEQRIS-JNK1_HS DASKRIS JNK3_ RN DPAKBIS-JNK2_HS DPDKRIS B0218_HS DPDRRPT-F4 2G8.3_CE DPDRRIS-P3 8