Mol Biol Rep DOI 10.1007/s11033-014-3728-y
Phylogenetic analysis of STK gene family and Usp domain in maize Ting Zhou • Mingxia Fan • Muhammad Irfan He Wang • Dongxu Wang • Lin Wang • Chunyu Zhang • Lin Feng
•
Received: 11 January 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Serine and threonine kinase STK1 and STK2 play an important regulatory role in the process of pollen development in maize. Six homologous sequences which were similar with STK1 and STK2 having more than 80 % similarity were found at NCBI, and they all belong to STK gene family. Phylogenetic analysis showed that STK family in maize might belong to RLK family. In STK family, gene duplication event was occurred during evolutionary process, and experienced purifying selection after gene duplication and the time of gene duplication was about 12 million years ago. The domains of STK family belongs to single transmembrane protein, which have intracellular conserved kinase catalytic domain and extracellular receptor domain on N-terminal. The evolution of intracellular selection was faster than extracellular selection, and positive selection or weak purifying selection play an important role. Analyzing its unique Usp domain we found that it was located between sensor domain at N-terminal and catalytic domain at C-terminal, which belongs to hydrophobic protein with several phosphorylation sites, acting on serine and threonine protein phosphorylation. The kinship of Usp domain in STK family was close to 35-like protein containing U-box domain, predicting that they might belong to the same family with a
Ting Zhou, Mingxia Fan have contributed equally and considered joint first author. T. Zhou M. Fan M. Irfan H. Wang D. Wang L. Wang C. Zhang (&) L. Feng (&) Biotechnology and Bioscience College, Shenyang Agricultural University, Shenyang 110866, China e-mail: [email protected] L. Feng e-mail: [email protected]
similar structure and function, so that we can predict the function of Usp domain in STK family. Keywords Receptor like kinase Phylogeny Ka/Ks Protein structure Usp domain Abbreviations STK Serine and threonine kinase RLK Receptor like kinase Usp Universal stress proteins
Introduction Serine/threonine kinases (STK) are kind of the receptorlike protein kinases (RLKs), which phosphorylate serine and threonine residues [1], changing the conformation of functional protein [2], leading to changes in protein activity and protein nature by catalyzing phosphorylation of various functional protein and further regulating protein biological activity [3]. It also plays an important role in regulating cell proliferation [4], differentiation, apoptosis, and embryonic development of life activities within the cell [5], and also involved in growth and various stress responses of plants [6]. Plant RLK is a kind of STK characterized by single transmembrane, N terminal signal peptide, receptor domain, transmembrane domain and protein kinase catalytic domain, playing an important signal transduction roles in plants [7]. The extracellular signal molecules bind receptor domains when they are recognized by N terminal signal peptides, and then are transferred into cells by transmembrane domains. After received by intracellular
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protein kinase catalytic domains, these extracellular signal molecules open or shutdown the downstream target protein, and trigger a cascade of cellular signals through phosphorylation or dephosphorylation [8]. A Bz chain gene was found on the short arm of chromosome 9 in maize working on anthocyanin biosynthesis gene Bronze in Dr. Dooner’s laboratory. Its encoded protein has a serine/threonine protein kinase catalytic domain, thus named it serine/threonine protein kinase gene 1(stk1) [9]. At the same time, a gene was also found in maize on the long arm of chromosome 4 which also encodes serine/ threonine protein kinase catalytic domain, named stk2. Huang and Dooner found that stk1 and stk2 were expressed in mature tassel and stk2 was also expressed in pollen, both of them were not expressed in other tissues. stk1 and stk2 mutants self progeny phenotype had slower pollen tube growth, stk1 is more significant which indicated that stk1 may play a more important role in the process of pollen development [10]. STK is an important defense signal transduction protein, its serine/threonine domain is an important part of the tomato Pto gene which not only interacts with the avirulence proteins from Pseudomonas syringae, but also acts as the medium of signal transduction [11]. The protein encoded by rice bacterial blight resistance gene Xa21 also contains a serine/threonine protein kinase domain [12]. Universal stress proteins (Usp) are multiple genes encoding resistance-associated proteins found widely in plants [13].When the plants are under duress by the adverse external conditions, DNA is damaged [14] or any other factors retards plant growth, up-regulated gene expression of Usp [15], enhanced cellular resistance [16], improve the content of the cytoplasmic phosphate, make serine and threonine phosphorylation, make cells in a relatively stable state [17]. Here we predicted the molecular evolutionary relationships and the structure and function of its unique Usp domain of STK gene family members through STK gene family and its domain in maize. This study could provide the basis for further study of evolutionary relatedness and biological function at the genetic level, support domain mediates signal transduction at the molecular level and reliable data sources for the use in agricultural production of STK gene family.
Materials and methods Retrieval of amino acid sequence of STK in maize Amino acid sequences of STK1 and STK2 in maize were obtained from NCBI, the accession number was AAK73111 and AFW64201. Six genes were obtained from
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maize genome by BlastP at NCBI which were more than 80 % similar with STK1 and STK2, the accession numbers were NP001183778, AAV64214, ACR38284, ACA21852, AFW64200 and AFW64201.1 respectively. In order to facilitate the analysis, stk1,stk2, stk3, stk4, stk5, stk6, stk7, stk8 were used instead of these eight candidate sequences in the following article. Amino acid sequences of STK1 and STK2 were used as the search object and then 34 highly similar sequences were obtained by BLASTp. In the same way, the sequence of serine/threonine protein kinase (STK) domain was used as the search object and then 36 highly similar sequences containing STK domain were obtained in maize genome. In addition, 17 highly similar Usp domain-containing genes were also obtained when Usp domain was used as the search object. Sequence analysis and phylogenetic of STK in maize Sequences were analyzed using ClustalX2.0 software for multiple sequence alignment by Neighbor-Joining method using default parameters. Goldview5.0 software was used to output results. Phylogenetic tree of 34 highly similar candidate sequences from 12 species, 36 highly similar candidate sequences containing STK domain and 17 highly similar candidate sequences containing Usp domain was constructed using MEGA 5 software on a set of 1,000 bootstrap replicates. Protein characterization of STKs in maize Signal peptide segment was predicted by SignalP V3.0 (http://www.cbs.dtu.dk/services/SignalP/) and subcellular localization was predicted by Predictprotein (https://www. predictprotein.org) and NetNES(http://www.cbs.dtu.dk/ser vices/NetNES/) was used to predict the signal site of leucine. Selection pressure and time of gene duplication Paralogs were inferred according to the results of phylogenetic tree among STK gene family members, vacancies were removed by multiple sequences, then KaKs_Calculator was used to calculate the rate of synonymous substitution (Ks) and antisense replacement (Ka). When Ka/ Ks [ 1, which indicates that paralogs experienced positive selection after replication. If Ka/Ks \ 1, which indicates that they experienced purifying selection after replication. If Ka/Ks & 1, this is the result of natural selection or neutral selection [18]. According to molecular clock theory, during the evolution the time variation of Ks between duplicate genes were similar [19], so Ks was used to estimate the time of gene duplication event and calculated as T = Ks/2c, c represents for molecular replacement rate, and its value is 6.5 9 10-9 [20].
Mol Biol Rep Table 1 STK gene family members in maize Name
Serial number (NCBI)
AA length
E-value
Domain
Chromosomal location
stk1
AAK73111
788
3.36E-16
Usp; PKinase
9
stk2
AFW64201
761
6.82E-15
Usp; PKinase
4
stk3
NP_001183778.1
787
3.36E-16
Usp; PKinase
9
-16
stk4
AAV64214.1
767
3.36E
Usp; PKinase
9
stk5
ACR38284.1
520
3.36E-16
PKinase
9
stk6
ACA21852.1
789
6.04E-10
Usp; PKinase
9
stk7
AFW64200.1
761
6.88E-15
PKinase
4
stk8
AFW64201.1
565
6.88E-15
Usp; PKinase
4
Functional prediction of Usp domain in STK family SMART (http://smart.embl-heidelberg.de) was used to predict the protein domain of STK family members from maize genome. ProtScale (http://www.expasy.org/cgi-binprotscale.pl) was used to analyze hydrophobic of Usp domain from eight STK family members. NetPhos (http:// www.cbs.dtu.dk/services/NetPhos/) was used to predict the posttranslational modification of Usp domain.
Results and discussion stk gene in maize genome and multiple sequence alignment Six amino acid sequences from maize genome which were highly homologous with STK1 and STK2 were obtained by BLASTp through NCBI, named stk3, stk4, stk5, stk6, stk7 and stk8 respectively. stk2, stk7 and stk8 were located on the chromosome 4 and stk1, stk3, stk4, stk5 and stk6 were located on chromosome 9 according to GRAMENE(http:// www.gramene.org/). Predicting the domains of eight candidate sequences found that they all had the kinase catalytic domain and six of them had the Usp domain except STK5 and STK7 (Table 1). It indicated that these eight candidate sequences might have the similar functions and belong to the same STK family. ClustalX2 was used for multiple sequence alignment with eight candidate sequences of STK gene family in maize (Fig. 1). The similarity among STK3, STK4, STK6 and STK1 was greater than 96 %. Even though STK5 and STK7 have large fragment deletion, but they also have a high similarity with STK1, STK3, STK4 and STK6, which was about 70–95 %. The amino acid sequence of STK2 has the highest similarity with STK7 and STK8 which was greater than 99 %. Overall, there were a lot of highly conserved residues between eight candidate sequences, marked by ‘‘*’’, which indicate that there were high degree of sequence similarity between family members and
remain highly conservative in evolution. The lack of Usp domain might be the reason of large fragment deletion on the N terminal of STK5 and STK7. Overall, eight sequences had a high similarity among each other, some were even more than 99 %, which indicated that they had strong conservation during evolution and might belong to the same STK family. All the members contained kinase catalytic domain and Usp domain while STK5 and STK7 lack Usp domain. The similarity of domains in STK family indicated the similarity of their functions. We found the characteristics of stk have been established before species differentiation and expanded according to species-specific manner after species differentiation through analyzing the evolutionary relationship between STK family members and its homologous genes in other species. Phylogenetic analysis of STK gene family in maize Thirty-four amino acid sequences which were highly homologous with STK1 and STK2 were obtained by BlastP at NCBI from 12 species, which were Arabidopsis, rice, maize, sorghum, castor, grape, barley, soybean, Camellia sinensis, Bristlegrass, Brachypodium and Alfalfa. In order to determine the molecular evolution of STK gene family, MEGA5.0 was used for construction of phylogenetic tree using random sampling repeat of 1,000 times (Fig. 2).The homologous sequences were divided into six clusters, each cluster had higher bootstrap support and each cluster member was not completely clustered according to the origin of species. This showed that independent replication existed in the evolutionary process and characteristics of gene family were established before species differentiation. While some were clustered according to the origin of species, indicated that stk was already existed before species differentiation. Here the phylogenetic tree had identified some homologous genes, nine pairs of orthologous genes, which were XP003631778 and XP003527814, EEE65245.1 and XP003572885, XP003564167 and BAK06108, XP004954165 and EEC74143, NP001048340
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Mol Biol Rep Fig. 1 Multiple sequence alignment of STK gene family. Asterisk indicated the amino acid identity and colon showed the amino acid similarity, dot represented low conservative residue. The red line and the green line represented the location of Usp domain and kinase domain respectively
Usp domain
Kinase domain
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Mol Biol Rep Fig. 2 Phylogenetic tree of STK gene family. Brackets in different colors represented different branches
and BAK07305, XP002437997 and XP002513301, XP002452944 and EAY99981, CAB87673 and ACE75946, XP003588643 and stk5. Five pairs of paralogs, which were mainly focus on STK family, expect XP003524426 and XP003530200. So many paralogs existed in STK family indicated that it expanded according to species-specific manner. STK family members can be divided into two classes according to their relative relationships, namely STK1, STK3, STK4, STK6 and STK8 were classified as a class, STK5 and STK7 were another class. The former one had close genetic relationship with XP004964592 containing U-box domain of Brochypodium distachyon, they might have similar function, but it lacked U-Box structure, this might because the U-Box structure was missing during the evolution. The later had close genetic relationship with XP003588643 containing kinase catalytic domain which was similar with Usp structure in soybean and Brochypodium disachyon, might belong to the same subfamily and play important role in serine and threonine phosphorylation [21]. So we predicted these two might encode two different proteins. Thirty-six candidate sequences from maize with STK domain were selected and constructed a phylogenetic tree (Fig. 3). The phylogenetic tree showed that STK family was similar with RLK family according to the evolutionary distance, such as NP001151988 and ADM88869, they might belong to the same family and had the similar structure and function.
In this study, the result was consistent with the classification of Hanks [22] and Walker [23] which acted on plant protein kinase based on the sequence similarity of catalytic region. Group I AGC, can receive signals of second messenger directly or indirectly, Group II CaMPK, dependents on Ca2?/CaM or activated by SNF1/AMP, regulated by second messenger, Group III CMGL, locate downstream of phosphorylation system, can not be regulated by second messenger directly, its representative kinase are CDK, MAPK, GSK3, CKI and CKII. Group IV are the kinase except the above three, like RLK. This result showed that STK family members were more inclined to RLK family. Their evolutionary distances were very close, predicting that they might belong to the same subfamily with a similar structure and function. So STK family members might play an important role in cell metabolism [24], phosphorylation [25] and plant signal transmission [26]. Protein characterization The signal peptide on N terminal sequences of STK family was predicted by SignalP v3.0 (Fig. 4). The signal peptide cleavage sites of STK1, STK2, STK3, STK4, STK6 and STK8 and were located between 15th and 16th amino acid. Namely STK2 and STK8 were between G and G, STK3, STK4, STK1 and STK6 were between D and L. The signal peptide cleavage sites of STK5 and
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Mol Biol Rep Fig. 3 Phylogenetic tree of serine/threonine protein kinase family in maize
Fig. 4 Prediction of signal peptide of STK gene family. X axis represented the number of amino acid and Y axis represented the value of Cscore, Sscore and Yscore. The lines in different colors represented S value and Y value of different members, the red line represented C value. The black arrow showed signal peptide cleavage sites of eight members. Sscore: each amino acid corresponding to one S value which was higher in signal peptide region. Cscore: the value of
splice site, each amino acid had a C value, which was the highest at splice site. Yscore: Y-max was a parameter comprehensive considered by S value and C value, it was more accurate than considering C value alone. Because there might be more than one higher site in a series of C values, but only one cleavage site. The splice site was predicted by Y-max value when S value was at steep position and had a high C value site
STK7 were located between 13th, 14th and 18th, 19th amino acids respectively, that was between F-E and A-K respectively.
We found that signal peptide of STK family were all located in the first 20 amino acids on N-terminal, which could form two spiral hairpin structure and insert into
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Fig. 5 a The Ka/Ks ratios of usp domain for stk paralogous genes pairs in maize. b The Ka/Ks ratios of kinase domain for stk paralogous genes pairs in maize. c Ka/Ks ratios of whole sequence for stk paralogous pairs of genes in maize
endoplasmic reticulum and its lumen, playing an important role in protein synthesis and expression through predicting their signal peptide. We found that the content of serine was very high, when predicted the protein structure of STK family, which demonstrated (Fig. 2) that STK family belongs to the serine/threonine receptor kinase family. All the STK family members had leucinerich signal sites which was the characteristic of RLK family [27]. It is the cell membrane where STK family members were located and their structures could be predicted by Predictprotein software. The content of serine in each member was the highest and some were even several times higher than other kinds. STK family members had the leucine-rich nuclear export signals, which were predicted by the NetNES software, this was one of the characteristics of RLK [28]. Leucine-rich nuclear export signal sites of stk1, stk3, stk4 and stk6 were located in 328 and 330 amino acids, and stk4 was located in 61 and 63 while the other members of STK family were located in 318 and 320 amino acids.
Ka/Ks calculation and time of gene duplication Research showed that positive Darwinian selection is the main force of functional divergence of gene [29]. Ka/Ks of paralogs, kinase catalytic domain and Usp domain of STK family members were used to research whether positive selection could cause changes of gene function after gene duplication (Fig. 5).The results showed that Ka/Ks of all the family members were less than 1, among them stk3stk4 was close to 1. Even though stk5 and stk7 did not belong to paralogous genes, but they were in the same subfamily on the phylogenetic tree and the distance of evolution was short, its ka/ks [ 1 (Fig. 5c). Analyzing Ka, Ks value of Usp domain section of all the paralogs, we found that ka/ks was close to 1, among them stk3-stk4 showed ka/ks [ 1(Fig. 5a). Most of ka/ks value of kinase catalytic domain were greater than 1, only stk1-stk4 showed ka/ks \ 1 and stk1-stk3 showed ka/ks & 1 (Fig. 5b). Ka/Ks value of stk3-stk4 and stk2-stk8 were all greater than 1 when compared with Usp domain and kinase catalytic domain (stk2-stk8 showed ka/ks & 1 in Usp
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domain), but the ka/ks of whole section were less than 1. This indicated that ka/ks of stk3-stk4 and stk2-stk8 in domains were higher. The ka/ks outside the domains section were significantly lower than domain section which indicated that the evolution of domain sections was faster. Positive selection played an important role in protein function after gene duplication [30]. Generally, nucleotide changes without amino acid changes called synonymous mutation (Ks), nucleotide changes with amino acid changes called non-synonymous mutation (Ka). Usually Ks was not affected by natural selection, but this affects Ka [31]. The ratio of Ka/Ks was molecular evolution parameter for balancing the selection pressure which could judge whether there was selection pressure acting on the protein coded by genes [32]. In this study all the paralogous of STK family showed ka/ks \ 1, indicated that genes experienced purifying selection after replication. The number of ka/ks [ 1 in kinase catalytic domain was more than the number in Usp domain, because Usp domain belongs to extracellular domain and kinase catalytic domain belongs to intracellular domain. Ka/Ks of intracellular domain were significantly higher than that of extracellular domain, which indicated intracellular domain evolved more quickly than extracellular domain. Positive selection or weaker purifying selection played an important role in the evolution of kinase catalytic domain, while the evolution of Usp domain was mainly affected by weaker purifying selection. Overall, Ka/Ks of the whole sequence were lower than 1, and the Ka/Ks of domains were greater than 1 or approximate to 1. This showed that domains were more easily affected by positive selection or weaker purifying selection and their evolutionary speed was quick, so positive selection played an important role in the process of evolution of the domains in STK family. Among paralogs, positively selected sites existed in most of duplicate genes, even though some did not have positively selected sites but their Ka/Ks values were close to 1, which indicated that positive selection and natural selection played an important role in functional differences of STK gene family. In order to further study the evolution of species-specific in STK family, we estimated their time of gene duplication occurred (Table 2).The Ks value of paralogs pairs stk1stk3, stk1-stk4, stk2-stk8 were broadly similar, which ranged from 1.613 to 1.6189, while Ks value of stk3-stk4 was slightly smaller, which was 1.07638. Time of gene duplication occurred of four paralogs pairs (stk1-stk3, stk1-stk4, stk2-stk8, stk3-stk4) were calculated by the formula T = Ks/2c, which were 13 million years, 12.78 million years, 12.4 million years and 8.82 million years ago respectively. Generally speaking, the time of gene duplication occurred for stk1-stk3, stk1-stk4, stk2-stk8 were similar. This research revealed that the time of paralogs stk3-stk4 was later, and its evolution speed was faster.
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Table 2 Ka/Ks ratios for all paralogous stk genes and estimation of the absolute date for large-scale duplication event in STK gene family in maize Duplicated Pair
Ka
Ks
Ka/Ks
stk3-stk4
0.972121
1.07638
0.903137
Date (million years) 8.28
stk1-stk3
0.913147
1.68982
0.540381
13
stk1-stk4
0.916248
1.66173
0.551381
12.78
stk2-stk8
0.897705
1.61314
0.556495
12.4
Note Ks the number of synonymous mutation SNP/the number of synonymous sites (Single Nucleotide Polymorphism SNP), Ka the number of synonymous mutation SNP/the number of nonsynonymous sites, the number of synonymous mutation SNP Rsynonymous SNP, the number of nonsynonymous mutation SNP Rnonsynonymous SNP, the number of synonymous sites Rsynonymous sites, the number of nonsynonymous sites Rnonsynonymous sites
Analysis of domain of STK gene family Serine/threonine kinase domain Domains of STK family members were predicted by SMART, the results showed that STK catalytic domain existed in all the members, and there were ATP binding site, substrate binding site, active site and activation loop in their domains (Fig. 6). There was a STK domain on C-terminal in the intracellular and the N terminal domain in the extracellular, which was consistent with the characteristics of RLK gene family [33]. STK gene family in maize (except STK5 and STK7) belongs to transmembrane protein, their amino acid sites of transmembrane helix region were located between 389 and 406. There were receptor domain on N-terminal, transmembrane domain consisted of 18 amino acids and STK catalytic domain. These characteristics tended that STK family belongs to RLK family, which was proved by phylogenetic tree in Fig. 3. Analysis of Usp domain Usp domain existed in all the members except STK5 and STK7 predicted by SMART online software. It was located between sensor domain on N-terminal and catalytic domain on C-terminal with a size of 130 amino acids which was encoded by multiple genes. Usp domain could act on the phosphorylation of serine and threonine and play an important role in cell stable growth. STK family members belong to single transmembrane protein except STK5 and STK7, predicted by Hmmtop online software. The amino acid sites of transmembrane helix section were located at 138–155 which was overlapped with the end of Usp domain and linked intracellular and extracellular together. Usp domain was existed in STK family generally according to the study, which was located between
Mol Biol Rep
Fig. 6 The domain of STK family (except stk5 and stk7). The red part on N terminal was Usp domain showed by green triangle whose length was from 17 to 156. The yellow part on N terminal was signal peptide showed by blue triangle whose size was from 7 to 29. The green part was transmembrane domain showed by red triangle,
whose length was from 152 to 169. Kinase domain was located on C-terminal containing ATP binding site showed in blue part, activation loop structure showed in orange triangle, whose size was from 447 to 683
Fig. 7 Hydrophobic analysis of Usp. X axis represented the position of amino acid, Y axis represented the score of hydrophobicity or hydrophilicity. The min score was -2.7 at the position of 10th and the max score was 1.656 at the position of 64th amino acid
sensor domain on N-terminal and catalytic domain on C-terminal, encoded by multiple genes, and the size was about 130 amino acids. The transmembrane helix region of STK family were overlapped with the end of Usp domain, which linked extracellular and intracellular together (Fig. 6) and helped the signal enter into intracellular, played a role in signal transmission [34].
Hydrophobic analysis of Usp domain ProteiScale was used to analyze the hydrophobicity of Usp domain (Fig. 7). The hydrophilic aspartic acid located on the tenth position was the strongest, so the possibility of acting as the antigen epitope was highest, and the hydrophobicity of leucine located on the sixty-fourth position was highest. Overall there were so many hydrophobic and hydrophilic regions on Usp domain. The distribution of hydrophobic and hydrophilic regions on the first 80 amino acids were more homogeneous, but hydrophobic region was the main part at the back portion, which located between 18–19, 26–34, 42–46, 60–66, 88–110, 114–116 and 118–120. So we could infer that the Usp might belong to hydrophobic protein according to hydrophobic analysis of Usp domain.
Transmembrane helix consisted of transmembrane protein in hydrophobic environment was most stable because of hydrogen bond which could achieve maximum link in hydrophobic environment. Because transmembrane domains coincided with the end of Usp domain, so Usp with hydrophobic is conducive to maintain the stability of transmembrane structure. The result of post-translational modifications about Usp domain showed that there were eight phosphorylation sites with the score greater than 0.5, namely phosphorylation might occur on the sites of 4 serine, 2 threonine and 2 tryptophan after Usp was translated into amino acids. Post translational modification of Usp domain Post-translational modification of Usp domain protein was predicted by NetPhos2.0 Server (Fig. 8). There were eight amino acids whose scores were higher than 0.5, four of them were serine and the position on peptide chain was 11, 40, 88 and 106. Their predictive values were 0.825, 0.884, 0.929, and 0.649 respectively. Another two were threonine, whose positions were 28 and 39, their predictive values were 0.972 and 0.614 respectively. The last two were tryptophan whose positions were 17 and 49 having predictive values of 0.529 and 0.744 respectively. This
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Mol Biol Rep Fig. 8 Post-translational modification of Usp domain. X axis represented the location of amino acid, Y axis represented the score of threshold. The lines in blue, green, red and gray represented serine, threonine, tyrosine and threshold respectively, if the score was higher than threshold, there would have a phosphorylation site
Fig. 9 Phylogenetic tree analysis of USP domain in multi species. 17 highly similar sequences containing Usp domain from different spaces at NCBI were used to build the neighbor-joining tree by MEGA5.0, random sampling repeat 1,000 times. Bootstrap length represented evolutionary distance, the greater the number (bootstrap values) was the closer the distance was. Brackets in different colors represented different branches
indicated that Usp phosphorylation could occur on serine, threonine and tryptophan sites. Analysis of homology of Usp family Seventeen sequences containing Usp domain of five different species which were highly similar with Usp domain of STK family were used to build the phylogenetic tree by MEGA5.0 (Fig. 9). The tree was divided into five clusters, and the members of STK family were divided into the same cluster. There was little difference between STK1, STK3, STK4 and STK6, also STK2 and STK8 in the process of evolution. STK1, STK3, STK4 belong to paralogs relationships also STK2 and STK8. The number and the sort of motifs they contain were the same which indicated that
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they might have common ancestral genes. After species differentiation Usp domain gene of STK family expanded according to species-specific manner. The phylogenetic relationship between 35-like protein containing U-box domain and STK family was similar; they might belong to the same family and have the similar structure and function. Each cluster member were not clustered according to species source indicated that the characteristics of Usp domain had been established before species differentiation. The rice EAY9998 was placed in a single cluster which indicated its Usp domain had significant differences with other members and expressed different functions. Because Usp domain was existing in all the members in the tree, speculated that before species differentiation they should belong to the same family and have common ancestral
Mol Biol Rep
genes. So the function of Usp domain in STK family can be predicted according to the function expressed by Usp domain in other species whose genetic relationship were close to STK family. The relationship between 35-like protein containing U-box domain and STK family was similar; they might belong to the same family and have the similar structure and function. The Usp domain of STK family could be predicted by the function expressed by Usp domain in other families which was similar with STK family, like 35-like protein. The Usp was produced as a result of the gene expression under adverse conditions which switched off some normal genes expression [35]. Usp expression can be induced in some conditions no matter the stimulation conditions were physical, chemical or biological factors [36]. In this sense it was adaption initiative and self-defense capabilities produced by the plant for changing external environment [37]. Usp can maintain the basic viability of plants and involve in plant defense mechanisms [38] as an ATP-binding protein, such as the transportation of chloroplasts, plant hormones, secondary metabolites, stomata movement [39] and pathogen infection [40]. Usp might control the porosity in plant leaves and participate in material transportation regulating in other tissues which played a defensive role [41]. Study showed that the growth of E. coli cells were inhibited when in the condition of Usp gene inactivation, while cell growth entered into a capture period when Usp gene overexpression [42]. Usp had a high expression in legume nodulation after inoculating legume with rhizobium [43]. Upland GUSP1 and GUSP2 had a high expression in its stems, roots, leaves under the condition of drought [44]. Usp gene was found to be up regulated after post-ripening treatment in tomato by Ethylene [45]. Mushegian and Koonin predicted that Usp might have DNA binding specificity and protect the DNA [46]. Usp can induce expression when plants were in stress conditions, so the resistance of Usp was important for plant growth and development [47]. According to the analysis above we found that STK family members might also have the function expressed by Usp, namely it can protect the cells from DNA damage, maintain cell homeostasis and participate in stress responses.
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Phylogenetic analysis of STK gene family and Usp domain in maize Ting Zhou • Mingxia Fan • Muhammad Irfan He Wang • Dongxu Wang • Lin Wang • Chunyu Zhang • Lin Feng
•
Received: 11 January 2014 / Accepted: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Serine and threonine kinase STK1 and STK2 play an important regulatory role in the process of pollen development in maize. Six homologous sequences which were similar with STK1 and STK2 having more than 80 % similarity were found at NCBI, and they all belong to STK gene family. Phylogenetic analysis showed that STK family in maize might belong to RLK family. In STK family, gene duplication event was occurred during evolutionary process, and experienced purifying selection after gene duplication and the time of gene duplication was about 12 million years ago. The domains of STK family belongs to single transmembrane protein, which have intracellular conserved kinase catalytic domain and extracellular receptor domain on N-terminal. The evolution of intracellular selection was faster than extracellular selection, and positive selection or weak purifying selection play an important role. Analyzing its unique Usp domain we found that it was located between sensor domain at N-terminal and catalytic domain at C-terminal, which belongs to hydrophobic protein with several phosphorylation sites, acting on serine and threonine protein phosphorylation. The kinship of Usp domain in STK family was close to 35-like protein containing U-box domain, predicting that they might belong to the same family with a
Ting Zhou, Mingxia Fan have contributed equally and considered joint first author. T. Zhou M. Fan M. Irfan H. Wang D. Wang L. Wang C. Zhang (&) L. Feng (&) Biotechnology and Bioscience College, Shenyang Agricultural University, Shenyang 110866, China e-mail: [email protected] L. Feng e-mail: [email protected]
similar structure and function, so that we can predict the function of Usp domain in STK family. Keywords Receptor like kinase Phylogeny Ka/Ks Protein structure Usp domain Abbreviations STK Serine and threonine kinase RLK Receptor like kinase Usp Universal stress proteins
Introduction Serine/threonine kinases (STK) are kind of the receptorlike protein kinases (RLKs), which phosphorylate serine and threonine residues [1], changing the conformation of functional protein [2], leading to changes in protein activity and protein nature by catalyzing phosphorylation of various functional protein and further regulating protein biological activity [3]. It also plays an important role in regulating cell proliferation [4], differentiation, apoptosis, and embryonic development of life activities within the cell [5], and also involved in growth and various stress responses of plants [6]. Plant RLK is a kind of STK characterized by single transmembrane, N terminal signal peptide, receptor domain, transmembrane domain and protein kinase catalytic domain, playing an important signal transduction roles in plants [7]. The extracellular signal molecules bind receptor domains when they are recognized by N terminal signal peptides, and then are transferred into cells by transmembrane domains. After received by intracellular
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protein kinase catalytic domains, these extracellular signal molecules open or shutdown the downstream target protein, and trigger a cascade of cellular signals through phosphorylation or dephosphorylation [8]. A Bz chain gene was found on the short arm of chromosome 9 in maize working on anthocyanin biosynthesis gene Bronze in Dr. Dooner’s laboratory. Its encoded protein has a serine/threonine protein kinase catalytic domain, thus named it serine/threonine protein kinase gene 1(stk1) [9]. At the same time, a gene was also found in maize on the long arm of chromosome 4 which also encodes serine/ threonine protein kinase catalytic domain, named stk2. Huang and Dooner found that stk1 and stk2 were expressed in mature tassel and stk2 was also expressed in pollen, both of them were not expressed in other tissues. stk1 and stk2 mutants self progeny phenotype had slower pollen tube growth, stk1 is more significant which indicated that stk1 may play a more important role in the process of pollen development [10]. STK is an important defense signal transduction protein, its serine/threonine domain is an important part of the tomato Pto gene which not only interacts with the avirulence proteins from Pseudomonas syringae, but also acts as the medium of signal transduction [11]. The protein encoded by rice bacterial blight resistance gene Xa21 also contains a serine/threonine protein kinase domain [12]. Universal stress proteins (Usp) are multiple genes encoding resistance-associated proteins found widely in plants [13].When the plants are under duress by the adverse external conditions, DNA is damaged [14] or any other factors retards plant growth, up-regulated gene expression of Usp [15], enhanced cellular resistance [16], improve the content of the cytoplasmic phosphate, make serine and threonine phosphorylation, make cells in a relatively stable state [17]. Here we predicted the molecular evolutionary relationships and the structure and function of its unique Usp domain of STK gene family members through STK gene family and its domain in maize. This study could provide the basis for further study of evolutionary relatedness and biological function at the genetic level, support domain mediates signal transduction at the molecular level and reliable data sources for the use in agricultural production of STK gene family.
Materials and methods Retrieval of amino acid sequence of STK in maize Amino acid sequences of STK1 and STK2 in maize were obtained from NCBI, the accession number was AAK73111 and AFW64201. Six genes were obtained from
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maize genome by BlastP at NCBI which were more than 80 % similar with STK1 and STK2, the accession numbers were NP001183778, AAV64214, ACR38284, ACA21852, AFW64200 and AFW64201.1 respectively. In order to facilitate the analysis, stk1,stk2, stk3, stk4, stk5, stk6, stk7, stk8 were used instead of these eight candidate sequences in the following article. Amino acid sequences of STK1 and STK2 were used as the search object and then 34 highly similar sequences were obtained by BLASTp. In the same way, the sequence of serine/threonine protein kinase (STK) domain was used as the search object and then 36 highly similar sequences containing STK domain were obtained in maize genome. In addition, 17 highly similar Usp domain-containing genes were also obtained when Usp domain was used as the search object. Sequence analysis and phylogenetic of STK in maize Sequences were analyzed using ClustalX2.0 software for multiple sequence alignment by Neighbor-Joining method using default parameters. Goldview5.0 software was used to output results. Phylogenetic tree of 34 highly similar candidate sequences from 12 species, 36 highly similar candidate sequences containing STK domain and 17 highly similar candidate sequences containing Usp domain was constructed using MEGA 5 software on a set of 1,000 bootstrap replicates. Protein characterization of STKs in maize Signal peptide segment was predicted by SignalP V3.0 (http://www.cbs.dtu.dk/services/SignalP/) and subcellular localization was predicted by Predictprotein (https://www. predictprotein.org) and NetNES(http://www.cbs.dtu.dk/ser vices/NetNES/) was used to predict the signal site of leucine. Selection pressure and time of gene duplication Paralogs were inferred according to the results of phylogenetic tree among STK gene family members, vacancies were removed by multiple sequences, then KaKs_Calculator was used to calculate the rate of synonymous substitution (Ks) and antisense replacement (Ka). When Ka/ Ks [ 1, which indicates that paralogs experienced positive selection after replication. If Ka/Ks \ 1, which indicates that they experienced purifying selection after replication. If Ka/Ks & 1, this is the result of natural selection or neutral selection [18]. According to molecular clock theory, during the evolution the time variation of Ks between duplicate genes were similar [19], so Ks was used to estimate the time of gene duplication event and calculated as T = Ks/2c, c represents for molecular replacement rate, and its value is 6.5 9 10-9 [20].
Mol Biol Rep Table 1 STK gene family members in maize Name
Serial number (NCBI)
AA length
E-value
Domain
Chromosomal location
stk1
AAK73111
788
3.36E-16
Usp; PKinase
9
stk2
AFW64201
761
6.82E-15
Usp; PKinase
4
stk3
NP_001183778.1
787
3.36E-16
Usp; PKinase
9
-16
stk4
AAV64214.1
767
3.36E
Usp; PKinase
9
stk5
ACR38284.1
520
3.36E-16
PKinase
9
stk6
ACA21852.1
789
6.04E-10
Usp; PKinase
9
stk7
AFW64200.1
761
6.88E-15
PKinase
4
stk8
AFW64201.1
565
6.88E-15
Usp; PKinase
4
Functional prediction of Usp domain in STK family SMART (http://smart.embl-heidelberg.de) was used to predict the protein domain of STK family members from maize genome. ProtScale (http://www.expasy.org/cgi-binprotscale.pl) was used to analyze hydrophobic of Usp domain from eight STK family members. NetPhos (http:// www.cbs.dtu.dk/services/NetPhos/) was used to predict the posttranslational modification of Usp domain.
Results and discussion stk gene in maize genome and multiple sequence alignment Six amino acid sequences from maize genome which were highly homologous with STK1 and STK2 were obtained by BLASTp through NCBI, named stk3, stk4, stk5, stk6, stk7 and stk8 respectively. stk2, stk7 and stk8 were located on the chromosome 4 and stk1, stk3, stk4, stk5 and stk6 were located on chromosome 9 according to GRAMENE(http:// www.gramene.org/). Predicting the domains of eight candidate sequences found that they all had the kinase catalytic domain and six of them had the Usp domain except STK5 and STK7 (Table 1). It indicated that these eight candidate sequences might have the similar functions and belong to the same STK family. ClustalX2 was used for multiple sequence alignment with eight candidate sequences of STK gene family in maize (Fig. 1). The similarity among STK3, STK4, STK6 and STK1 was greater than 96 %. Even though STK5 and STK7 have large fragment deletion, but they also have a high similarity with STK1, STK3, STK4 and STK6, which was about 70–95 %. The amino acid sequence of STK2 has the highest similarity with STK7 and STK8 which was greater than 99 %. Overall, there were a lot of highly conserved residues between eight candidate sequences, marked by ‘‘*’’, which indicate that there were high degree of sequence similarity between family members and
remain highly conservative in evolution. The lack of Usp domain might be the reason of large fragment deletion on the N terminal of STK5 and STK7. Overall, eight sequences had a high similarity among each other, some were even more than 99 %, which indicated that they had strong conservation during evolution and might belong to the same STK family. All the members contained kinase catalytic domain and Usp domain while STK5 and STK7 lack Usp domain. The similarity of domains in STK family indicated the similarity of their functions. We found the characteristics of stk have been established before species differentiation and expanded according to species-specific manner after species differentiation through analyzing the evolutionary relationship between STK family members and its homologous genes in other species. Phylogenetic analysis of STK gene family in maize Thirty-four amino acid sequences which were highly homologous with STK1 and STK2 were obtained by BlastP at NCBI from 12 species, which were Arabidopsis, rice, maize, sorghum, castor, grape, barley, soybean, Camellia sinensis, Bristlegrass, Brachypodium and Alfalfa. In order to determine the molecular evolution of STK gene family, MEGA5.0 was used for construction of phylogenetic tree using random sampling repeat of 1,000 times (Fig. 2).The homologous sequences were divided into six clusters, each cluster had higher bootstrap support and each cluster member was not completely clustered according to the origin of species. This showed that independent replication existed in the evolutionary process and characteristics of gene family were established before species differentiation. While some were clustered according to the origin of species, indicated that stk was already existed before species differentiation. Here the phylogenetic tree had identified some homologous genes, nine pairs of orthologous genes, which were XP003631778 and XP003527814, EEE65245.1 and XP003572885, XP003564167 and BAK06108, XP004954165 and EEC74143, NP001048340
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Mol Biol Rep Fig. 1 Multiple sequence alignment of STK gene family. Asterisk indicated the amino acid identity and colon showed the amino acid similarity, dot represented low conservative residue. The red line and the green line represented the location of Usp domain and kinase domain respectively
Usp domain
Kinase domain
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Mol Biol Rep Fig. 2 Phylogenetic tree of STK gene family. Brackets in different colors represented different branches
and BAK07305, XP002437997 and XP002513301, XP002452944 and EAY99981, CAB87673 and ACE75946, XP003588643 and stk5. Five pairs of paralogs, which were mainly focus on STK family, expect XP003524426 and XP003530200. So many paralogs existed in STK family indicated that it expanded according to species-specific manner. STK family members can be divided into two classes according to their relative relationships, namely STK1, STK3, STK4, STK6 and STK8 were classified as a class, STK5 and STK7 were another class. The former one had close genetic relationship with XP004964592 containing U-box domain of Brochypodium distachyon, they might have similar function, but it lacked U-Box structure, this might because the U-Box structure was missing during the evolution. The later had close genetic relationship with XP003588643 containing kinase catalytic domain which was similar with Usp structure in soybean and Brochypodium disachyon, might belong to the same subfamily and play important role in serine and threonine phosphorylation [21]. So we predicted these two might encode two different proteins. Thirty-six candidate sequences from maize with STK domain were selected and constructed a phylogenetic tree (Fig. 3). The phylogenetic tree showed that STK family was similar with RLK family according to the evolutionary distance, such as NP001151988 and ADM88869, they might belong to the same family and had the similar structure and function.
In this study, the result was consistent with the classification of Hanks [22] and Walker [23] which acted on plant protein kinase based on the sequence similarity of catalytic region. Group I AGC, can receive signals of second messenger directly or indirectly, Group II CaMPK, dependents on Ca2?/CaM or activated by SNF1/AMP, regulated by second messenger, Group III CMGL, locate downstream of phosphorylation system, can not be regulated by second messenger directly, its representative kinase are CDK, MAPK, GSK3, CKI and CKII. Group IV are the kinase except the above three, like RLK. This result showed that STK family members were more inclined to RLK family. Their evolutionary distances were very close, predicting that they might belong to the same subfamily with a similar structure and function. So STK family members might play an important role in cell metabolism [24], phosphorylation [25] and plant signal transmission [26]. Protein characterization The signal peptide on N terminal sequences of STK family was predicted by SignalP v3.0 (Fig. 4). The signal peptide cleavage sites of STK1, STK2, STK3, STK4, STK6 and STK8 and were located between 15th and 16th amino acid. Namely STK2 and STK8 were between G and G, STK3, STK4, STK1 and STK6 were between D and L. The signal peptide cleavage sites of STK5 and
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Mol Biol Rep Fig. 3 Phylogenetic tree of serine/threonine protein kinase family in maize
Fig. 4 Prediction of signal peptide of STK gene family. X axis represented the number of amino acid and Y axis represented the value of Cscore, Sscore and Yscore. The lines in different colors represented S value and Y value of different members, the red line represented C value. The black arrow showed signal peptide cleavage sites of eight members. Sscore: each amino acid corresponding to one S value which was higher in signal peptide region. Cscore: the value of
splice site, each amino acid had a C value, which was the highest at splice site. Yscore: Y-max was a parameter comprehensive considered by S value and C value, it was more accurate than considering C value alone. Because there might be more than one higher site in a series of C values, but only one cleavage site. The splice site was predicted by Y-max value when S value was at steep position and had a high C value site
STK7 were located between 13th, 14th and 18th, 19th amino acids respectively, that was between F-E and A-K respectively.
We found that signal peptide of STK family were all located in the first 20 amino acids on N-terminal, which could form two spiral hairpin structure and insert into
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Fig. 5 a The Ka/Ks ratios of usp domain for stk paralogous genes pairs in maize. b The Ka/Ks ratios of kinase domain for stk paralogous genes pairs in maize. c Ka/Ks ratios of whole sequence for stk paralogous pairs of genes in maize
endoplasmic reticulum and its lumen, playing an important role in protein synthesis and expression through predicting their signal peptide. We found that the content of serine was very high, when predicted the protein structure of STK family, which demonstrated (Fig. 2) that STK family belongs to the serine/threonine receptor kinase family. All the STK family members had leucinerich signal sites which was the characteristic of RLK family [27]. It is the cell membrane where STK family members were located and their structures could be predicted by Predictprotein software. The content of serine in each member was the highest and some were even several times higher than other kinds. STK family members had the leucine-rich nuclear export signals, which were predicted by the NetNES software, this was one of the characteristics of RLK [28]. Leucine-rich nuclear export signal sites of stk1, stk3, stk4 and stk6 were located in 328 and 330 amino acids, and stk4 was located in 61 and 63 while the other members of STK family were located in 318 and 320 amino acids.
Ka/Ks calculation and time of gene duplication Research showed that positive Darwinian selection is the main force of functional divergence of gene [29]. Ka/Ks of paralogs, kinase catalytic domain and Usp domain of STK family members were used to research whether positive selection could cause changes of gene function after gene duplication (Fig. 5).The results showed that Ka/Ks of all the family members were less than 1, among them stk3stk4 was close to 1. Even though stk5 and stk7 did not belong to paralogous genes, but they were in the same subfamily on the phylogenetic tree and the distance of evolution was short, its ka/ks [ 1 (Fig. 5c). Analyzing Ka, Ks value of Usp domain section of all the paralogs, we found that ka/ks was close to 1, among them stk3-stk4 showed ka/ks [ 1(Fig. 5a). Most of ka/ks value of kinase catalytic domain were greater than 1, only stk1-stk4 showed ka/ks \ 1 and stk1-stk3 showed ka/ks & 1 (Fig. 5b). Ka/Ks value of stk3-stk4 and stk2-stk8 were all greater than 1 when compared with Usp domain and kinase catalytic domain (stk2-stk8 showed ka/ks & 1 in Usp
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domain), but the ka/ks of whole section were less than 1. This indicated that ka/ks of stk3-stk4 and stk2-stk8 in domains were higher. The ka/ks outside the domains section were significantly lower than domain section which indicated that the evolution of domain sections was faster. Positive selection played an important role in protein function after gene duplication [30]. Generally, nucleotide changes without amino acid changes called synonymous mutation (Ks), nucleotide changes with amino acid changes called non-synonymous mutation (Ka). Usually Ks was not affected by natural selection, but this affects Ka [31]. The ratio of Ka/Ks was molecular evolution parameter for balancing the selection pressure which could judge whether there was selection pressure acting on the protein coded by genes [32]. In this study all the paralogous of STK family showed ka/ks \ 1, indicated that genes experienced purifying selection after replication. The number of ka/ks [ 1 in kinase catalytic domain was more than the number in Usp domain, because Usp domain belongs to extracellular domain and kinase catalytic domain belongs to intracellular domain. Ka/Ks of intracellular domain were significantly higher than that of extracellular domain, which indicated intracellular domain evolved more quickly than extracellular domain. Positive selection or weaker purifying selection played an important role in the evolution of kinase catalytic domain, while the evolution of Usp domain was mainly affected by weaker purifying selection. Overall, Ka/Ks of the whole sequence were lower than 1, and the Ka/Ks of domains were greater than 1 or approximate to 1. This showed that domains were more easily affected by positive selection or weaker purifying selection and their evolutionary speed was quick, so positive selection played an important role in the process of evolution of the domains in STK family. Among paralogs, positively selected sites existed in most of duplicate genes, even though some did not have positively selected sites but their Ka/Ks values were close to 1, which indicated that positive selection and natural selection played an important role in functional differences of STK gene family. In order to further study the evolution of species-specific in STK family, we estimated their time of gene duplication occurred (Table 2).The Ks value of paralogs pairs stk1stk3, stk1-stk4, stk2-stk8 were broadly similar, which ranged from 1.613 to 1.6189, while Ks value of stk3-stk4 was slightly smaller, which was 1.07638. Time of gene duplication occurred of four paralogs pairs (stk1-stk3, stk1-stk4, stk2-stk8, stk3-stk4) were calculated by the formula T = Ks/2c, which were 13 million years, 12.78 million years, 12.4 million years and 8.82 million years ago respectively. Generally speaking, the time of gene duplication occurred for stk1-stk3, stk1-stk4, stk2-stk8 were similar. This research revealed that the time of paralogs stk3-stk4 was later, and its evolution speed was faster.
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Table 2 Ka/Ks ratios for all paralogous stk genes and estimation of the absolute date for large-scale duplication event in STK gene family in maize Duplicated Pair
Ka
Ks
Ka/Ks
stk3-stk4
0.972121
1.07638
0.903137
Date (million years) 8.28
stk1-stk3
0.913147
1.68982
0.540381
13
stk1-stk4
0.916248
1.66173
0.551381
12.78
stk2-stk8
0.897705
1.61314
0.556495
12.4
Note Ks the number of synonymous mutation SNP/the number of synonymous sites (Single Nucleotide Polymorphism SNP), Ka the number of synonymous mutation SNP/the number of nonsynonymous sites, the number of synonymous mutation SNP Rsynonymous SNP, the number of nonsynonymous mutation SNP Rnonsynonymous SNP, the number of synonymous sites Rsynonymous sites, the number of nonsynonymous sites Rnonsynonymous sites
Analysis of domain of STK gene family Serine/threonine kinase domain Domains of STK family members were predicted by SMART, the results showed that STK catalytic domain existed in all the members, and there were ATP binding site, substrate binding site, active site and activation loop in their domains (Fig. 6). There was a STK domain on C-terminal in the intracellular and the N terminal domain in the extracellular, which was consistent with the characteristics of RLK gene family [33]. STK gene family in maize (except STK5 and STK7) belongs to transmembrane protein, their amino acid sites of transmembrane helix region were located between 389 and 406. There were receptor domain on N-terminal, transmembrane domain consisted of 18 amino acids and STK catalytic domain. These characteristics tended that STK family belongs to RLK family, which was proved by phylogenetic tree in Fig. 3. Analysis of Usp domain Usp domain existed in all the members except STK5 and STK7 predicted by SMART online software. It was located between sensor domain on N-terminal and catalytic domain on C-terminal with a size of 130 amino acids which was encoded by multiple genes. Usp domain could act on the phosphorylation of serine and threonine and play an important role in cell stable growth. STK family members belong to single transmembrane protein except STK5 and STK7, predicted by Hmmtop online software. The amino acid sites of transmembrane helix section were located at 138–155 which was overlapped with the end of Usp domain and linked intracellular and extracellular together. Usp domain was existed in STK family generally according to the study, which was located between
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Fig. 6 The domain of STK family (except stk5 and stk7). The red part on N terminal was Usp domain showed by green triangle whose length was from 17 to 156. The yellow part on N terminal was signal peptide showed by blue triangle whose size was from 7 to 29. The green part was transmembrane domain showed by red triangle,
whose length was from 152 to 169. Kinase domain was located on C-terminal containing ATP binding site showed in blue part, activation loop structure showed in orange triangle, whose size was from 447 to 683
Fig. 7 Hydrophobic analysis of Usp. X axis represented the position of amino acid, Y axis represented the score of hydrophobicity or hydrophilicity. The min score was -2.7 at the position of 10th and the max score was 1.656 at the position of 64th amino acid
sensor domain on N-terminal and catalytic domain on C-terminal, encoded by multiple genes, and the size was about 130 amino acids. The transmembrane helix region of STK family were overlapped with the end of Usp domain, which linked extracellular and intracellular together (Fig. 6) and helped the signal enter into intracellular, played a role in signal transmission [34].
Hydrophobic analysis of Usp domain ProteiScale was used to analyze the hydrophobicity of Usp domain (Fig. 7). The hydrophilic aspartic acid located on the tenth position was the strongest, so the possibility of acting as the antigen epitope was highest, and the hydrophobicity of leucine located on the sixty-fourth position was highest. Overall there were so many hydrophobic and hydrophilic regions on Usp domain. The distribution of hydrophobic and hydrophilic regions on the first 80 amino acids were more homogeneous, but hydrophobic region was the main part at the back portion, which located between 18–19, 26–34, 42–46, 60–66, 88–110, 114–116 and 118–120. So we could infer that the Usp might belong to hydrophobic protein according to hydrophobic analysis of Usp domain.
Transmembrane helix consisted of transmembrane protein in hydrophobic environment was most stable because of hydrogen bond which could achieve maximum link in hydrophobic environment. Because transmembrane domains coincided with the end of Usp domain, so Usp with hydrophobic is conducive to maintain the stability of transmembrane structure. The result of post-translational modifications about Usp domain showed that there were eight phosphorylation sites with the score greater than 0.5, namely phosphorylation might occur on the sites of 4 serine, 2 threonine and 2 tryptophan after Usp was translated into amino acids. Post translational modification of Usp domain Post-translational modification of Usp domain protein was predicted by NetPhos2.0 Server (Fig. 8). There were eight amino acids whose scores were higher than 0.5, four of them were serine and the position on peptide chain was 11, 40, 88 and 106. Their predictive values were 0.825, 0.884, 0.929, and 0.649 respectively. Another two were threonine, whose positions were 28 and 39, their predictive values were 0.972 and 0.614 respectively. The last two were tryptophan whose positions were 17 and 49 having predictive values of 0.529 and 0.744 respectively. This
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Mol Biol Rep Fig. 8 Post-translational modification of Usp domain. X axis represented the location of amino acid, Y axis represented the score of threshold. The lines in blue, green, red and gray represented serine, threonine, tyrosine and threshold respectively, if the score was higher than threshold, there would have a phosphorylation site
Fig. 9 Phylogenetic tree analysis of USP domain in multi species. 17 highly similar sequences containing Usp domain from different spaces at NCBI were used to build the neighbor-joining tree by MEGA5.0, random sampling repeat 1,000 times. Bootstrap length represented evolutionary distance, the greater the number (bootstrap values) was the closer the distance was. Brackets in different colors represented different branches
indicated that Usp phosphorylation could occur on serine, threonine and tryptophan sites. Analysis of homology of Usp family Seventeen sequences containing Usp domain of five different species which were highly similar with Usp domain of STK family were used to build the phylogenetic tree by MEGA5.0 (Fig. 9). The tree was divided into five clusters, and the members of STK family were divided into the same cluster. There was little difference between STK1, STK3, STK4 and STK6, also STK2 and STK8 in the process of evolution. STK1, STK3, STK4 belong to paralogs relationships also STK2 and STK8. The number and the sort of motifs they contain were the same which indicated that
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they might have common ancestral genes. After species differentiation Usp domain gene of STK family expanded according to species-specific manner. The phylogenetic relationship between 35-like protein containing U-box domain and STK family was similar; they might belong to the same family and have the similar structure and function. Each cluster member were not clustered according to species source indicated that the characteristics of Usp domain had been established before species differentiation. The rice EAY9998 was placed in a single cluster which indicated its Usp domain had significant differences with other members and expressed different functions. Because Usp domain was existing in all the members in the tree, speculated that before species differentiation they should belong to the same family and have common ancestral
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genes. So the function of Usp domain in STK family can be predicted according to the function expressed by Usp domain in other species whose genetic relationship were close to STK family. The relationship between 35-like protein containing U-box domain and STK family was similar; they might belong to the same family and have the similar structure and function. The Usp domain of STK family could be predicted by the function expressed by Usp domain in other families which was similar with STK family, like 35-like protein. The Usp was produced as a result of the gene expression under adverse conditions which switched off some normal genes expression [35]. Usp expression can be induced in some conditions no matter the stimulation conditions were physical, chemical or biological factors [36]. In this sense it was adaption initiative and self-defense capabilities produced by the plant for changing external environment [37]. Usp can maintain the basic viability of plants and involve in plant defense mechanisms [38] as an ATP-binding protein, such as the transportation of chloroplasts, plant hormones, secondary metabolites, stomata movement [39] and pathogen infection [40]. Usp might control the porosity in plant leaves and participate in material transportation regulating in other tissues which played a defensive role [41]. Study showed that the growth of E. coli cells were inhibited when in the condition of Usp gene inactivation, while cell growth entered into a capture period when Usp gene overexpression [42]. Usp had a high expression in legume nodulation after inoculating legume with rhizobium [43]. Upland GUSP1 and GUSP2 had a high expression in its stems, roots, leaves under the condition of drought [44]. Usp gene was found to be up regulated after post-ripening treatment in tomato by Ethylene [45]. Mushegian and Koonin predicted that Usp might have DNA binding specificity and protect the DNA [46]. Usp can induce expression when plants were in stress conditions, so the resistance of Usp was important for plant growth and development [47]. According to the analysis above we found that STK family members might also have the function expressed by Usp, namely it can protect the cells from DNA damage, maintain cell homeostasis and participate in stress responses.
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