Identifying and Characterizing a Novel Protein Kinase STK35L1 and Deciphering Its Orthologs and Close-Homologs in Vertebrates

Background The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4 ) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria. Methodology/Principal Findings In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes. Conclusions/Significance We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.


Introduction
Protein kinases mediate signal transduction in eukaryotic cells and are involved in regulating all cellular processes such as transcription, translation, cell cycle progression, cytoskeleton rearrangements, migration, apoptosis and differentiation. Eukaryotic protein kinases that phosphorylate serine/threonine (Ser/Thr) or tyrosine (Tyr) residues are the largest superfamily of enzymes, comprising ,1.7% of all human genes [1]. Since completion of the first draft of the human genome sequence, various groups estimated the number of human kinases as 448, 510, and 518 depending on methods and data set used in the analysis [1][2][3]. Based on the current kinase databases (www.kinase.com; http:// hodgkin.mbu.iisc.ernet.in/,king/), and a study by Stevan Hanks, the estimated number of human eukaryotic kinases (ePKs) is now 478 [4]. Most protein kinases have regulatory domains located Cterminal and/or N-terminal to the kinase domain. Despite the differences in their substrate specificities, the kinase domain of Ser/Thr-and Tyr-kinases is highly conserved and composed of two lobes with an overall length of roughly 275 amino acids [5]. The catalytic domain is characterized by a series of short sequence motifs, which define 11 sub domains and serve as key elements in the catalytic core of the kinase domain [6]. These motifs in combination with the overall catalytic domain sequence can be used to identify genes in genomes that encode protein kinases by various approaches such as sequence alignments and hidden Markov model searches. Such analysis and corresponding cDNA sequence information permits prediction of the number of protein kinases in different genomes [1]. The human kinome has over 100 uncharacterized kinases with unknown substrates and biological functions.
STK35 and its homolog PDIK1L (PDLIM1 (CLP36) Interacting Kinase 1 Like) shares 69% protein sequence identity in kinase domain. They are members of the NKF4 (New Kinase Family 4) Ser/Thr kinases (STK) family and classified in group ''Other'' in the human kinome [www.kinase.com; 1,7,8]. In Kinomer database, the best match for the STK35 kinase sequence is within the group TKL (Tyrosine Kinase Like) [9]. STK35 has also been named as Clik1 (CLP36 Interacting Kinase 1) based on one study that showed an association of STK35 with CLP36 after overexpression of both proteins in osteosarcoma cells [7]. CLP36 is an a-actinin binding protein, which contains a LIM-and a PDZ-domain and is mainly localized on stress fibers [10]. Therefore, a possible function of STK35 in the regulation of the actin cytoskeleton was proposed [7].
The biological function and substrates of STK35 are not known. The STK35 gene was found upregulated specifically in colorectum cancer [11]. The expression of STK35 gene was altered in a rodent model of Parkinson disease [12]. A kinomewide RNAi screen revealed that STK35 silencing was among top five hits leading to reduced infection of hepatocytes by Plasmodium berghei sporozoites [13]. These studies suggest that STK35 may play a role in various human diseases deserving immediate attention by scientists working in different fields of biology and medicine.
In the present study, we describe the correct genomic organization and coding sequence of STK35, now renamed STK35L1, which was localized in the nucleus and nucleolus. We identified a new kinase subfamily containing three STK35L genes conserved in vertebrates. The present comprehensive study will provide a platform to further analyze the functional role and regulation of the STK35L kinases.

EGFP-STK35 co-expressed with CLP36 does not translocate to stress fibers in endothelial cells
Previously, it has been shown that myc-tagged Clik1 (STK35) translocated from the nucleus to actin stress fibers upon coexpression with EGFP-CLP36 in U2OS osteosarcoma cells [7]. To test whether the translocation of STK35 to actin stress fibers could be observed in endothelial cells, we transfected endothelial cells with EGFP-STK35 plasmid alone or together with mRFP-CLP36 plasmid. In cells transfected with EGFP-STK35 only, we found the EGFP-STK35 protein predominantly nuclear with a faint cytoplasmic localization ( Figure 1A). Unexpectedly, the coexpression of EGFP-STK35 and mRFP-CLP36 did not lead to translocation of STK35 to the cytoplasm and stress fibers ( Figure 1B1 and 1B2) as reported previously. Moreover, we could not observe the presence of CLP36 in EGFP-STK35 immunoprecipitates from endothelial cells (data not shown). These data suggest that CLP36 interaction with STK35 does not occur ubiquitously. Therefore, the former designation of this kinase STK35 was used.

Expression of STK35 RNA in various cell types
To investigate STK35 mRNA expression in different cell types, total RNA was isolated from endothelial, HEK and HeLa cells and analyzed by RT-PCR. A fragment of the expected size for STK35 (273 bp) was amplified with STK35 specific primers (Table S1) in all cell types (Figure 2A). The correct sequence for STK35 fragment was confirmed by DNA-sequencing of the amplified product. Hence, endothelial, HeLa, and HEK cells express a transcript for STK35. By quantitative RT-PCR, we found that STK35 is equally expressed in these cell types (data not shown). To exclude a false positive amplification of possible DNA contamination of the isolated RNA, the primers for STK35 were designed to bind exactly at the exon-intron boundaries. No additional amplification of a genomic fragment of STK35 including the intron (13.3 kbp) was obtained in addition to the fragment derived from the STK35 mRNA transcript. In order to obtain further information about the expression profile of STK35 in human tissues and cell lines, we searched the Unigene database for Expressed Sequence Tags (ESTs) of STK35. 217 ESTs were found for the human STK35 gene, which are present in most of the tissues such as testis, ovary, skin, brain, heart, liver, and eye. To analyze the relative expression of STK35 RNA in various human tissues, we obtained the gene expression profile of STK35 from the SymAtlas database (http://symatlas.gnf.org) [14]. STK35 is expressed in all 79 human tissues studied. In testis and CD56+ NK cells, the expression was higher ( Figure S1).

Expression of STK35 in various cell types at protein level
To analyze the expression of endogenous STK35 protein in cells, rabbit polyclonal peptide antibodies against a C-terminal STK35 peptide were raised. To test the specificity of the antibody, cell lysates of untransfected and endothelial cells transiently transfected with plasmids encoding for EGFP-tagged STK35 proteins were used as negative and positive controls respectively. The antibody recognized the over-expressed proteins (,72 kDa for EGFP-STK35) and a prominent band at about 58 kDa specifically whereas the pre-immune serum of the same rabbit did not detect these bands (data not shown). Importantly, we could not observe in transfected and non-transfected cells a band at 44.6 kDa, the theoretical calculated molecular weight of endogenous STK35. During the course of the study a commercial polyclonal goat anti-STK35 peptide antibody against the Cterminal amino acid sequence (ELETRMDQVTCAA) of STK35 became available. This antibody recognized the EGFP-STK35 fusion proteins which contain the C-terminal part of the protein specifically (EGFP-STK35, EGFP-STK35D1-63) but not a truncated construct lacking this sequence (EGFP-STK35D72-400) ( Figure 2B). The blot incubated with mouse anti-EGFP antibody clearly showed a band of this truncated construct confirming its expression in cells ( Figure 2B). Beside the detection of the EGFPtagged STK35 proteins, again a band of 58 kDa was specifically recognized by the goat anti-STK35 antibody but not by the anti-EGFP antibody ( Figure 2B). This indicates that this 58 kDa band could represent endogenous STK35 protein.
To analyze further, nuclear lysates of endothelial, HEK and HeLa cells were prepared. A prominent band at 58 kDa was recognized by the anti-STK35 antibody in nuclear extracts of HEK, HeLa and endothelial cell ( Figure 2C) and macrophages (data not shown). In HeLa cells two additional bands at about 52 kDa and 48 kDa were detected. The control blot probed with goat IgG did not show any bands. The theoretical molecular weight of STK35 (44.6 kDa) is significantly less than the molecular weight of the endogenous protein (58 kDa) recognized by these two different antibodies. Posttranslational modification as explanation for such a large mobility shift from 45 kDa to 58 kDa seemed to us unlikely. Moreover, overexpressed EGFP-tagged STK35 would be expected to undergo the same posttranslational modifications as endogenous STK35 in endothelial cells but this was not the case: the detected molecular weight of this tagged protein corresponded exactly to its calculated molecular weight. We also examined the possibilities of longer STK35 gene splice variants from data set of Ensembl and NCBI human build. However, no splice variants co-relation can be established. Therefore, we undertook a careful genome analysis of the STK35 locus.

Identification of STK35 protein coding region in different genomes
To identify the open reading frame of the STK35 gene, we analyzed the genomic sequence of the STK35 locus in different genomes from different representative species of vertebrate phyla using various gene structure prediction tools. First we used the ElDorado tool of Genomatix GmbH (http://www.genomatix.de/). We used STK35 cDNA (NM_080836) sequence for complete analysis of the STK35 locus in human genome. The STK35 gene is located on chromosome 20 subregion p13 ( Figure 3A). The chromosomal region NC_000020 between 2.013.772 and 2.063.771 (50.000 bp) was analyzed. Three transcription start regions (TSR_2.030.439-484, TSR_2.030.648-690 and TSR_2.031.306-335) were found based on CAGE (Cap-analysis gene expression) tags profile. CAGE is a high-throughput method to measure expression levels of a gene and is an essential resource for profiling transcriptional starting sites [15,16]. The transcription start region TSR_2.030.439-484 was 1035 bp upstream of the previously defined STK35 start codon. This region was highly conserved among mammals ( Figure 3B). We investigated this region for coding sequences manually and by using different gene prediction software such as FGENESH (www.softberry.com). We identified a start codon surrounded by a Kozak consensus sequence and an open reading frame encoding a protein of 534 amino acids instead of the 401 amino acids predicted earlier for STK35 ( Figure 3C and 3D). The theoretical molecular weight of the predicted protein was 58 kDa which matched completely the molecular weight of the endogenous protein on the immunoblots. Furthermore, we found that the new STK35 gene has 3 coding exons ( Figure 3C). The newly identified STK35 gene structure is maintained from fish to mammals ( Figure 4). In contrast, the previously identified protein start codon of the STK35 gene is found in the middle of second exon.   To investigate the presence of mRNA transcripts of the potentially longer form of STK35 experimentally, endothelial cell total RNA was isolated and analyzed by RT-PCR. The primers were designed to span the introns boundaries and to combine the extended predicted sequence with the published sequence of STK35 ( Figure 3E). The RT-PCR results show an expression of mRNA transcripts of the newly identified STK35 gene including exon 1 ( Figure 3E). The correct sequences of these PCR-amplified products were confirmed by DNA-sequencing (data not shown). Possible genomic DNA contamination was excluded by using negative control (reverse transcriptase polymerase was excluded during RT-PCR) of RT-PCR samples ( Figure S3). This data indicates that the longer form of STK35 is expressed in mammals. Hence, based on our finding we termed the full length gene of STK35 as STK35L (STK35 Long). Since it represents the first member of NKF4 family, it is named STK35L1.

Knockdown of endogenous STK35L1 expression by siRNA
In order to prove that the endogenous 58 kDa protein band is STK35L1, we used the siRNA approach to knockdown the expression of endogenous STK35L1. We designed siRNA directed against three different regions of STK35L1 gene (Table S1). Indeed, the expression of 58 kDa band was highly reduced in endothelial cells (by ,80%) after transfecting a pool of the three siRNA oligonucleotides ( Figure 5). One siRNA oligonucleotide (siSTK35L_1111) could downregulate 70% of the 58 kDa protein, whereas the others (siSTK35L_839 and siNSTK35L_5816) were less effective (data not shown). These data confirm that the endogenous 58 kDa protein is STK35L1.

Subcellular distribution of STK35L1
To analyze the subcellular distribution of STK35L1, EGFP-STK35L1 plasmids were transfected into endothelial cells and analyzed by fluorescent microscopy. The protein accumulated primarily in the nucleus. Moreover, EGFP-STK35L1 was concentrated in dense bodies in the nucleus ( Figure 6, white arrow), that were easily identified as nucleoli by phase contrast microscopy and exclusion of Hoechst DNA staining dye ( Figure 6). The construct deleted of the first 196 N-terminal amino acids (EGFP-Kinase), containing only the kinase domain, diffusely distributed throughout the cytoplasm and the nucleus ( Figure 6). These data suggest that the N-terminus of STK35L has a functional nuclear and nucleolar localization signal.

Identification of STK35L1 orthologous set and gene conservation in vertebrates
Gene synteny is a useful tool for predicting functional interaction and relatedness among genes within cluster. Selective processes are essential to preserve the organization of these clusters in closely related species. In the human genome, the STK35L1 gene is localized on chromosome 20. On further inspecting this gene in different vertebrates genome (Table S2), we found that this gene is maintained in fish, Xenopus and in all mammals. To ensure orthology of this gene, syntenies amongst different vertebrates were created. From human to fish, the STK35L1 gene is flanked by the marker gene PYDN (prodynorphin) in an inverse manner ( Figure 7). Moreover, the STK35L1-PYDN region has tandem duplication resulting in two copies of these genes in the Danio genome. These data infer that flanking marker genes are conserved in micro-syntenic environment corresponding to STK35L1 synteny. STK35L1 orthologs and species-specific paralogs are listed in Table S3. Interestingly, the PDYN-STK35L1 locus is not found in Gallus genome using PDYN as marker, since PDYN was not detectable in the same genomic locus. However, we could identify the STK35L1 locus by using some other conserved markers (data not shown). By homology search in Gallus genome, we found that the STK35L1 gene was truncated and the exon 1 and 2 were missing. The expression of the truncated STK35L1 gene in Gallus is supported by an mRNA sequence submitted in the gene bank (BU403355).

Identification of STK35L2 orthologous set in vertebrates
STK35L2 (previously known as PDIK1L) is localized on chromosome 1 in human. In mammals, STK35L2 was conserved differently: the locus is flanked by a marker tetrad EXTL1-TRIM63-PHARAH2-STMN1 (Table S4 for markers details) on one side and the marker gene LIN28 on the other side ( Figure 8). However, in frog and chicken genomes, this gene was only flanked by tetrad EXTL1-TRIM63-PHARAH2-STMN1 on one side. In fish, the marker sets were not totally conserved: on one side, the CITED4 gene was present, on the other side, two additional flanking genes-MANEAL and SF3A3 (Table S4) were present in inverse orientation to the conserved LIN28. STMN1 gene is maintained from human to G. asculeatus within an 800 kb fragment. Jointly, LIN28 and STMN1 aided in deciding orthology of STK35L2 gene locus. Hence, this suggests that STK35L2 has an independent origin from STK35L1. The human orthologs of STK35L1and STK35L2 were maintained from fish to humans. STK35L2 orthologs and species-specific paralogs are listed in Table S3.

Identification of the novel homolog STK35L3 in vertebrates and characterizing its orthologous set in different vertebrates
During investigation of STK35L1 and STK35L2 genes from different vertebrates, we found that some vertebrates possess a novel STK35L like gene. This gene is not reported in the literature by today. Since two homologs of the STK35L1 gene are already known, we named this gene as STK35L3. Astonishingly, this gene is maintained from fish to mammals as listed in Table 1 with accession ids and genomic location.
To unravel the origin of STK35L3 gene, the micro-genomic organization flanking the STK35L3 gene was analyzed (Figure 9). In M. domestica (opossum) genome, STK35L3 is gene flanked by PNOC and ELP3 (Table S4 for markers details) in a ,380 kb region on chromosome 1. A similar locus is conserved in a ,120 kb fragment on scaffold_470 in frog genome. However in fish genomes, this locus contains two additional conserved markers ANKRD5 and EMILIN1 (Table S4) flanking in inverse orientation. Conservation of locus of STK35L3 suggest that the STK35L3 gene is found in orthologous condition from fish to mammals, and that the STK35L3 does not result by tandem duplication of STK35L genes as they are found in distinct syntenic organization. On further investigating this gene in different mammals, we found that this gene is lost in eutherian (''placental'') mammals (such as human and mouse in Figure 9), whereas it is retained in metatherian (''marsupial'') mammals (as in opossum).This suggests that STK35L3 gene in eutherians is lost after metatherian\eutherian divergence ,173-190 MY ago [17,18,19]. Hence, we corroborate the finding of novel STK35L3 ortholog in marsupial mammals, chicken, frog and at least in five fish genomes.

Evolution of STK35L1 and its homologs
Our data clearly demonstrated that there are distinct orthologous sets of STK35L1 and its homologs in different vertebrate species. Since these are maintained from fish to human, this indicates that an ancestor gene might be present before the emergence of vertebrates or that these genes arose at the point of vertebrate origin. To identify an ancestor gene of STK35L1, homology searches in different invertebrate genomes were carried out. By searching invertebrate model genomes (Table S2) such as Branchiostoma floridea (lancelets) [20], Strongylocentrotus purpuratus (sea urchins) [21], Drosophila melanogaster (flies) [22], and Caenorhabditis elegans (worms) [23], no genuine homologs were found, although these species possess many eukaryotic protein kinases. However, we identified a STK35L1 like gene in two genomes of sea squirts -Ciona intestinalis (JGI Ciona genome accession id -fge-nesh3_pg.C_chr_07q001099/Ensembl -ENSCINP00000006268) and Ciona savignyi (Ensembl-ENSCSAV00000013846) with iden-tity of ,45% with human STK35L1 ( Figure S2). Moreover, in a previous study on the sea urchin kinome [24], STK35 kinase gene (SPU_003710) had been reported. However by careful comparison of the protein sequence of sea urchin kinase with the STK35L1 protein, we found that the sea urchin kinase shared only 10% identity with STK35L1 in the kinase domain. To confirm whether the sea urchin kinase is clustered together with STK35L1 homologs or not in the evolutionary tree, we created a phylogenetic tree of randomly selected representative kinases with STK35 homologs, sea urchin and Ciona STK35L1 like kinases ( Figure 10) by using neighboring joining method [25,26]. The status of the sea urchin kinase being STK35 is highly questionable as it did not cluster along with STK35L1 homologs in this phylogenetic tree with a statically significant value (the bootstrap value is below 60%). This sea urchin kinase randomly located as other representative kinases in this tree. In contrast the STK35 like gene in Ciona species is grouped with STK35L1 homologs with a bootstrap value of 100%. These data suggest that Ciona kinase but not sea urchin kinase is the closest homlog of the vertebrate STK35L1.
To compare the evolution of STK35L1, STK35L2, STK35L3 on a time scale, a time-wise gene distribution of these STK35L1 homologs was created ( Figure 11). This finding indicates that STK35L1 homologs were present from 550 MY ago with separation of sea squirt lineage from vertebrate lineage. Notably, the STK33 is the closest homolog of the STK35L1gene (28% identity of the kinase domain) in comparison to other kinases. An ancestor gene of STK33 is found in sea anemone genome [27] from the beginning of metazoan dating ,700 MY ago.

Gene structures of STK35L1 homologs
Gene structures attribute phylogenetic evidence to genes and aid in assigning origin of given genes. To compare gene structures, exon-introns architectures of different STK35L1 homologs were determined. STK35L1 homologs have variable exon-intron organization patterns (Figure 12). STK35L1 and STK35L2 genes in different vertebrates possess gene structures with 'three exons/ two introns' ( Figure 12A) and 'two exons/one intron' (Figure 12B) patterns, respectively. STK35L3 from marsupials, chicken and frog possess a 'three exons/two introns' (Figure 12C) pattern. However STK35L3 from fish carries a discernable 'four exons/three introns' gene structure pattern, indicating intron loss from fish to human. A nine exons/eight introns gene structure pattern is found in ancestor gene of STK35L1 from Ciona ( Figure 12D), suggesting that the vertebrate STK35L1 gene arose by intron losses event at the point of vertebrate emergence. The analysis of exon-intron structures has proved to be a valuable method for determining phylogenic relatedness of STK35L1 homologs as it suggests independent origin of these orthologous sets of STK35L1 homologs. Otherwise, a conservation of the intron-exon organization among these three types of genes would have been maintained. Additionally, the gene structure patterns of STK35L1 homologs do not resemble that of the closest homologs of serine threonine kinases (such as STK33/STK38/STK40). This corroborates that STK35L1 homologs do not share orthology with other serine threonine kinases.

Discussion
In the present study we found that the previously published coding sequence of the STK35 gene (code for 401 amino acids) is incomplete. The newly identified sequence of STK35 gene codes for a protein of 534 amino acids which was termed as STK35L1. By western blot analysis of endothelial cell lysates using two different specific polyclonal anti-STK35 antibodies, we found that STK35L1 was expressed in all mammalian cells studied. By using the siRNA approach, we could confirm that the 58 kDa protein is endogenously expressed STK35L1 protein. In contrast, no evidence for the expression of STK35 was obtained by western blotting using the antibodies directed against the C-terminal peptide of STK35.
Endogenous STK35L1 was found to be localized in the nucleus. Moreover, EGFP-STK35L1 was also localized in the nucleolus suggesting a role in gene regulation. A previous report suggested that STK35 may have role in actin cytoskeleton rearrangement as the interaction of the co-transfected STK35 and CLP36 proteins led to relocalization of Clik1 from the nucleus to actin stress fibers  [3]. In endothelial cells, this result could not be confirmed. Recent studies suggest that STK35 could play a role in various diseases [11,12,13].
The identification of the full protein coding gene sequence is obviously crucial for the determination of the biological function of the kinase. Therefore, we analyzed the STK35 locus in human genome by using various approaches. Based on CAGE tags, we found three transcription start regions within a conserved region of 1035 bp upstream of the previously defined STK35 start codon. CAGE is a high-throughput method to measure expression levels  by counting large amounts of sequenced capped 59 ends of transcripts, termed CAGE tags [15,16]. CAGE tags are an essential resource for profiling transcriptional starting sites and can be used for profiling gene expressions by counting CAGE tags associated with genes. By analyzing the transcription start region TSR_2.030.439-484 we found a start codon AUG surrounded by a Kozak sequence upstream 990 bp of the previously published start codon. The translation initiation sites in eukaryotic mRNAs are reached via a scanning mechanism which predicts that translation should start at the AUG codon nearest the 59 end of the mRNA [28]. So translation initiation from the previously known start codon which is present in the middle of the second exon is most unlikely. Indeed, this novel open reading frame coded for a protein of 58 kDa, exactly the size of the protein band recognized by the antibody on immunoblot. Moreover, RT-PCR analysis confirmed the presence of the 58 kDa transcript of STK35L1 in endothelial cells. Three orthologous sets of STK35L1 homologs were identified based on syntenic and gene structure pattern data. STK35L1 is conserved in vertebrates. An exception was chicken that had a truncated mRNA starting at the third exon translating only for a partial kinase domain. The present version of the Gallus genomic sequence is unable to solve this issue; it can be only clarified with further improvement of genomic assembly of chicken. STK35L2 gene is present in all vertebrates in conserved microenvironment without any exceptions. One of the most exciting outcomes of the present analysis is the identification of a third member in this family as STK35L3. Its orthologs are found from fish to marsupial mammal. However, this gene is lost in placental mammals after divergence of marsupial and placental mammals at ,173-190 MY ago. Placental mammals have a conserved syntenic organization, but no STK35L3 gene. It gives a hint that this gene was lost due to positive selection and might be involved in physiology related to laying eggs or egg development.
The three sets of STK35L1 homologs have independent gene structure patterns, further supporting their orthologous status. Any other known ser/thr kinase family member does not share these patterns of exon-intron organization. An ancestor of the STK35L1 is identified in two Ciona species that are the closest living relatives of vertebrates [29]. Additionally, in our analysis we do not find any evidence to support the presence of STK35 in sea urchin. Furthermore, no genuine homologs of STK35L1 in worms, flies, echinoderms and lancelets were found even though these groups of invertebrate models possess easily identifiable ser/thr kinases. Thus, it is most likely that the STK35L1 homologs evolved before the divergence of the vertebrate lineage/Ciona lineage about 550 MY ago. Furthermore, this comprehensive study will provide a platform to further analyze the functional role and regulation of the STK35L1 kinase, and gene transcription in various physiological and patho-physiological conditions.

RT-PCR
Total RNA was isolated from Endothelial HeLa and HEK cells, using RNeasy mini kit (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized with Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany) using random hexamer primers as per manufacturer's protocol. cDNAs from these cell types were subjected for gene expression analysis using primers. These primers were designed from different regions of the STK35L1 gene (Table S1).

Construction of the expression plasmids
The full-length coding sequence of STK35 and ST35L1 was amplified by PCR from a cDNA pool of human umbilical vein endothelial cell total RNA. The PCR-amplified product of STK35 was cloned into XhoI and HindIII sites of pEGFP-C1 vector (Clontech) to obtain STK35 fused with EGFP. The full length of STK35L1 gene was obtained using a multistep cloning strategy. In the first step PCR, we amplified 39 end of STK35L1 (bp 400-1605) using primers Clik1_long400F and STK35R_HINDIII (Table S1). In second step, the 59 end (520 bp) of STK35L1 gene was amplified using 1_F and 520_R. In third step, the PCR fragments from first step and second step were mixed in 1:3 molar ratios respectively and used as PCR template for amplification of the full length STK35L1gene using 1_F and STK35R_HINDIII primers. The deletion mutants of EGFP-STK35 were generated by Quik-Change II site-directed mutagenesis kit (Stratagene, La Jolla, CA) as per the manufacturer's instruction. CLP36 gene was amplified as describe previously [5] and cloned into EcoR1 and Sal1 sites of pDsRed-Monomer-N1 (Clontech). All the constructs were confirmed by DNA sequencing (MWG Biotech AG, Ebersberg, Germany).

Cell culture and transfection
Human umbilical vein endothelial cells were obtained and cultured as described previously [30]. Briefly, cells harvested from umbilical cords were plated onto collagen-coated (room temperature, 75 mg/ml collagen G; Biochrom, Berlin, Germany) plastic culture flasks and were cultured at 5% CO 2 , and 37uC in complete endothelial growth medium (Promo Cell, Heidelberg, Germany). In all experiments, human umbilical vein endothelial cells up to third passage were used. HeLa and HEK cells were grown in OptiMEM medium (Invitrogen) supplemented with 10% fetal calf serum.
Endothelial cells were transfected using the nucleofaction transfection technology developed by Amaxa GmbH as per manufacturer's instructions. Figure 10. Evolutionary relationships of STK35L1 and its homologs with selected kinases. The previously published [24] probable STK35 gene of Sea urchin is not STK35 gene as do not cluster with STK35 like genes from other genomes with 60% bootstrap values. In contrast, Ciona STK35 gene is clustered with STK35 like genes. The evolutionary history of selected kinases was inferred using the Neighbor-Joining method [26]. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of these selected kinases [25]. Branches corresponding to partitions reproduced in less than 60% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated kinases clustered together in the bootstrap test (1,000 replicates) are shown above the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 227 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4. Outgroup is yeast STE11 gene choose from www.kinase.com. SCER-yeast, HSAP-human, DRER-zebrafish, MDOMopossum, CINT-sea squirt, SPUR-sea urchin. doi:10.1371/journal.pone.0006981.g010

Confocal microscopy
After 8-10 h of transfection, cells were washed fixed with 3.7% formaldehyde in PBS for 10 minutes at 4uC. For staining of the DNA, cells were incubated with Hoechst 33258 dye solution (1 mg/ml) in PBS for 10 minutes. Cells were observed with a Zeiss LSM510 confocal laser-scanning microscope. The microscope function was controlled by a light manager through the software LSM 510 META.

Western blot analysis
Transfected or untransfected endothelial cells or nuclear lysates (prepared by using nuclei isolation kit from sigma as per manufacturer's protocol) of HEK, HeLa and endothelial cells were dissolved in an equal volume of 26 Laemmli buffer. Equal amounts of proteins in the samples were subjected to SDS-PAGE and then transferred to nitrocellulose membrane at 200 mAmp for 60 min at 4uC using the Mini Trans-Blot electrophoresis cell (Bio-Rad). Membranes were blocked with 5% (w/v) nonfat milk and incubated with the respective primary and secondary antibodies. The dilutions of the primary antibodies were: anti-STK35 (1:250) and anti-EGFP (1:250). The dilution of the secondary antibody (horseradish peroxidase-conjugated) was 1:5000. The membranes were developed with SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to Hyperfilm (Amersham Biosciences).

Data sources of genomic, cDNA, and protein sequences
The genomic DNA/cDNA/protein sequences from different organisms were extracted via different types of BLAST [31] searches using human STK35L1/PDIK1L as query sequence from the different genomic databases (Table S2). The newly identified cDNA sequences of STK35L1 and STK35L3 are deposited in Genbank. GenBank accession numbers of STK35L1 and STK35L3 nucleotide sequences are GQ281297 and GQ281298 respectively. Ensembl accession identifiers of STK35L1, STK35L2 and STK35L3 genes in different organisms are mention in Table S3.

Sequence alignment of protein kinases
Protein alignments of protein kinases were generated with CLUSTALX 1.83 [38,39]. The alignments were edited and visualized different sequence characteristics using GENEDOC [40].

Sequence similarities comparisons between two species
Pairwise protein alignments for STK35L1 and its exons in two species were carried out by Emboss tool 'needle'at EBI (http:// www.ebi.ac.uk/Tools/emboss/align/index.html).

Phylogenetic tree analysis
All phylogeny trees were created with full length sequences of representative serine threonine kinases from different organisms with help of Neighbor Join (NJ) method [26] using ''MEGA 4'', a molecular evolutionary genetics analysis software [41].