# Classification of Nonenzymatic Homologues of Protein Kinases

Authors: K. Anamika; K. R. Abhinandan; K. Deshmukh; N. Srinivasan
Journal: Comparative and Functional Genomics
 (2009)

---

## Abstract

Protein Kinase-Like Non-kinases (PKLNKs), which are closely related to protein kinases,
lack the crucial catalytic aspartate in the catalytic loop, and hence cannot function as
protein kinase, have been analysed. Using various sensitive sequence analysis methods, we
have recognized 82 PKLNKs from four higher eukaryotic organisms, namely,Homo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. On the basis of
their domain combination and function, PKLNKs have been classified mainly into four
categories: (1) Ligand binding PKLNKs, (2) PKLNKs with extracellular protein-protein
interaction domain, (3) PKLNKs involved in dimerization, and (4) PKLNKs with cytoplasmic
protein-protein interaction module. While members of the first two classes of PKLNKs
have transmembrane domain tethered to the PKLNK domain, members of the other two
classes of PKLNKs are cytoplasmic in nature. The current classification scheme hopes to
provide a convenient framework to classify the PKLNKs from other eukaryotes which
would be helpful in deciphering their roles in cellular processes.


---

## Body

## 1. Introduction

It is  now  well known that enzymes, in their role as biocatalysts, are the most important control points in the living organisms, and the catalytic residues of an enzyme are key to its molecular function. Bartlett and colleagues [1] have described pairs of active and inactive enzyme homologues having same structural scaffold but different functions. Catalytically inactive enzyme homologues are represented in a large variety of enzyme families with families of signaling enzymes having high number of enzymatically inactive members [2].Phosphorylation by Ser/Thr/Tyr protein kinases plays a crucial role in cellular signal transduction. A canonical kinase domain consists of 12 subdomains containing few conserved residues of functional importance. Subdomains I, II, VIB, and VII are considered to be the most important ones. Subdomain I includesβ-turn structure with 2 or 3 glycine residues (G-X-G-X-X-G) while subdomain II comprises an invariant lysine participating in anchoring and orienting the ATP (Adenosine Tri Phosphate). Subdomain VIB contains catalytic loop with a key aspartate [D] residue that mediates the transfer of a phosphate group from ATP to the appropriate substrate. The D residue of DFG motif in subdomain VII ligates Mg2+ which in turn interacts with β and γ phosphates of ATP [3]. Roles of these residues in protein kinases are well established. The catalytic residues of the protein kinases are usually highly conserved to maintain their ability for efficient cellular signal transduction. However, there have been few reports of proteins with substitutions/deletion at essential catalytic sites. Among these functionally important residues in a Ser/Thr/Tyr kinase, the aspartate residue in subdomain VIB acting as catalytic base seems to be most important as we are not aware of a properly functional kinase which lacks this residue.Although the importance of protein kinases has long been recognized, studies on protein kinase homologues lacking catalytic residue/residues are more recent. Several studies on repertoire of kinases in various organisms have revealed presence of enzymatically inactive homologues of protein kinases [4–6] which lack catalytic function and instead serve as scaffolds or kinase substrates. Boudeau and colleagues have discussed roles of human kinase-like proteins in regulating diverse cellular processes [7].Despite considerable sequence similarity to enzymatically active protein kinases, Protein Kinase-Like Nonkinase (PKLNK—also referred as Kinase Homology Domain—KHD in some of the earlier publications) domains lacking key residues thought to have regulatory roles. Some examples of proteins containing such domains which lack catalytic base aspartate are a PKLNK domain tethered to a tyrosine kinase domain in Janus Kinase (JAK) and membrane guanylyl cyclases (or particulate guanylyl cyclase) in which a regulatory PKLNK domain is situated N-terminal to the guanylyl cyclase domain [8–10]. PKLNK domain in JAK is thethered to functional kinase domain; however in guanylyl cyclases (GC), a functional kinase domain is absent, and the PKLNK is tethered to a cyclase domain.  PKLNK domain of Guanylyl cyclase-A serves as an important mediator in transducing the ligand-induced signals to activate the catalytic cyclase domain of the receptor. Deletion of PKLNK domain from GC-A, -B, and -C resulted in constitutive activation of these enzymes [11, 12] and is shown to act as a repressor of the catalytic domain in the basal state [13]. The PKLNK of guanylyl cyclase-A (Natriuretic peptide receptor A) is more closely related to protein tyrosine kinase than protein serine/threonine kinase [11, 12, 14]. PKLNK in receptor guanylyl cyclase provides a critical structural link between the extracellular domain and the catalytic domain in regulating the activity of this family of receptor. Modeling of  the PKLNK of human GC-C indicates that it can adopt a structure similar to that of tyrosine kinases [15]. There are many other protein kinase-like domains which lack other catalytically important residues, though playing important role as regulatory proteins, for example, “dead” RTK-ErbB3 [16], OTK (Off Track Kinase), WNK (with no lysine kinase), Tribbles, giant muscle protein titin (present in vertebrates), HER3, CCK-4 (Colon Carcinoma Kinase-4), Eph (Erythropoietin-producing hepatocyte) family of receptor tyrosine kinase, h-Ryk/d-Derailed, integrin-linked kinase (ILK) [17], and so forth. Recently, crystal structure of first PKLNK, VRK3 (a member of the vaccinia-related kinase family), which lacks aspartate in the catalytic loop has been reported [18] which revealed that it cannot bind ATP because of residue substitutions in the binding pocket, compared to ATP binding homologues. However, VRK3 still shares prominent structural similarity with enzymatically active protein kinase.In the past, our group has reported presence of ABC1, RIO1, and kinases in archaea and bacteria that share significant similarity with Ser/Thr/Tyr kinase family [19]. The sequences of these protein kinases were examined for the presence of catalytic aspartate in the catalytic loop. Sixteen prokaryotes have been predicted to have at least one member lacking catalytic aspartate, and the total number of such sequences is 23. This study indicates that PKLNK has been evolved much before the divergence of prokaryote and eukaryote.In the current analysis, we present a detailed analysis of the PKLNKs from four completely sequenced higher eukaryotes, namely,Homo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster. An attempt has been made to classify these PKLNKs based upon their amino acid sequences and domain tethering preference in order to understand molecular basis of evolution and functions of these proteins.


## 2. Materials and Methods

In order to identify the repertoire of PKLNKs in various eukaryotic organisms PSI-BLAST [20] search was performed using traditional protein kinases as queries against the Nonredundant Data Base (NRDB) which is a database of protein amino acid sequences maintained at NCBI, USA. Hits were analyzed for the absence of catalytic base aspartate in the catalytic loop. In the PKLNKs, lacking catalytic aspartate, we further looked for the presence of other key-residues such as glycine in glycine-rich motif in the subdomain II, lysine and glutamic acid in the subdomain III, and DFG motif in subdomain VII. However we have considered all the kinase-like sequences lacking the catalytic base (Asp) residue for the present analysis.The subfamily classification of these PKLNKs and recognition of other domains in the PKLNK domain containing multidomain proteins have been made using the procedures and protocols developed earlier in our group in connection with analysis of kinomes [4, 6, 21]. We have essentially employed multiple sensitive search and analysis methods like PSI-BLAST [20], RPS-BLAST [22], and HMMer [23] which match sequences to Hidden Markov Models (HMMs) of various families in Pfam (release 23) [24] to identify various domains in the multidomain sequences. Procedure such as PSI-BLAST has been used to detect homologues of noncatalytic kinase like domains using an E-value cut-off of 0.0001 that has been previously bench marked [25]. Hits lacking significant sequence similarity with the query have been further examined manually.In the current analysis, we have identified a total 82 PKLNKs in the four organisms. CD-hits program [26, 27] was used in order to eliminate redundant sequences, which are indicated by 100% sequence identity. So the data set is devoid of redundant sequences.CLUSTALW [28] has been used to align the nonenzymatic domains of 82 PKLNKs (see Table 1 in Supplementary Material available online at doi: 10.1155/2009/365637). Further, catalytic domain of the protein kinase and PKLNK domain from mouse have been aligned, and  MEGA [29] was used to generate phylogenetic dendrograms.Domain assignment to the other regions apart from the noncatalytic kinase domain of these PKLNKs has been carried out using HMMer search methods by querying each of the PKLNK against the 10340 protein families HMMs available in the Pfam database (http://pfam.sanger.ac.uk/). MulPSSM (Multiple PSSM) [30] approach was used further to assign domain to the region which has not been assigned using HMMer approach. Trans-membrane regions were detected using TMHMM [31].


## 3. Results and Discussion

In the current analysis, we have identified 82 PKLNKs. The main criteria used to detect these PKLNKs involve ensuring acceptable e-value with protein kinases and absence of catalytic base residue (Asp). There are 31 PKLNKs identified inHomo sapiens (Table 1), 18 PKLNKs in Drosophila melanogaster (Table 2), 13 PKLNKs in Rattus norvegicus (Table 3), and 20 PKLNKs in Mus musculus (Table 4). Although the catalytic Asp is absent in these sequences, we looked for the presence or absence of other key residues, characteristic of functional protein kinases, in the 82 identified PKLNKs. Glycine rich loop in the subdomain I (displaying consensus sequence G-X-G-X-X-G) contains at least two glycine residues in 26 gene products (see Supplementary Table 1). The phosphorylation of the activation segment is required for the activation of most protein kinases that contain an Arginine (R) preceding the catalytic base aspartate. We have essentially looked for the H-R-X motif (where X can be any residue but cannot be D) in all the 82 PKLNKs. There are 18 gene products which have “R” of H-R-X motif conserved (see Supplementary Table 1). We further checked for the presence of DFG and APE motifs in the activation loop and found that these motifs are not completely conserved in 82 PKLNKs identified so far. There are 12 and 49 protein gene products which have DFG and APE motif conserved, respectively, (see Supplementary Table 1).Table 1
List of 31 human PKLNKs is represented in the table. Substitutions of various important residues in the motifs which are generally conserved in the functional protein kinase are shown for each human PKLNK. “-” indicates deletion.
Gene code“HRD” motif in the catalytic loopActivation loop startActivation loop endgi|119534|sp|P21860HRNDFGALEgi|6005792|ref|NP_009130.1CGSDFAPEEgi|4758292|ref|NP_004436.1HRSRLGAPEgi|4758606|ref|NP_004508.1PRH- - -APEgi|14042287|dbj|BAB55185.1HGN- -SAPEgi|15779207|gb|AAH14662.1HNN- - GAPEgi|7020363|dbj|BAA91097.1YGHDLE- - -gi|7243101|dbj|BAA92598.1HGNGFDAPEgi|4504217|ref|NP_000171.1HGRDHGAPEgi|14041796|dbj|BAB55454.1HNN- - GA- -gi|18676872|dbj|BAB85045.1HRAKFGAPEgi|17368698|sp|Q9BXU1HGSDF DAPEgi|18386335|gb|AAB19934.2HGRDFGAPEgi|12052916|emb|CAB66632.1HRNDFH- - -gi|22760572|dbj|BAC11248.1HRNDFHAPEgi|4580422|ref|NP_003986.2HGSDYGAPEgi|13027388|ref|NP_061041.2HRS- - GSPEgi|22749323|ref|NP_689862.1HGKGFESP-gi|22760645|dbj|BAC11278.1HRNDFHAPEgi|16306492|ref|NP_203698.1Deletion- - -SPEgi|115430241HNN- - GA- -gi|134152694HGRDYGAPEgi|31982929- - -GYGSMDgi|34191428HRND- -APEgi|40254426HGNDYGAPEgi|57997202HR-SPGAPEgi|58530886HSN- - -PEDENSP00000222246HGNDPGAPEENSP00000264818HGNDPGAPEENSP00000294423HGNDPGAPEENSP00000371067HGNDPGPPETable 2
List of 18 drosophila PKLNKs. Substitutions of various important residues in the motifs which are generally conserved in the functional protein kinase are shown for each drosophila PKLNK. “-” indicates deletion.
Gene code“HRD” motif in the catalytic loopActivation loop startActivation loop endgi|17368346|sp|P83097HRQVFGAPEgi|21626698|gb|AAF47134.2HMGDFGSPEgi|17862032|gb|AAL39493.1HGSNFSAPEgi|7301824|gb|AAF56933.1HNN- - -SPEgi|7291217|gb|AAF46649.1HGKDFG- - -gi|7294467|gb|AAF49811.1HGADFGAPEgi|21357711|ref|NP_647767.1- - -- - -- - -gi|5052670|gb|AAD38665.1HGN- - -APEgi|21627748|gb|AAM68879.1YGH- - -AIEgi|22945875|gb|AAN10635.1HGH- - G- - -gi|23093524|gb|AAN11824.1HGADFGAPEgi|23171402|gb|AAF55244.2HGNDFGAPEgi|21358011|ref|NP_651655.1HRNDFCAPEgi|113194917|gb|AAF51744.3- - -DLGAPEgi|24667933|ref|NP_525001.2H- -- - -SPEgi|28380344|gb|AAF53079.3HGNDFGAPEgi|45446806|gb|AAF45995.2- - -- - -APEgi|24641273HNYDPGRNLTable 3
List of 13 rat PKLNKs. Substitutions of various important residues in the motifs which are generally conserved in the functional protein kinase are shown for each rat PKLNK. “-” indicates deletion.
Gene code“HRD” motif in the catalytic loopActivation loop startActivation loop endgi|19173772|ref|NP_596900.1PRH- - -APEgi|13027458|ref|NP_076490.1HRNDFHAPEgi|16758684|ref|NP_446283.1HGRDYGAPEgi|477540|pir||A49183HGNDYGAPEgi|13242283|ref|NP_077356.1HGRDHGAPEgi|204270|gb|AAA41202.1HGNDYGAPEgi|18543337|ref|NP_570093.1HGRDHGAPEgi|16758694|ref|NP_446290.1HGSDYGAPEgi|109476840|ref|XP_001061647.1HGNDPGAPEgi|149044565|gb|EDL97824.1HGNDPGAPEgi|2288925|emb|CAA04187.1HGNDLGAPEgi|2499669|sp|Q62689.1HGNDPGPPEgi|2499671|sp|Q63272.1HGNDPGAPETable 4
List of 20 mouse PKLNKs. Substitutions of various important residues in the motifs which are generally conserved in the functional protein kinase are shown for each mouse PKLNK. “-” indicates deletion.
Gene code“HRD” motif in the catalytic loopActivation loop startActivation loop endgi|12839087|dbj|BAB24429.1HRLDFGCPEgi|21594381|gb|AAH31924.1HRARLGAPEgi|21703091|gb|AAM74471.1HGN- - -- - -gi|21704242|ref|NP_663596.1HRNDFHAPEgi|21707860|gb|AAH34064.1HGRDFGAPEgi|12858445|dbj|BAB31320.1HRNGFESPEgi|20829352|ref|XP_129532.1HNN- - GSPEgi|12963867|ref|NP_076401.1HNN- - GPPEgi|20349229|ref|XP_111982.1HGRDYGAPEgi|20826487|ref|XP_131378.1HGSDYGAPEgi|20895826|ref|XP_139682.1- - -DFGAPQgi|20869393|ref|XP_127567.1YGHD LE- - -gi|158635954|ref|NP_113563.2HRS- - -APEgi|159110415|ref|NP_032218.2HGRDHGAPEgi|28529710|ref|XP_142224.2HGRDYGAPEgi|28804246|emb|CAD29448.2CGNDFAPEEgi|111607496|ref|NP_666257.2HGNDPGAPEgi|114326478|ref|NP_001041642.1HGNDPGPPEgi|133922607|ref|NP_061263.2HGNDPGAPEgi|156630890|sp|Q62137.2HGNDPGAPEThough these PKLNKs lack the crucial aspartate in the catalytic loop, they are closely related to the functional protein kinases, in terms of the sequence similarity. Table5 provides information on the closest protein kinase subfamily to which these PKLNKs belong to. Many of the PKLNKs are closely related to tyrosine kinase or tyrosine kinase-like group. Further, phylogenetic tree has been constructed considering PKLNK domain and catalytic domain of protein kinase subfamilies of mouse to which these PKLNKs from mouse are closely related (Figure 1). It has been observed that most of the PKLNKs from mouse are grouping to protein kinase subfamilies to which they closely belong to. This information provides a hint about the nearest evolutionary relation between PKLNKs and protein kinases. However, there are two PKLNKs from mouse, one of which is closely related to Tyrosine kinase-like group (gi|6005792), and the other one (gi|158635954) is not closely related to any of the known protein kinase subfamilies which are not grouping with their closest kinase subfamilies (Figure 1) suggesting that these two PKLNKs are evolutionary quite diverged.Table 5
List of PKLNK analysed. Information on number of residues and the nearest protein kinase subfamily to which they belong to has also been provided. Abbreviations followed in the table are RGC, Receptor guanylate cyclase; CAMK1, Ca2+/Calmodulin dependent protein kinase 1; TSSK, Testis-specific serine/Threonine kinase; MLCK, Myosin light chain kinase; Eph, Ephrin receptor; EGFR, Epidermal growth factor receptor; JakA, Janus kinase A; MLK, Mixed lineage kinase; Slob, SLOw Border; NRBP, Nuclear receptor binding protein; TBCK, TBC domain-containing kinase; VRK, Vaccinia-related kinase; IRAK, IL1 receptor associated kinase; CDK, Cyclin dependent kinase; MAPK, Mitogen activated kinase; WNK, With no K (Lysine).
PKLNK gene accession codeNumber of residuesClosest  subfamily of protein kinasegi|40254426|ref|NP_000897.2|1061RGCgi|477540|pir||A49183333RGCgi|4580422|ref|NP_003986.2|1047RGCgi|20826487|ref|XP_131378.1|477RGCgi|16758694|ref|NP_446290.1|1047RGCgi|204270|gb|AAA41202.1|1057RGCgi|23093524|gb|AAN11824.1|AE003537_31272RGCgi|7294467|gb|AAF49811.1|1172RGCgi|7291217|gb|AAF46649.1|1076RGCgi|23171402|gb|AAF55244.2|1417RGCgi|4504217|ref|NP_000171.1|1103RGCgi|159110415|ref|NP_032218.2|1108RGCgi|13242283|ref|NP_077356.1|1108RGCgi|134152694|ref|NP_001513.2|1108RGCgi|28529710|ref|XP_142224.2|1372RGCgi|20349229|ref|XP_111982.1|625RGCgi|16758684|ref|NP_446283.1|1108RGCgi|18543337|ref|NP_570093.1|1110RGCgi|28380344|gb|AAF53079.3|1163RGCgi|18386335|gb|AAB19934.2|1073RGCgi|21707860|gb|AAH34064.1|754RGCgi|22760572|dbj|BAC11248.1|501CAMK1gi|22760645|dbj|BAC11278.1|470CAMK1gi|12052916|emb|CAB66632.1|473CAMK1gi|21704242|ref|NP_663596.1|512CAMK1gi|13027458|ref|NP_076490.1|504CAMK1gi|12839087|dbj|BAB24429.1|292TSSKgi|21626698|gb|AAF47134.2|3197MLCKgi|4758292|ref|NP_004436.1|1006Ephgi|21594381|gb|AAH31924.1|1014Ephgi|17368346|sp|P83097|WSCK_DROME809Ephgi|119534|sp|P21860.1|ERBB3_HUMAN1342EGFRgi|246412731177JakAENSP000002944231156JakAgi|133922607|ref|NP_061263.2|1184JakAgi|111607496|ref|NP_666257.2|1153JakAgi|149044565|gb|EDL97824.1|1153JakAgi|109476840|ref|XP_001061647.1|1198JakAgi|2288925|emb|CAA04187.1|866JakAENSP000003710671132JakAgi|114326478|ref|NP_001041642.1|1132JakAgi|2499669|sp|Q62689.1|JAK2_RAT1132JakAgi|156630890|sp|Q62137.2|JAK3_MOUSE1100JakAgi|2499671|sp|Q63272.1|JAK3_RAT1100JakAENSP000002222461131JakAENSP000002648181187JakAgi|5052670|gb|AAD38665.1|AF145690_1637NRBPgi|14042287|dbj|BAB55185.1|535NRBPgi|21358011|ref|NP_651655.1|835SCY1gi|7301824|gb|AAF56933.1|873SCY1gi|14041796|dbj|BAB55454.1|707SCY1gi|115430241|ref|NP_065731.3|808SCY1gi|12963867|ref|NP_076401.1|806SCY1gi|15779207|gb|AAH14662.1|688SCY1gi|7243101|dbj|BAA92598.1|796SCY1gi|20829352|ref|XP_129532.1|735SCY1gi|113194917|gb|AAF51744.3|2352WNKgi|21627748|gb|AAM68879.1|646Slobgi|22945875|gb|AAN10635.1|AE003618_7638Slobgi|7020363|dbj|BAA91097.1|649Slobgi|20869393|ref|XP_127567.1|582Slobgi|45446806|gb|AAF45995.2|819TBCKgi|18676872|dbj|BAB85045.1|893TBCKgi|31982929|ref|NP_057524.2|474VRKgi|21703091|gb|AAM74471.1|453VRKgi|24667933|ref|NP_525001.2|448MLKgi|4758606|ref|NP_004508.1|452MLKgi|19173772|ref|NP_596900.1|452MLKgi|58530886|ref|NP_001561.3|625IRAKgi|6005792|ref|NP_009130.1|596IRAKgi|28804246|emb|CAD29448.2|609IRAKgi|12858445|dbj|BAB31320.1|472Dicty4gi|22749323|ref|NP_689862.1|471Dicty4gi|16306492|ref|NP_203698.1|240CDKgi|20895826|ref|XP_139682.1|216MAPKgi|17862032|gb|AAL39493.1|346STE20gi|13027388|ref|NP_061041.2|418STE20gi|17368698|sp|Q9BXU1|STK31_HUMAN1019Unclassified protein kinasegi|21357711|ref|NP_647767.1|790Unclassified protein kinasegi|34191428|gb|AAH36504.2|700Unclassified protein kinasegi|57997202|emb|CAD38856.2|1491Unclassified protein kinasegi|158635954|ref|NP_113563.2|1450Unclassified protein kinaseFigure 1
Phylogenetic tree representing catalytic kinase domain and PKLNK domains of mouse. Abbreviations followed in the figure are Pkinase, Protein kinase; PKLNK, Protein kinase-like nonkinase.


### 3.1. Accessory Domains Tethered to the PKLNKs

In the multidomain proteins a given domain acts in conjunction with other domains which are tethered together in the same polypeptide chain and help in their regulation. Absence of catalytic aspartate in the active site reflects the noncatalytic activity of PKLNKs, but domain organization of a protein can give clues about putative functional roles. Domains tethered to PKLNKs suggest that though these PKLNKs lack key catalytic residues, they are involved in protein-protein interactions and might have important regulatory roles in signal transduction pathway. In the current analysis we have identified 19 different types of domains tethered to the PKLNK domain. Except 24 PKLNKs, majority of PKLNKs identified have accessory domains tethered to them (Table6). The Pfam domains tethered to PKLNK domain and their frequency of occurrence are represented in Table 7. As can be seen in Table 7, the most commonly tethered domains are ANF receptor domain, Transmembrane domain and Guanylate cyclase domains. Interestingly most of the time it has been observed that all the three domains are present in the same polypeptide. There are some domain families which occur in repeats like Immunoglobulin I-set domain and HEAT domain which are mainly involved in cell-cell recognition, and protein-protein interactions, respectively, have also been found tethered to the PKLNK domain. Prediction of transmembrane domain has revealed occurrence of receptor PKLNKs which have most  of the time single pass transmembrane region. Interestingly a drosophila protein (gi|21626698) has two PKLNK domains, many I-set (Immunoglobulin) repeats and fn3 domains which has been observed for the first time (Figure 2(a)) and not seen in any functional protein kinase. Our study has revealed that these two PKLNK domains are closely related to myosin light chain kinase subfamily of calcium/calmodulin dependent kinase group. There are a few PKLNKs which are closely related to receptor guanylate cyclase family of protein kinase which is characterized by extracellular ANF receptor domain. Interestingly some of these PKLNKs which are closely related to receptor guanylate cyclase subfamily of protein kinase do not have extracellular domain predicted in the N-terminal (Figure 2(b)) suggesting evolutionary paradigm.Table 6
List of 82 PKLNKs identified from human, mouse, rat, and drosophila. Their gene accession code and domain architecture are also provided. Abbreviations followed in the table are SH2, Src homology 2; PKLNK, Protein kinase-like nonkinase, Pkinase, Protein kinase; Guanylate_cyc, Guanylate cyclase; TM, Transmembrane; Ank, Ankyrin; SAM, Sterile alpha motif; PX, Phox; I-set, Immunoglobulin; fn3, Fibronectin type III; Ephrin_lbd, Ephrin receptor ligand binding domain.
Gene accession codeDomain name, boundary, and E-valueENSP00000222246SH2,  377   457  0.0066 * PKLNK,  822   1071  2e-27 *ENSP00000264818PKLNK,  589   866  4.9e-10 * Pkinase,  897   1172  1.2e-44 *ENSP00000294423SH2,  441   526  0.0016 * PKLNK,  583   847  1e-12 * Pkinase,  877   1151  2.1e-40 *ENSP00000371067SH2,  401   481  0.00012 * PKLNK,  545   805  2.6e-10 * Pkinase,  849   1123  4.4e-42 *gi|109476840|ref|XP_001061647.1|SH2,  486   570  0.0016 * PKLNK,  627   889  1.5e-12 * Pkinase,  919   1193  3.5e-39 *gi|111607496|ref|NP_666257.2|SH2,  441   525  0.0038 * PKLNK,  582   844  1.8e-12 * Pkinase,  874   1148  4.5e-39 *gi|113194917|gb|AAF51744.3|PKLNK,  444   650  2.4e-21 *gi|114326478|ref|NP_001041642.1|SH2,  401   481  0.00014 * PKLNK,  545   805  1.5e-09 * Pkinase,  849   1123  5.2e-43 *gi|115430241|ref|NP_065731.3|PKLNK, 29  259  4e-88 * HEAT,  383   419  0.0067 * HEAT,  501   537  3.2e-05 *gi|119534|sp|P21860.1|ERBB3_HUMANRecep_L_domain,  55   167  6.7e-45 * Furin-like,  180   332  3.4e-79 * Recep_L_domain,  353   474  3.4e-46 *  TM, o644  666i * PKLNK,  709   965  1.9e-24 *gi|12052916|emb|CAB66632.1|PKLNK,  24   258  2.2e-38 *gi|12839087|dbj|BAB24429.1|PKLNK,  25   289  6e-55 *gi|12858445|dbj|BAB31320.1|PKLNK,  195   461  5e-11 *gi|12963867|ref|NP_076401.1|PKLNK,  29   301  0.00098 * HEAT,  383   419  0.014 * HEAT,  501   537  0.00015 *gi|13027388|ref|NP_061041.2|PKLNK,  58   369  2e-28 *gi|13027458|ref|NP_076490.1|PKLNK,  24   286  9.9e-70 *gi|13242283|ref|NP_077356.1|ANF_receptor,  75   411  5.3e-59 * TM, o468  490i * PKLNK,  520   808  1.8e-09 * Guanylate_cyc,  874   1061  2.2e-92 *gi|133922607|ref|NP_061263.2|PKLNK,  589   863  1.7e-10 * Pkinase,  894   1166  4.5e-43 *gi|134152694|ref|NP_001513.2|ANF_receptor,  71   412  3.1e-83 *  TM, o468  490i * PKLNK,  532   809  3.1e-14 * Guanylate_cyc,  875   1062  3.4e-92 *gi|14041796|dbj|BAB55454.1|PKLNK 29  259 9e-89 * HEAT,  383   419  0.0067 * HEAT,  501   537  3.2e-05 *gi|14042287|dbj|BAB55185.1|PKLNK,  81   327  3.2e-08 *gi|149044565|gb|EDL97824.1|SH2,  441   525  0.0016 * PKLNK,  582   844  1.5e-12 * Pkinase,  874   1148  3.5e-39 *gi|156630890|sp|Q62137.2|JAK3_MOUSEPKLNK,  818   1091  2.1e-37 *gi|15779207|gb|AAH14662.1|PKLNK,  18   245  0.00017 *gi|58635954|ref|NP_113563.2|Ank,  55   87  0.00011 * Ank,  88   120  0.018 * PKLNK,  251   503  2e-06 *gi|159110415|ref|NP_032218.2|ANF_receptor,  75   411  1.1e-56 * TM, o345  364i  * PKLNK,  520   811  2.3e-09 * Guanylate_cyc,  874   1061  7e-92 *gi|16306492|ref|NP_203698.1|PKLNK,  4   230  8.7e-56 *gi|16758684|ref|NP_446283.1|ANF_receptor,  71   412  2.8e-74 * TM, o468  490i * PKLNK,  532   809  8.6e-11 * Guanylate_cyc,  875   1062  2.2e-92 *gi|16758694|ref|NP_446290.1|ANF_receptor,  44   400  5.9e-75 *  TM, o456  478i * PKLNK,  534   786  5.3e-17 * Guanylate_cyc,  852   1038  5.6e-107 *gi|17368346|sp|P83097|WSCK_DROMETM, i12  34o * WSC,  42   115  3.8e-25 * fn3,  129   233  2e-05 *  TM, o422  444i * PKLNK,  511   768  2.6e-10 *gi|17368698|sp|Q9BXU1|STK31_HUMANTUDOR,  28   147  2e-31 * PKLNK,  739   972  3.6e-05 *gi|17862032|gb|AAL39493.1|PKLNK,  10   298  3.7e-11 *gi|18386335|gb|AAB19934.2|ANF_receptor,  53   386  3.9e-32 *  TM, o432  454i * PKLNK,  497   745  1.1e-09 * Guanylate_cyc,  815   1002  1.5e-104 *gi|18543337|ref|NP_570093.1|ANF_receptor,  88   422  7e-71 *  TM, o480  502i * PKLNK,  550   818  2.4e-11 * Guanylate_cyc,  884   1071  1.4e-90 *gi|18676872|dbj|BAB85045.1|PKLNK,  26   273  3.4e-18 * TBC,  463   673  1.1e-10 * Rhodanese,  776   883  9.2e-09 *gi|19173772|ref|NP_596900.1|Ank,  33   65  9.4e-09 * Ank,  66   98  1.6e-09 * Ank,  99   131  3.2e-07 * PKLNK,  193   449  1.5e-10 *gi|20349229|ref|XP_111982.1|PKLNK,  1   214  2e-08 * Guanylate_cyc,  306   521  1.2e-79 *gi|204270|gb|AAA41202.1|ANF_receptor,  50   412  1.3e-80 * PKLNK,  534   797  8.4e-13 * Guanylate_cyc,  863   1049  6e-109 *gi|20826487|ref|XP_131378.1|PKLNK,  1   216  7e-09 * Guanylate_cyc,  282   468  5.6e-107 *gi|20829352|ref|XP_129532.1|PKLNK,  18   245  8.9e-05 *gi|20869393|ref|XP_127567.1|PX,  17   122  2.4e-19 * PKLNK,  146   443  0.0012 *gi|20895826|ref|XP_139682.1|PKLNK,  4   206  3.7e-06 *gi|21357711|ref|NP_647767.1|PKLNK,  406   658  0.0018 *gi|21358011|ref|NP_651655.1|PKLNK,  31   314  5.7e-09 *gi|21594381|gb|AAH31924.1|TM, i13  32o * Ephrin_lbd,  34   227  4.5e-113 * fn3,  365   463  1.8e-06 * fn3,  481   565  1.3e-17 * TM, o590  612i * PKLNK,  663   908  7.4e-22 * SAM,  938   1005  1.6e-22 *gi|21626698|gb|AAF47134.2|I-set,  2   87  2.1e-06 * I-set,  102   195  6.9e-17 * I-set,  203   278  0.00011 * I-set,  292   382  7.6e-23 * I-set,  385   479  2.6e-05 * I-set,  483   574  2.6e-15 * I-set,  578   669  6.2e-11 * I-set,  673   765  7.1e-20 * I-set,  800   884  3.1e-10 * I-set,  893   993  0.00015 * I-set,  997   1087  2.5e-10 * I-set,  1092   1194  1.3e-05 * I-set,  1199   1290  2.1e-21 * I-set,  1297   1387  7.4e-24 *  I-set,  1394   1485  7.3e-23 * I-set,  1498   1587  4.6e-21 * I-set,  1594   1683  1.1e-18 * I-set,  1696   1786  1.1e-21 * fn3,  1812   1898  9.9e-07 * I-set,  1951   2042  0.00043 * I-set,  2046   2136  7.1e-12 * PKLNK,  2165   2419  2.5e-48 * I-set,  2633   2723  1.2e-17 * fn3,  2727   2809  1.2e-12 * PKLNK,  2876   3130  1.7e-34 *gi|21627748|gb|AAM68879.1|PX,  17   122  7.9e-15 * PKLNK,  146   435  0.00023 *gi|21703091|gb|AAM74471.1|PKLNK, 145 439  e-128  *gi|21704242|ref|NP_663596.1|PKLNK,  24   286  3.4e-70 *gi|21707860|gb|AAH34064.1|TM, o114  136i * PKLNK,  176   426  3.7e-10 *Guanylate_cyc,  496   683  2e-99 *gi|22749323|ref|NP_689862.1|PKLNK,  209   466  1e-13 *gi|22760572|dbj|BAC11248.1|PKLNK,  24   286  7.8e-67 *gi|22760645|dbj|BAC11278.1|PKLNK,  24   286  3.4e-70 *gi|2288925|emb|CAA04187.1|SH2,  313   397  0.00056 * PKLNK,  454   716  3.2e-14 *gi|22945875|gb|AAN10635.1|AE003618_7PKLNK, 304  520 *gi|23093524|gb|AAN11824.1|AE003537_3ANF_receptor,  64   447  8e-33 * PKLNK,  610   869  4.4e-16 * Guanylate_cyc,  935   1121  1.5e-89 *gi|23171402|gb|AAF55244.2|ANF_receptor,  107   473  1.2e-83 * PKLNK,  608   877  1.8e-15 * Guanylate_cyc,  944   1130  9.7e-103 *gi|24667933|ref|NP_525001.2|Ank,  33   65  1.6e-07 * Ank,  66   98  1.4e-06 * Ank,  99   131  1.5e-08 * PKLNK,  192   446  6.9e-09 *gi|2499669|sp|Q62689.1|JAK2_RATSH2,  401   481  8.6e-05 * PKLNK,  545   805  8.9e-10 * Pkinase,  849   1123  1.9e-43 *gi|2499671|sp|Q63272.1|JAK3_RATPKLNK,  818   1091  2.7e-38 *gi|28380344|gb|AAF53079.3|ANF_receptor,  57   398  2e-37 * PKLNK,  529   798  3e-11 * Guanylate_cyc,  864   1050  3.3e-88 *gi|28529710|ref|XP_142224.2|ANF_receptor,  166   499  3.7e-62 * PKLNK,  647   926  1.5e-10 * Guanylate_cyc,  1018   1229  2.4e-72 *gi|28804246|emb|CAD29448.2|Death,  26   106  5.1e-18 * PKLNK,  178   456  1e-17 *gi|31982929|ref|NP_057524.2|PKLNK,  202   460  0.0082 *gi|34191428|gb|AAH36504.2|PKLNK,  48   317  7.9e-14 *gi|40254426|ref|NP_000897.2|ANF_receptor,  54   416  2.7e-79 * PKLNK,  538   801  3.7e-11 * Guanylate_cyc,  867   1053  7e-110 *gi|4504217|ref|NP_000171.1|ANF_receptor,  72   408  1.3e-55 *  TM, o465  487i* PKLNK,  517   805  8.8e-10 * HNOBA,  718   870  0.009 * Guanylate_cyc,  871   1058  1.8e-89 *gi|45446806|gb|AAF45995.2|PKLNK,  18   263  1.2e-07 * TBC,  430   636  1.4e-08 *gi|4580422|ref|NP_003986.2|ANF_receptor,  44   400  2.7e-75 * PKLNK,  534   786  3.2e-18 * Guanylate_cyc,  852   1038  5.6e-107 *gi|4758292|ref|NP_004436.1|Ephrin_lbd,  18   217  4.5e-110 * fn3,  355   455  1.8e-06 * fn3,  473   557  3.2e-18 *  TM, o582  604i * PKLNK,  655   900  1.1e-22 * SAM,  930   997  6e-22 *gi|4758606|ref|NP_004508.1|Ank,  33   65  9.4e-09 * Ank,  66   98  1.6e-09 * Ank,  99   131  3.2e-07 * PKLNK,  193   449  1.3e-10 *gi|477540|pir||A49183PKLNK,  16   250  7.9e-07 *gi|5052670|gb|AAD38665.1|AF145690_1PKLNK,  122   375  4.7e-09 *gi|57997202|emb|CAD38856.2|Ank,  55   87  0.0025 * Ank,  88   120  0.0056 * PKLNK,  251   503  1.6e-06 *gi|58530886|ref|NP_001561.3|Death,  14   94  2.9e-16 * PKLNK,  210   496  2.6e-11 *gi|6005792|ref|NP_009130.1|Death,  26   106  4.4e-18 * PKLNK,  165   443  3e-12 *gi|7020363|dbj|BAA91097.1|PX,  17   122  1.3e-16 * PKLNK,  146   449  0.0028 *gi|7243101|dbj|BAA92598.1|PKLNK, 4  190  2e-63 *gi|7291217|gb|AAF46649.1|ANF_receptor,  107   481  1.4e-73 * PKLNK,  555   794  3.9e-10 * Guanylate_cyc,  860   1046  1.2e-99 *gi|7294467|gb|AAF49811.1|ANF_receptor,  64   423  3.9e-35 * PKLNK,  573   832  4.4e-16 * Guanylate_cyc,  898   1084  1.5e-89 *gi|7301824|gb|AAF56933.1|PKLNK, 28  268  2e-82 * HEAT,  383   419  0.0027 *gi|24641273PKLNK,  892   1151  1.4e-35 *Table 7
Frequency of domain families tethered to the PKLNKs.
Domain nameNumber of gene productsNumber of domainsANF receptor1717Guanylate cyclase2020Protein kinase99Ankyrin513Fibronectin 347Heat47Immunoglobulin I-set121Phox33TBC22Receptor L domain12Furin-like11Death33Ephrin receptor ligand binding domain22Sterile Alpha Motif22WSC11Rhodanese11Tudor11Transmembrane domain1315Unique domain architectures which are present in PKLNK and so far not reported in the protein kinase. Abbreviations followed in the figure are I-set, Immunoglobulin I-set; fn3, Fibronectin 3; PKLNK, Protein Kinase-Like Nonkinase.
(a)(b)(c)Based upon the broad function, the domains tethered to PKLNK can be functionally categorized into four categories:(1)
Domains which are mainly involved in ligand binding like ANF receptor, Receptor L domain, and Ephrin receptor ligand binding domain.There are 17 gene products which have domains architecture similar to ANP receptor [12] in which ANF_receptor is followed by PKLNK which is followed by Guanylate cyclase domain. The ANF receptor is an extracellular ligand binding domain in a wide range of receptors [33]. Guanylate cyclase catalyses the formation of cyclic GMP (cGMP) from GTP which acts as intracellular messenger and regulates various cellular processes like smooth muscle relaxation, retinal phototransduction, regulation of ion channels, and so forth [34, 35]. The ephrin receptor ligand-binding domain (EPH_lbd) which binds to ephrin is a large family of receptor tyrosine kinases. Biochemical studies suggest that the multimerization of EPH_lbd modulates the cellular response and acts on actin cytoskeleton [36].(2)
Domains which are extracellular and involved in protein-protein interactions like I-set (Immunoglobulin like domain) and Fn3 (Fibronectin type III) domains.(3)
Domains involved in dimerization like Death domain, SAM (Sterile Alpha Motif) domain, and Furin-like domain.Proteins containing death domains are well known to participate in the signaling events which regulate apoptosis [37] indicating role of PKLNK in apoptosis. Proteins containing SAM domains are involved in homo- and hetero oligomerization with other SAM domains and are involved in various developmental processes [38]. Furin-like domain is found tethered to receptor tyrosine kinase. It is rich in cysteine and involved in receptor aggregation.(4)
Domains involved in protein-protein interactions like Ank (Ankyrin repeats) and Heat repeats.Ank is one of the most common protein-protein interaction modules which occur in large number of functionally diverse proteins. PKLNKs containing Ank repeat are likely to play role in diverse functions like signal transduction, ion transportation, transcription initiation, and so forth. Heat domain is 30–40 amino acid tandemly repeated domain. PKLNK containing Heat domain might have role in intracellular transport processes.Apart from the domains discussed above there are some more accessory domains found tethered to the PKLNK domain which provide functional diversity to the PKLNKs. A human PKLNK (gi|18676872) has TBC domain and Rhodanese domain in the C-terminal. TBC domain is involved in GTPase signaling, and Rhodanese domain which shares evolutionary  relationship with large family of protein is involved in cyanide detoxification [39]. Another human PKLNK (gi|17368698) has TUDOR domain N-terminal to the PKLNK domain which indicates its role in RNA binding [40] which has so far not seen tethered with protein kinase (Figure 2(c)).A drosophila PKLNK (gi|17368346) has WSC domains N-terminal to the PKLNK domain which is likely to be an extracellular carbohydrate binding domains. At least three PKLNKs (gi|20869393, gi|21627748, gi|7020363) have PX domain N-terminal to the PKLNK domain which might have role in lipid signaling.Phylogenetic tree has been generated by considering the nonenzymatic PKLNK domains of these 82 PKLNKs (Figure3) in which interestingly we have observed some clusters having similar domain organization. Some of the frequently found tethered domains have been represented in various colours. It can be noticed that, in general, PKLNKs with similar domains tethered are clustered together in Figure 3. There are few PKLNKs which have only one domain, and other parts of the sequences have not been assigned to any other Pfam domains. There are 20 PKLNKs which have guanylate cyclase domains tethered in the C-terminus (represented in green in Figure 3). There are 13 PKLNK sequences which have SH2 (Src homology 2) domain in the N-terminus (represented in red in Figure 3). SH2 domain functions as regulatory module of intracellular signaling cascade by interacting with the phosphopeptide.  All of these SH2 containing PKLNKs except one (gi|2288925) have protein kinase domain tethered in the C-terminus. These 12 protein kinases domains are close homologues of protein tyrosine kinase 7 subfamily. This kind of domain architecture having SH2 domain followed by PKLNK which is followed by protein kinase domain has not been reported anywhere to the best of our knowledge. However, JAK1 (Janus kinase 1) has very similar domain combination in which apart from these three domains, FERM domain which, is involved in binding to cytokine receptors [32, 41] is present in the N-terminus [42].Figure 3
The dendrogram generated by considering the nonenzymatic protein kinase-like nonkinase domains. Various sequences having similar domain organization are represented in different colours: Green: PKLNK having Guanylate Cyclase (GC) domain in the C-terminus; Pink: Ankyrin repeat tethered to the PKLNK; Cyan: Ephrin_lbd, fn3, SterilAlpha Motif (SAM) tethered to the PKLNK; Purple: Death domain tethered to the PKLNK; Blue: Phox (PX) domain tethered to the PKLNK; Brown: Heat domain tethered to the PKLNK; Red: Src Homology 2 (SH2) tethered to the PKLNK.The biological function of these PKLNKs might be in the regulation of tyrosine protein kinase activity.Further, we have compared the domain structure of PKLNKs and their closest protein kinase subfamilies. Interestingly, we have observed that there are a few domain combinations which are unique to either PKLNKs or protein kinases. There are a few Pfam domains such as TUDOR and HNOBA (Heme NO binding associated) which have not been seen tethered to protein kinase domains so far. HNOBA domain is known to function as heme-dependent sensor for gaseous ligands and transduce diverse downstream signals across diverse organisms [43]. The domain structures which commonly occur between PKLNK and protein kinase have also been studied (Table 8).Table 8
Common and unique domain structures of PKLNKs and protein kinases encoded in the data set of genomes. Abbreviations followed in the table are Mad3_BUB1_I, Mad3/BUB1 homology region 1; Pkinase, Protein kinase; PKLNK, Protein kinase-like nonkinase; TM, Transmembrane; SH2, Src-homology 2; I-set, Immunoglobulin; Ank, Ankyrin; fn3, Fibronectin type III; Ephrin_lbd, Ephrin receptor ligand binding domain; SAM, Sterile alpha motif; HNOBA, Heme NO binding associated;  Guanylate_cyc, Guanylate cyclase.
Domain structure unique to PKLNKsDomain structure unique to protein kinasesDomain structure common to protein kinases and PKLNKsPKLNK, HEAT× 2TM, ANF_receptor, Pkinase, Guanylate_cycSH2, PKLNK, PkinaseAnk× 2, PKLNKI-set× 2, PkinaseRecep_L_domain, Furin-like,  Recep_L_domain, TM, PKLNKTUDOR, PKLNKMad3_BUB1_I, PkinasePKLNK, PkinaseANF_receptor, TM, PKLNK, HNOBA,  Guanylate_cycAnk× 3, PkinaseTM, WSC, fn3, TM, PKLNKANF_receptor, TM, PKLNK, Guanylate_cycPKLNK, TBC, RhodanesePKLNK, Guanylate_cycTM, Ephrin_lbd,  fn3× 2,  TM, PKLNK, SAMI-set× 18, fn3,  I-set × 2,  PKLNK,  I-set,  fn3, PKLNKTM, PKLNK,  Guanylate_cycSH2, PKLNKANF_receptor, PKLNK, Guanylate_cycPKLNK,  TBCEphrin_lbd,  fn3× 2, TM, PKLNK, SAMDeath, PKLNKPX,  PKLNKPKLNK, HEAT


### 3.2. Protein-Protein Interaction of Human PKLNKs

Understanding the biological roles of proteins in the cellular environment is the main aim of genome analysis. For almost all cellular processes in a living cell protein-protein interactions are of central importance. In the current section, we have focused on human PKLNKs. We have looked for the protein-protein interactions of PKLNKs using HPRD database (http://www.hprd.org/) [44]. At least 9 human PKLNKs are shown to interact with various other proteins (Table 9) and most of these proteins are signaling proteins and adapter proteins which module the cell signaling and play critical role in cell polarization, differentiation, cell adhesion, neuronal cell development, apoptosis, homeostasis, and so forth. Four of these nine PKLNKs which are closely related to receptor guanylate cyclase (RGC) family of protein kinase are reported to interact mainly with natriuretic peptide and guanylate cyclase. The protein-protein interaction informations obtained from HPRD emphasize role of PKLNKs in signaling.Table 9
List of PKLNKs which interact with a large number of proteins. The in vivo/in vitro protein-protein interaction data has been obtained from HPRD database [32].
PKLNKAccession code of the interacting proteinNameRoleNP_009130.1NP_001560Interleukin-1 receptor-associated kinase 1 isoform 1Partially responsible for IL1-induced upregulation of the transcription factor NF-kappa BNP_001561Interleukin-1 receptor-associated kinase 2Participate in the IL1-induced upregulation of NF-kappa BNP_002459Myeloid differentiation primary response gene 88Functions as adapter protein in the association of IL-1 receptor associated kinase (IRAK) with the IL-1 receptorNP_067681Toll-like receptor adaptor molecule 2Involved in Toll receptor siganlingNP_665802TNF receptor-associated factor 6Mediates signal transduction from members of the TNF receptor superfamily and from the members of the Toll/IL-1 familyNP_004436.1NP_001035090Myeloid/lymphoid or mixed-lineage leukemiaRegulates cell-cell adhesions downstream of Ras activationNP_002077Growth factor receptor-bound protein 2 isoform 1Binds epidermal growth factor receptor. Involved in signal transduction pathwayNP_002961Spermidine/spermine N1-acetyltransferaseInvolved in the catabolic pathway of plymine metabolismNP_004084Ephrin B2Mediates developmental events, especially in the nervous system and in erythropoiesisNP_004432Ephrin receptor EphB1 precursorMediates developmental events, especially in the nervous system and in erythropoiesisNP_005179Cas-Br-M (murine) ecotropic retroviral transforming sequenceAdaptor protein for receptor protein-tyrosine kinases, positively regulates receptor protein-tyrosine kinase ubiquitination in a manner dependent upon its variant SH2 and RING finger domainsNP_005198v-crk sarcoma virus CT10 oncogene homologPlays a role in fibroblast transformationNP_058431v-crk sarcoma virus CT10 oncogene homolog isoform aMember of an adapter protein family that binds to several tyrosine-phosphorylated proteinsNP_115500Haloacid dehalogenase-like hydrolase domain containing 2HydrolaseNP_004508.1AAM77350LIMS2Is a focal adhesion protein that associates with integrin-linked kinases and involved in protein-protein interactions at adhesion sites between cells and the extracellular matrixBAA18998Paxillin gammaA focal adhesion complex (FAC) which interacts with a wide array of molecules involved in managing the cells response to extracellular matrix components, growth factors, cell : cell interactions, and chemotatic signals.NP_000202Integrin, beta 2 precursorCell-surface protein participates in cell adhesion as well as cell-surface mediated signalingNP_000203Integrin beta chain, beta 3Participates in cell adhesion as well as cell-surface mediated signalingNP_001003828Parvin, beta isoform aActin-binding proteins associated with focal contactsNP_001744Caveolin 1Main component of the caveolae plasma membranes, links integrin subunits to the tyrosine kinase FYN which helps in cell cycle progressionNP_002084Glycogen synthase kinase 3 betaInvolved in energy metabolism, neuronal cell development, and body pattern formationNP_002471Protein phosphatase 1, regulatory (inhibitor) subunit 12ARegulates interaction of actin and myosin downstream of the guanosine triphosphatase RhoNP_0026043-phosphoinositide dependent protein kinase-1 isoform 1Phosphorylates and activates protein kinase B alpha and p70 S6 kinaseNP_004978LIM and senescent cell antigen-like domains 1Adaptor protein which may play role in integrin-mediated cell adhesion pr spreadingNP_005154v-akt murine thymoma viral oncogene homolog 1Mediator of growth factor-induced neuronal survivalNP_060692Parvin, alphaActin-binding protein associated with focal contactsNP_066932Thymosin, beta 4Plays a role in regulation of actin polymerization, cell proliferation, migration, and differentiationNP_071424Parvin, gammaActin-binding proteins associated with focal contactsNP_110395Integrin-linked kinase-associated protein phosphatase 2CRegulates the kinase activity of integrin-linked kinase and participate in Wnt signalingNP_391987Integrin beta 1 isoform 1C-1 precursorInvolved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response, and metastatic diffusion of tumor cells.NP_000171.1NP_000400Guanylate cyclase activator 1A (retina)Activator of guanylate cyclaseNP_002089Guanylate cyclase activator 1B (retina)Activator of guanylate cyclaseNP_006263S100 calcium-binding protein, betaInvolved in cell cycle progression and differentiationNP_009033Guanylate cyclase activator 2BActivator of guanylate cyclase receptorO75916Regulator of G-protein signaling 9Inhibits signal transduction by increasing the GTPase activity of G-protein alpha subunits, involved in phosphotransductionNP_003986.2NP_003986Natriuretic peptide receptor B precursorPrimary receptor for C-type natriuretic peptide, which upon ligand binding exhibits greatly increased guanylyl cyclase activityNP_077720Natriuretic peptide precursor CPossesses potent natriuretic, diurectic, and vasodilating activities and are implicated in body fluid homeostasis and blood pressure controlNP_061041.2NP_000446Serine/threonine protein kinase 11Regulates cell polarity and functions as a tumor suppressorNP_001158Baculoviral IAP repeat-containing protein 4Inhibits apoptosis through binding to tumor necrosis factor receptor-associated factors TRAF1 and TRAF2NP_057373Calcium binding protein 39Calcium bindingNP_663304Mitogen-activated protein kinase kinase kinase 7 isoform BMediates signal transduction induced by TGF beta and morphogenetic protein and controls variety of cell functions including transcription regulation and apoptosisNP_665802TNF receptor-associated factor 6Mediates signal transduction from members of the TNF receptor superfamily and from the members of the Toll/IL-1 familyNP_065731.3NP_004636CoilinThe protein encoded by this gene is an integral component of Cajal bodies (also called coiled bodies). Cajal bodies are nuclear suborganelles of varying number and composition that are involved in the posttranscriptional modification of small nuclear and small nucleolar RNAs.NP_036204CD93 antigen precursorInvolved in intercellular adhesion and in the clearance of apoptotic cellsNP_689494SCY1-like 1 binding protein 1Known to interact with SCY1-like family of kinase-like proteinsNP_001513.2NP_001513.2Guanylate cyclase 2FProbably plays a specific functional role in the rods and/or cones of photoreceptors.NP_002089Guanylate cyclase activator 1B (retina)Activator of guanylate cyclaseNP_000897.2NP_006249Protein kinase, cGMP-dependent, type I isoform 2Protein phosphorylationNP_002512Natriuretic peptide precursor B preproproteinFunctions as cardiac hormone, has role in natriuresis, diuresis, vasorlaxation inhibition of rennin, and aldosterone secretion, and has a key role in cardiovascular homeostasisNP_000897Natriuretic peptide receptor 1Membrane bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptidesNP_006163Natriuretic peptide precursor APotent natriuretic, diuretic, and vasodilating activities and are implicated in body fluid homeostasis and blood pressure controlNP_060649Activating transcription factor 7 interacting proteinModulates transcription regulation and chromatin formation


## 3.1. Accessory Domains Tethered to the PKLNKs

In the multidomain proteins a given domain acts in conjunction with other domains which are tethered together in the same polypeptide chain and help in their regulation. Absence of catalytic aspartate in the active site reflects the noncatalytic activity of PKLNKs, but domain organization of a protein can give clues about putative functional roles. Domains tethered to PKLNKs suggest that though these PKLNKs lack key catalytic residues, they are involved in protein-protein interactions and might have important regulatory roles in signal transduction pathway. In the current analysis we have identified 19 different types of domains tethered to the PKLNK domain. Except 24 PKLNKs, majority of PKLNKs identified have accessory domains tethered to them (Table6). The Pfam domains tethered to PKLNK domain and their frequency of occurrence are represented in Table 7. As can be seen in Table 7, the most commonly tethered domains are ANF receptor domain, Transmembrane domain and Guanylate cyclase domains. Interestingly most of the time it has been observed that all the three domains are present in the same polypeptide. There are some domain families which occur in repeats like Immunoglobulin I-set domain and HEAT domain which are mainly involved in cell-cell recognition, and protein-protein interactions, respectively, have also been found tethered to the PKLNK domain. Prediction of transmembrane domain has revealed occurrence of receptor PKLNKs which have most  of the time single pass transmembrane region. Interestingly a drosophila protein (gi|21626698) has two PKLNK domains, many I-set (Immunoglobulin) repeats and fn3 domains which has been observed for the first time (Figure 2(a)) and not seen in any functional protein kinase. Our study has revealed that these two PKLNK domains are closely related to myosin light chain kinase subfamily of calcium/calmodulin dependent kinase group. There are a few PKLNKs which are closely related to receptor guanylate cyclase family of protein kinase which is characterized by extracellular ANF receptor domain. Interestingly some of these PKLNKs which are closely related to receptor guanylate cyclase subfamily of protein kinase do not have extracellular domain predicted in the N-terminal (Figure 2(b)) suggesting evolutionary paradigm.Table 6
List of 82 PKLNKs identified from human, mouse, rat, and drosophila. Their gene accession code and domain architecture are also provided. Abbreviations followed in the table are SH2, Src homology 2; PKLNK, Protein kinase-like nonkinase, Pkinase, Protein kinase; Guanylate_cyc, Guanylate cyclase; TM, Transmembrane; Ank, Ankyrin; SAM, Sterile alpha motif; PX, Phox; I-set, Immunoglobulin; fn3, Fibronectin type III; Ephrin_lbd, Ephrin receptor ligand binding domain.
Gene accession codeDomain name, boundary, and E-valueENSP00000222246SH2,  377   457  0.0066 * PKLNK,  822   1071  2e-27 *ENSP00000264818PKLNK,  589   866  4.9e-10 * Pkinase,  897   1172  1.2e-44 *ENSP00000294423SH2,  441   526  0.0016 * PKLNK,  583   847  1e-12 * Pkinase,  877   1151  2.1e-40 *ENSP00000371067SH2,  401   481  0.00012 * PKLNK,  545   805  2.6e-10 * Pkinase,  849   1123  4.4e-42 *gi|109476840|ref|XP_001061647.1|SH2,  486   570  0.0016 * PKLNK,  627   889  1.5e-12 * Pkinase,  919   1193  3.5e-39 *gi|111607496|ref|NP_666257.2|SH2,  441   525  0.0038 * PKLNK,  582   844  1.8e-12 * Pkinase,  874   1148  4.5e-39 *gi|113194917|gb|AAF51744.3|PKLNK,  444   650  2.4e-21 *gi|114326478|ref|NP_001041642.1|SH2,  401   481  0.00014 * PKLNK,  545   805  1.5e-09 * Pkinase,  849   1123  5.2e-43 *gi|115430241|ref|NP_065731.3|PKLNK, 29  259  4e-88 * HEAT,  383   419  0.0067 * HEAT,  501   537  3.2e-05 *gi|119534|sp|P21860.1|ERBB3_HUMANRecep_L_domain,  55   167  6.7e-45 * Furin-like,  180   332  3.4e-79 * Recep_L_domain,  353   474  3.4e-46 *  TM, o644  666i * PKLNK,  709   965  1.9e-24 *gi|12052916|emb|CAB66632.1|PKLNK,  24   258  2.2e-38 *gi|12839087|dbj|BAB24429.1|PKLNK,  25   289  6e-55 *gi|12858445|dbj|BAB31320.1|PKLNK,  195   461  5e-11 *gi|12963867|ref|NP_076401.1|PKLNK,  29   301  0.00098 * HEAT,  383   419  0.014 * HEAT,  501   537  0.00015 *gi|13027388|ref|NP_061041.2|PKLNK,  58   369  2e-28 *gi|13027458|ref|NP_076490.1|PKLNK,  24   286  9.9e-70 *gi|13242283|ref|NP_077356.1|ANF_receptor,  75   411  5.3e-59 * TM, o468  490i * PKLNK,  520   808  1.8e-09 * Guanylate_cyc,  874   1061  2.2e-92 *gi|133922607|ref|NP_061263.2|PKLNK,  589   863  1.7e-10 * Pkinase,  894   1166  4.5e-43 *gi|134152694|ref|NP_001513.2|ANF_receptor,  71   412  3.1e-83 *  TM, o468  490i * PKLNK,  532   809  3.1e-14 * Guanylate_cyc,  875   1062  3.4e-92 *gi|14041796|dbj|BAB55454.1|PKLNK 29  259 9e-89 * HEAT,  383   419  0.0067 * HEAT,  501   537  3.2e-05 *gi|14042287|dbj|BAB55185.1|PKLNK,  81   327  3.2e-08 *gi|149044565|gb|EDL97824.1|SH2,  441   525  0.0016 * PKLNK,  582   844  1.5e-12 * Pkinase,  874   1148  3.5e-39 *gi|156630890|sp|Q62137.2|JAK3_MOUSEPKLNK,  818   1091  2.1e-37 *gi|15779207|gb|AAH14662.1|PKLNK,  18   245  0.00017 *gi|58635954|ref|NP_113563.2|Ank,  55   87  0.00011 * Ank,  88   120  0.018 * PKLNK,  251   503  2e-06 *gi|159110415|ref|NP_032218.2|ANF_receptor,  75   411  1.1e-56 * TM, o345  364i  * PKLNK,  520   811  2.3e-09 * Guanylate_cyc,  874   1061  7e-92 *gi|16306492|ref|NP_203698.1|PKLNK,  4   230  8.7e-56 *gi|16758684|ref|NP_446283.1|ANF_receptor,  71   412  2.8e-74 * TM, o468  490i * PKLNK,  532   809  8.6e-11 * Guanylate_cyc,  875   1062  2.2e-92 *gi|16758694|ref|NP_446290.1|ANF_receptor,  44   400  5.9e-75 *  TM, o456  478i * PKLNK,  534   786  5.3e-17 * Guanylate_cyc,  852   1038  5.6e-107 *gi|17368346|sp|P83097|WSCK_DROMETM, i12  34o * WSC,  42   115  3.8e-25 * fn3,  129   233  2e-05 *  TM, o422  444i * PKLNK,  511   768  2.6e-10 *gi|17368698|sp|Q9BXU1|STK31_HUMANTUDOR,  28   147  2e-31 * PKLNK,  739   972  3.6e-05 *gi|17862032|gb|AAL39493.1|PKLNK,  10   298  3.7e-11 *gi|18386335|gb|AAB19934.2|ANF_receptor,  53   386  3.9e-32 *  TM, o432  454i * PKLNK,  497   745  1.1e-09 * Guanylate_cyc,  815   1002  1.5e-104 *gi|18543337|ref|NP_570093.1|ANF_receptor,  88   422  7e-71 *  TM, o480  502i * PKLNK,  550   818  2.4e-11 * Guanylate_cyc,  884   1071  1.4e-90 *gi|18676872|dbj|BAB85045.1|PKLNK,  26   273  3.4e-18 * TBC,  463   673  1.1e-10 * Rhodanese,  776   883  9.2e-09 *gi|19173772|ref|NP_596900.1|Ank,  33   65  9.4e-09 * Ank,  66   98  1.6e-09 * Ank,  99   131  3.2e-07 * PKLNK,  193   449  1.5e-10 *gi|20349229|ref|XP_111982.1|PKLNK,  1   214  2e-08 * Guanylate_cyc,  306   521  1.2e-79 *gi|204270|gb|AAA41202.1|ANF_receptor,  50   412  1.3e-80 * PKLNK,  534   797  8.4e-13 * Guanylate_cyc,  863   1049  6e-109 *gi|20826487|ref|XP_131378.1|PKLNK,  1   216  7e-09 * Guanylate_cyc,  282   468  5.6e-107 *gi|20829352|ref|XP_129532.1|PKLNK,  18   245  8.9e-05 *gi|20869393|ref|XP_127567.1|PX,  17   122  2.4e-19 * PKLNK,  146   443  0.0012 *gi|20895826|ref|XP_139682.1|PKLNK,  4   206  3.7e-06 *gi|21357711|ref|NP_647767.1|PKLNK,  406   658  0.0018 *gi|21358011|ref|NP_651655.1|PKLNK,  31   314  5.7e-09 *gi|21594381|gb|AAH31924.1|TM, i13  32o * Ephrin_lbd,  34   227  4.5e-113 * fn3,  365   463  1.8e-06 * fn3,  481   565  1.3e-17 * TM, o590  612i * PKLNK,  663   908  7.4e-22 * SAM,  938   1005  1.6e-22 *gi|21626698|gb|AAF47134.2|I-set,  2   87  2.1e-06 * I-set,  102   195  6.9e-17 * I-set,  203   278  0.00011 * I-set,  292   382  7.6e-23 * I-set,  385   479  2.6e-05 * I-set,  483   574  2.6e-15 * I-set,  578   669  6.2e-11 * I-set,  673   765  7.1e-20 * I-set,  800   884  3.1e-10 * I-set,  893   993  0.00015 * I-set,  997   1087  2.5e-10 * I-set,  1092   1194  1.3e-05 * I-set,  1199   1290  2.1e-21 * I-set,  1297   1387  7.4e-24 *  I-set,  1394   1485  7.3e-23 * I-set,  1498   1587  4.6e-21 * I-set,  1594   1683  1.1e-18 * I-set,  1696   1786  1.1e-21 * fn3,  1812   1898  9.9e-07 * I-set,  1951   2042  0.00043 * I-set,  2046   2136  7.1e-12 * PKLNK,  2165   2419  2.5e-48 * I-set,  2633   2723  1.2e-17 * fn3,  2727   2809  1.2e-12 * PKLNK,  2876   3130  1.7e-34 *gi|21627748|gb|AAM68879.1|PX,  17   122  7.9e-15 * PKLNK,  146   435  0.00023 *gi|21703091|gb|AAM74471.1|PKLNK, 145 439  e-128  *gi|21704242|ref|NP_663596.1|PKLNK,  24   286  3.4e-70 *gi|21707860|gb|AAH34064.1|TM, o114  136i * PKLNK,  176   426  3.7e-10 *Guanylate_cyc,  496   683  2e-99 *gi|22749323|ref|NP_689862.1|PKLNK,  209   466  1e-13 *gi|22760572|dbj|BAC11248.1|PKLNK,  24   286  7.8e-67 *gi|22760645|dbj|BAC11278.1|PKLNK,  24   286  3.4e-70 *gi|2288925|emb|CAA04187.1|SH2,  313   397  0.00056 * PKLNK,  454   716  3.2e-14 *gi|22945875|gb|AAN10635.1|AE003618_7PKLNK, 304  520 *gi|23093524|gb|AAN11824.1|AE003537_3ANF_receptor,  64   447  8e-33 * PKLNK,  610   869  4.4e-16 * Guanylate_cyc,  935   1121  1.5e-89 *gi|23171402|gb|AAF55244.2|ANF_receptor,  107   473  1.2e-83 * PKLNK,  608   877  1.8e-15 * Guanylate_cyc,  944   1130  9.7e-103 *gi|24667933|ref|NP_525001.2|Ank,  33   65  1.6e-07 * Ank,  66   98  1.4e-06 * Ank,  99   131  1.5e-08 * PKLNK,  192   446  6.9e-09 *gi|2499669|sp|Q62689.1|JAK2_RATSH2,  401   481  8.6e-05 * PKLNK,  545   805  8.9e-10 * Pkinase,  849   1123  1.9e-43 *gi|2499671|sp|Q63272.1|JAK3_RATPKLNK,  818   1091  2.7e-38 *gi|28380344|gb|AAF53079.3|ANF_receptor,  57   398  2e-37 * PKLNK,  529   798  3e-11 * Guanylate_cyc,  864   1050  3.3e-88 *gi|28529710|ref|XP_142224.2|ANF_receptor,  166   499  3.7e-62 * PKLNK,  647   926  1.5e-10 * Guanylate_cyc,  1018   1229  2.4e-72 *gi|28804246|emb|CAD29448.2|Death,  26   106  5.1e-18 * PKLNK,  178   456  1e-17 *gi|31982929|ref|NP_057524.2|PKLNK,  202   460  0.0082 *gi|34191428|gb|AAH36504.2|PKLNK,  48   317  7.9e-14 *gi|40254426|ref|NP_000897.2|ANF_receptor,  54   416  2.7e-79 * PKLNK,  538   801  3.7e-11 * Guanylate_cyc,  867   1053  7e-110 *gi|4504217|ref|NP_000171.1|ANF_receptor,  72   408  1.3e-55 *  TM, o465  487i* PKLNK,  517   805  8.8e-10 * HNOBA,  718   870  0.009 * Guanylate_cyc,  871   1058  1.8e-89 *gi|45446806|gb|AAF45995.2|PKLNK,  18   263  1.2e-07 * TBC,  430   636  1.4e-08 *gi|4580422|ref|NP_003986.2|ANF_receptor,  44   400  2.7e-75 * PKLNK,  534   786  3.2e-18 * Guanylate_cyc,  852   1038  5.6e-107 *gi|4758292|ref|NP_004436.1|Ephrin_lbd,  18   217  4.5e-110 * fn3,  355   455  1.8e-06 * fn3,  473   557  3.2e-18 *  TM, o582  604i * PKLNK,  655   900  1.1e-22 * SAM,  930   997  6e-22 *gi|4758606|ref|NP_004508.1|Ank,  33   65  9.4e-09 * Ank,  66   98  1.6e-09 * Ank,  99   131  3.2e-07 * PKLNK,  193   449  1.3e-10 *gi|477540|pir||A49183PKLNK,  16   250  7.9e-07 *gi|5052670|gb|AAD38665.1|AF145690_1PKLNK,  122   375  4.7e-09 *gi|57997202|emb|CAD38856.2|Ank,  55   87  0.0025 * Ank,  88   120  0.0056 * PKLNK,  251   503  1.6e-06 *gi|58530886|ref|NP_001561.3|Death,  14   94  2.9e-16 * PKLNK,  210   496  2.6e-11 *gi|6005792|ref|NP_009130.1|Death,  26   106  4.4e-18 * PKLNK,  165   443  3e-12 *gi|7020363|dbj|BAA91097.1|PX,  17   122  1.3e-16 * PKLNK,  146   449  0.0028 *gi|7243101|dbj|BAA92598.1|PKLNK, 4  190  2e-63 *gi|7291217|gb|AAF46649.1|ANF_receptor,  107   481  1.4e-73 * PKLNK,  555   794  3.9e-10 * Guanylate_cyc,  860   1046  1.2e-99 *gi|7294467|gb|AAF49811.1|ANF_receptor,  64   423  3.9e-35 * PKLNK,  573   832  4.4e-16 * Guanylate_cyc,  898   1084  1.5e-89 *gi|7301824|gb|AAF56933.1|PKLNK, 28  268  2e-82 * HEAT,  383   419  0.0027 *gi|24641273PKLNK,  892   1151  1.4e-35 *Table 7
Frequency of domain families tethered to the PKLNKs.
Domain nameNumber of gene productsNumber of domainsANF receptor1717Guanylate cyclase2020Protein kinase99Ankyrin513Fibronectin 347Heat47Immunoglobulin I-set121Phox33TBC22Receptor L domain12Furin-like11Death33Ephrin receptor ligand binding domain22Sterile Alpha Motif22WSC11Rhodanese11Tudor11Transmembrane domain1315Unique domain architectures which are present in PKLNK and so far not reported in the protein kinase. Abbreviations followed in the figure are I-set, Immunoglobulin I-set; fn3, Fibronectin 3; PKLNK, Protein Kinase-Like Nonkinase.
(a)(b)(c)Based upon the broad function, the domains tethered to PKLNK can be functionally categorized into four categories:(1)
Domains which are mainly involved in ligand binding like ANF receptor, Receptor L domain, and Ephrin receptor ligand binding domain.There are 17 gene products which have domains architecture similar to ANP receptor [12] in which ANF_receptor is followed by PKLNK which is followed by Guanylate cyclase domain. The ANF receptor is an extracellular ligand binding domain in a wide range of receptors [33]. Guanylate cyclase catalyses the formation of cyclic GMP (cGMP) from GTP which acts as intracellular messenger and regulates various cellular processes like smooth muscle relaxation, retinal phototransduction, regulation of ion channels, and so forth [34, 35]. The ephrin receptor ligand-binding domain (EPH_lbd) which binds to ephrin is a large family of receptor tyrosine kinases. Biochemical studies suggest that the multimerization of EPH_lbd modulates the cellular response and acts on actin cytoskeleton [36].(2)
Domains which are extracellular and involved in protein-protein interactions like I-set (Immunoglobulin like domain) and Fn3 (Fibronectin type III) domains.(3)
Domains involved in dimerization like Death domain, SAM (Sterile Alpha Motif) domain, and Furin-like domain.Proteins containing death domains are well known to participate in the signaling events which regulate apoptosis [37] indicating role of PKLNK in apoptosis. Proteins containing SAM domains are involved in homo- and hetero oligomerization with other SAM domains and are involved in various developmental processes [38]. Furin-like domain is found tethered to receptor tyrosine kinase. It is rich in cysteine and involved in receptor aggregation.(4)
Domains involved in protein-protein interactions like Ank (Ankyrin repeats) and Heat repeats.Ank is one of the most common protein-protein interaction modules which occur in large number of functionally diverse proteins. PKLNKs containing Ank repeat are likely to play role in diverse functions like signal transduction, ion transportation, transcription initiation, and so forth. Heat domain is 30–40 amino acid tandemly repeated domain. PKLNK containing Heat domain might have role in intracellular transport processes.Apart from the domains discussed above there are some more accessory domains found tethered to the PKLNK domain which provide functional diversity to the PKLNKs. A human PKLNK (gi|18676872) has TBC domain and Rhodanese domain in the C-terminal. TBC domain is involved in GTPase signaling, and Rhodanese domain which shares evolutionary  relationship with large family of protein is involved in cyanide detoxification [39]. Another human PKLNK (gi|17368698) has TUDOR domain N-terminal to the PKLNK domain which indicates its role in RNA binding [40] which has so far not seen tethered with protein kinase (Figure 2(c)).A drosophila PKLNK (gi|17368346) has WSC domains N-terminal to the PKLNK domain which is likely to be an extracellular carbohydrate binding domains. At least three PKLNKs (gi|20869393, gi|21627748, gi|7020363) have PX domain N-terminal to the PKLNK domain which might have role in lipid signaling.Phylogenetic tree has been generated by considering the nonenzymatic PKLNK domains of these 82 PKLNKs (Figure3) in which interestingly we have observed some clusters having similar domain organization. Some of the frequently found tethered domains have been represented in various colours. It can be noticed that, in general, PKLNKs with similar domains tethered are clustered together in Figure 3. There are few PKLNKs which have only one domain, and other parts of the sequences have not been assigned to any other Pfam domains. There are 20 PKLNKs which have guanylate cyclase domains tethered in the C-terminus (represented in green in Figure 3). There are 13 PKLNK sequences which have SH2 (Src homology 2) domain in the N-terminus (represented in red in Figure 3). SH2 domain functions as regulatory module of intracellular signaling cascade by interacting with the phosphopeptide.  All of these SH2 containing PKLNKs except one (gi|2288925) have protein kinase domain tethered in the C-terminus. These 12 protein kinases domains are close homologues of protein tyrosine kinase 7 subfamily. This kind of domain architecture having SH2 domain followed by PKLNK which is followed by protein kinase domain has not been reported anywhere to the best of our knowledge. However, JAK1 (Janus kinase 1) has very similar domain combination in which apart from these three domains, FERM domain which, is involved in binding to cytokine receptors [32, 41] is present in the N-terminus [42].Figure 3
The dendrogram generated by considering the nonenzymatic protein kinase-like nonkinase domains. Various sequences having similar domain organization are represented in different colours: Green: PKLNK having Guanylate Cyclase (GC) domain in the C-terminus; Pink: Ankyrin repeat tethered to the PKLNK; Cyan: Ephrin_lbd, fn3, SterilAlpha Motif (SAM) tethered to the PKLNK; Purple: Death domain tethered to the PKLNK; Blue: Phox (PX) domain tethered to the PKLNK; Brown: Heat domain tethered to the PKLNK; Red: Src Homology 2 (SH2) tethered to the PKLNK.The biological function of these PKLNKs might be in the regulation of tyrosine protein kinase activity.Further, we have compared the domain structure of PKLNKs and their closest protein kinase subfamilies. Interestingly, we have observed that there are a few domain combinations which are unique to either PKLNKs or protein kinases. There are a few Pfam domains such as TUDOR and HNOBA (Heme NO binding associated) which have not been seen tethered to protein kinase domains so far. HNOBA domain is known to function as heme-dependent sensor for gaseous ligands and transduce diverse downstream signals across diverse organisms [43]. The domain structures which commonly occur between PKLNK and protein kinase have also been studied (Table 8).Table 8
Common and unique domain structures of PKLNKs and protein kinases encoded in the data set of genomes. Abbreviations followed in the table are Mad3_BUB1_I, Mad3/BUB1 homology region 1; Pkinase, Protein kinase; PKLNK, Protein kinase-like nonkinase; TM, Transmembrane; SH2, Src-homology 2; I-set, Immunoglobulin; Ank, Ankyrin; fn3, Fibronectin type III; Ephrin_lbd, Ephrin receptor ligand binding domain; SAM, Sterile alpha motif; HNOBA, Heme NO binding associated;  Guanylate_cyc, Guanylate cyclase.
Domain structure unique to PKLNKsDomain structure unique to protein kinasesDomain structure common to protein kinases and PKLNKsPKLNK, HEAT× 2TM, ANF_receptor, Pkinase, Guanylate_cycSH2, PKLNK, PkinaseAnk× 2, PKLNKI-set× 2, PkinaseRecep_L_domain, Furin-like,  Recep_L_domain, TM, PKLNKTUDOR, PKLNKMad3_BUB1_I, PkinasePKLNK, PkinaseANF_receptor, TM, PKLNK, HNOBA,  Guanylate_cycAnk× 3, PkinaseTM, WSC, fn3, TM, PKLNKANF_receptor, TM, PKLNK, Guanylate_cycPKLNK, TBC, RhodanesePKLNK, Guanylate_cycTM, Ephrin_lbd,  fn3× 2,  TM, PKLNK, SAMI-set× 18, fn3,  I-set × 2,  PKLNK,  I-set,  fn3, PKLNKTM, PKLNK,  Guanylate_cycSH2, PKLNKANF_receptor, PKLNK, Guanylate_cycPKLNK,  TBCEphrin_lbd,  fn3× 2, TM, PKLNK, SAMDeath, PKLNKPX,  PKLNKPKLNK, HEAT


## 3.2. Protein-Protein Interaction of Human PKLNKs

Understanding the biological roles of proteins in the cellular environment is the main aim of genome analysis. For almost all cellular processes in a living cell protein-protein interactions are of central importance. In the current section, we have focused on human PKLNKs. We have looked for the protein-protein interactions of PKLNKs using HPRD database (http://www.hprd.org/) [44]. At least 9 human PKLNKs are shown to interact with various other proteins (Table 9) and most of these proteins are signaling proteins and adapter proteins which module the cell signaling and play critical role in cell polarization, differentiation, cell adhesion, neuronal cell development, apoptosis, homeostasis, and so forth. Four of these nine PKLNKs which are closely related to receptor guanylate cyclase (RGC) family of protein kinase are reported to interact mainly with natriuretic peptide and guanylate cyclase. The protein-protein interaction informations obtained from HPRD emphasize role of PKLNKs in signaling.Table 9
List of PKLNKs which interact with a large number of proteins. The in vivo/in vitro protein-protein interaction data has been obtained from HPRD database [32].
PKLNKAccession code of the interacting proteinNameRoleNP_009130.1NP_001560Interleukin-1 receptor-associated kinase 1 isoform 1Partially responsible for IL1-induced upregulation of the transcription factor NF-kappa BNP_001561Interleukin-1 receptor-associated kinase 2Participate in the IL1-induced upregulation of NF-kappa BNP_002459Myeloid differentiation primary response gene 88Functions as adapter protein in the association of IL-1 receptor associated kinase (IRAK) with the IL-1 receptorNP_067681Toll-like receptor adaptor molecule 2Involved in Toll receptor siganlingNP_665802TNF receptor-associated factor 6Mediates signal transduction from members of the TNF receptor superfamily and from the members of the Toll/IL-1 familyNP_004436.1NP_001035090Myeloid/lymphoid or mixed-lineage leukemiaRegulates cell-cell adhesions downstream of Ras activationNP_002077Growth factor receptor-bound protein 2 isoform 1Binds epidermal growth factor receptor. Involved in signal transduction pathwayNP_002961Spermidine/spermine N1-acetyltransferaseInvolved in the catabolic pathway of plymine metabolismNP_004084Ephrin B2Mediates developmental events, especially in the nervous system and in erythropoiesisNP_004432Ephrin receptor EphB1 precursorMediates developmental events, especially in the nervous system and in erythropoiesisNP_005179Cas-Br-M (murine) ecotropic retroviral transforming sequenceAdaptor protein for receptor protein-tyrosine kinases, positively regulates receptor protein-tyrosine kinase ubiquitination in a manner dependent upon its variant SH2 and RING finger domainsNP_005198v-crk sarcoma virus CT10 oncogene homologPlays a role in fibroblast transformationNP_058431v-crk sarcoma virus CT10 oncogene homolog isoform aMember of an adapter protein family that binds to several tyrosine-phosphorylated proteinsNP_115500Haloacid dehalogenase-like hydrolase domain containing 2HydrolaseNP_004508.1AAM77350LIMS2Is a focal adhesion protein that associates with integrin-linked kinases and involved in protein-protein interactions at adhesion sites between cells and the extracellular matrixBAA18998Paxillin gammaA focal adhesion complex (FAC) which interacts with a wide array of molecules involved in managing the cells response to extracellular matrix components, growth factors, cell : cell interactions, and chemotatic signals.NP_000202Integrin, beta 2 precursorCell-surface protein participates in cell adhesion as well as cell-surface mediated signalingNP_000203Integrin beta chain, beta 3Participates in cell adhesion as well as cell-surface mediated signalingNP_001003828Parvin, beta isoform aActin-binding proteins associated with focal contactsNP_001744Caveolin 1Main component of the caveolae plasma membranes, links integrin subunits to the tyrosine kinase FYN which helps in cell cycle progressionNP_002084Glycogen synthase kinase 3 betaInvolved in energy metabolism, neuronal cell development, and body pattern formationNP_002471Protein phosphatase 1, regulatory (inhibitor) subunit 12ARegulates interaction of actin and myosin downstream of the guanosine triphosphatase RhoNP_0026043-phosphoinositide dependent protein kinase-1 isoform 1Phosphorylates and activates protein kinase B alpha and p70 S6 kinaseNP_004978LIM and senescent cell antigen-like domains 1Adaptor protein which may play role in integrin-mediated cell adhesion pr spreadingNP_005154v-akt murine thymoma viral oncogene homolog 1Mediator of growth factor-induced neuronal survivalNP_060692Parvin, alphaActin-binding protein associated with focal contactsNP_066932Thymosin, beta 4Plays a role in regulation of actin polymerization, cell proliferation, migration, and differentiationNP_071424Parvin, gammaActin-binding proteins associated with focal contactsNP_110395Integrin-linked kinase-associated protein phosphatase 2CRegulates the kinase activity of integrin-linked kinase and participate in Wnt signalingNP_391987Integrin beta 1 isoform 1C-1 precursorInvolved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response, and metastatic diffusion of tumor cells.NP_000171.1NP_000400Guanylate cyclase activator 1A (retina)Activator of guanylate cyclaseNP_002089Guanylate cyclase activator 1B (retina)Activator of guanylate cyclaseNP_006263S100 calcium-binding protein, betaInvolved in cell cycle progression and differentiationNP_009033Guanylate cyclase activator 2BActivator of guanylate cyclase receptorO75916Regulator of G-protein signaling 9Inhibits signal transduction by increasing the GTPase activity of G-protein alpha subunits, involved in phosphotransductionNP_003986.2NP_003986Natriuretic peptide receptor B precursorPrimary receptor for C-type natriuretic peptide, which upon ligand binding exhibits greatly increased guanylyl cyclase activityNP_077720Natriuretic peptide precursor CPossesses potent natriuretic, diurectic, and vasodilating activities and are implicated in body fluid homeostasis and blood pressure controlNP_061041.2NP_000446Serine/threonine protein kinase 11Regulates cell polarity and functions as a tumor suppressorNP_001158Baculoviral IAP repeat-containing protein 4Inhibits apoptosis through binding to tumor necrosis factor receptor-associated factors TRAF1 and TRAF2NP_057373Calcium binding protein 39Calcium bindingNP_663304Mitogen-activated protein kinase kinase kinase 7 isoform BMediates signal transduction induced by TGF beta and morphogenetic protein and controls variety of cell functions including transcription regulation and apoptosisNP_665802TNF receptor-associated factor 6Mediates signal transduction from members of the TNF receptor superfamily and from the members of the Toll/IL-1 familyNP_065731.3NP_004636CoilinThe protein encoded by this gene is an integral component of Cajal bodies (also called coiled bodies). Cajal bodies are nuclear suborganelles of varying number and composition that are involved in the posttranscriptional modification of small nuclear and small nucleolar RNAs.NP_036204CD93 antigen precursorInvolved in intercellular adhesion and in the clearance of apoptotic cellsNP_689494SCY1-like 1 binding protein 1Known to interact with SCY1-like family of kinase-like proteinsNP_001513.2NP_001513.2Guanylate cyclase 2FProbably plays a specific functional role in the rods and/or cones of photoreceptors.NP_002089Guanylate cyclase activator 1B (retina)Activator of guanylate cyclaseNP_000897.2NP_006249Protein kinase, cGMP-dependent, type I isoform 2Protein phosphorylationNP_002512Natriuretic peptide precursor B preproproteinFunctions as cardiac hormone, has role in natriuresis, diuresis, vasorlaxation inhibition of rennin, and aldosterone secretion, and has a key role in cardiovascular homeostasisNP_000897Natriuretic peptide receptor 1Membrane bound guanylate cyclase that serves as the receptor for both atrial and brain natriuretic peptidesNP_006163Natriuretic peptide precursor APotent natriuretic, diuretic, and vasodilating activities and are implicated in body fluid homeostasis and blood pressure controlNP_060649Activating transcription factor 7 interacting proteinModulates transcription regulation and chromatin formation


## 4. Conclusions

This work represents functional analysis of noncatalytic PKLNKs across a data set of 82 PKLNKs from four higher eukaryotes. Our analysis has indicated that existence of noncatalytic PKLNKs is quite common. The fact that noncatalytic PKLNKs are well conserved betweenHomo sapiens, Mus musculus, Rattus norvegicus, and Drosophila melanogaster strongly argues against pseudogenes, as otherwise these would have been lost during the evolutionary time. Our study on PKLNKs suggests that most noncatalytic PKLNKs are derived from active protein kinase ancestors and have lost one or more of the critical catalytic residues within the active site which provides new insight into nature’s way of eliciting new functions of PKLNKs. Based upon the domain tethering preferences we have classified PKLNKs into four main classes in which members of the two classes are receptor PKLNKs which are mainly involved in ligand binding and protein-protein interaction extracellularly while other two classes of PKLNKs have members which are cytoplasmic, and they are mainly involved in dimerization and protein-protein interaction in the cytoplasm. The phylogenetic analysis reveals function-based clustering of these PKLNKs. Conservation of some of the modular organization across the four organisms suggests their central role in the eukaryotic signaling pathway. Since many of the PKLNKs have other domains tethered to them and are involved in protein-protein interactions, one can speculate that though the kinase-like domain is nonenzymatic, they might have role in regulation and scaffolding. Some of these catalytically inactive members of PKLNKs which are close homologues of the receptor tyrosine kinase are shown to be over-expressed in cancer cells. Additional studies are required to determine precise function and role of these PKLNKs in tumorgenesis and its usefulness in the diagnosis of tumors. Domain organization of these PKLNKs revealed that some of the PKLNKs have new and hence unique domain organization so far not seen in any other family of gene products. 3D structure and biochemical analysis can further determine and explore the functional role of these PKLNKs. The presence of putative PKLNK in higher eukaryotes indicates that we have more to learn about cellular signaling involving these noncatalytic domains. Evolutionary history of these PKLNKs would be of particular interest. It is hoped that this analysis will provide a better understanding about the frequent occurrence of PKLNKs in different organisms and hence their function.



---

*Source: 365637-2009-09-28.xml*