LOK Is a Novel Mouse STE20-like Protein Kinase That Is Expressed Predominantly in Lymphocytes*

We have identified a new gene, designatedlok (lymphocyte-oriented kinase), that encodes a 966-amino acid protein kinase whose catalytic domain at the N terminus shows homology to that of the STE20 family members involved in mitogen-activated protein (MAP) kinase cascades. The non-catalytic domain of LOK does not have any similarity to that of other known members of the family. There is a proline-rich motif with Src homology region 3 binding potential, followed by a long coiled-coil structure at the C terminus. LOK is expressed as a 130-kDa protein, which was detected predominantly in lymphoid organs such as spleen, thymus, and bone marrow, in contrast to other mammalian members of the STE20 family. LOK phosphorylated itself as well as substrates such as myelin basic protein and histone IIA on serine and threonine residues but not on tyrosine residues, establishing LOK as a novel serine/threonine kinase. When coexpressed in COS7 cells with the known MAP kinase isoforms (ERK, JNK, and p38), LOK activated none of them in contrast to PAK- and GCK-related kinases. These results suggest that LOK could be involved in a novel signaling pathway in lymphocytes, which is distinct from the known MAP kinase cascades.

Lymphocytes play a central role in the immune system. They originate from pluripotent hematopoietic stem cells and differentiate in primary lymphoid organs such as bone marrow and thymus. In these organs the interaction of lymphocyte precursor cells with stromal cells through various adhesion molecules and cytokines such as interleukin-7 has been shown to elicit signals for the proliferation and differentiation of precursor cells (1,2). Recent studies have revealed that pre-B/pre-T cell receptors expressed on lymphocyte precursors also transduce signals for the regulation of their proliferation and differentiation as well as the rearrangement of genes coding for antigen receptors (3,4).
Ample evidence suggests that an array of protein kinases, both tyrosine kinases and serine/threonine kinases, are involved in the signal transduction for the differentiation of lymphocyte precursors as well as the activation of mature lympho-cytes (5). Therefore, we have studied what kinds of protein kinases were expressed and functioned in early lymphocyte precursor cells. In this process we identified and isolated a novel protein kinase gene, lok, which is the focus of this report. lok was found to encode a serine/threonine kinase that belongs to the STE20 family (6). STE20 is well characterized as a component of the MAP 1 kinase cascade for the signal transduction via the G-protein-linked pheromone receptor in yeast (7). Genetic epistasis placed STE20 at the head of the cascade of kinases. STE20 phosphorylates STE11, which acts as a MAP-KKK for STE7. STE7 in turn acts as a MAPKK for FUS3 and KSS1 MAP kinases (8). Recently, several mammalian homologues of STE20 have been identified and shown to be involved in MAP kinase cascades activated by extracellular stimuli such as cytokines, mitogens and cellular stresses (6,9). The present study shows that LOK is a unique member of the STE20 family in terms of its expression pattern among tissues and the distinctive structure of its non-catalytic domain.

EXPERIMENTAL PROCEDURES
Isolation and Sequence Analysis of lok cDNA Clone-A pair of degenerate oligonucleotide primers for RT-PCR was synthesized which corresponded to the two conserved amino acid sequences in the catalytic domains of various protein kinases, IHRDL and DVWSFG: 5Ј-CG-GATCCAC(A/C)GNG A(C/T)(C/T)T-3Ј and 5Ј-GGAATTCCA(A/T)AG-GACCA(C/G)AC(A/G)TC-3Ј, having an BamHI and EcoRI site at the 5Ј end (underlined), respectively, as described by Wilks (10). RNA was prepared from NFS5.3 pre-B cells, and subjected to RT-PCR with the primers. The RT-PCR products of the expected size (about 210 base pairs) were cloned between the BamHI and EcoRI sites of pBluescript and then sequenced. Nucleotide sequencing of the cDNA inserts from 21 randomly chosen clones resulted in the identification of 6 repetitive clones containing a cDNA for a novel kinase, which we designated LOK. To obtain a full-length cDNA, a cDNA library was prepared from mouse thymus by using gt22 (Stratagene) and screened by the cDNA fragment.
Preparation of Polyclonal Antibody against LOK-The GST-LOK plasmid was constructed by inserting the fragment of the lok cDNA encoding a sequence from Leu-357 to Lys-778 into the pGEX4T-3 expression vector (Pharmacia Biotech Inc.). The GST-fusion proteins was expressed in Escherichia coli XL1-blue in the presence of 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside, purified and used to immunize * This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan, the Uehara Memorial Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D89728.
Transient Expression and Immune Complex Kinase Assays-For the kinase assay of LOK (Fig. 5), each plasmid DNA was transfected into COS7 cells by the calcium phosphate method. The cells were lysed after 48 h of transfection in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 20 mM EDTA, 1 mM Na 3 VO 4 , 10 mM NaF, and 50 units/ml aprotinin. For the co-transfection experiment (Fig. 6), COS7 cells were transiently transfected using LipofectAMINE (Life Technologies Inc.) and, after 24 h of transfection, cells were lysed as described previously (19). Cell lysates were precleared with protein A-Sepharose 4B (Pharmacia) for 30 min and incubated with anti-FLAG (M2, Eastman Kodak Co.), anti-LOK antibody, or anti-HA (12CA5) for 2 h at 4°C. The immune complexes were precipitated with protein A-Sepharose, washed with lysis buffer, and then washed with kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl 2 , and 3 mM MnCl 2 ). To assay the kinase activity of LOK, the immunoprecipitates were incubated with 20 M cold ATP and [␥-32 P]ATP with/without substrates for 20 min at 30°C. The substrates include casein, histone IIA, and myelin basic protein (MBP). To assay the activity of MAPK, JNK/SAKP, or p38, samples were incubated for 30 min at 30°C with 3 g of MBP, c-Jun, or ATF2 as described previously (20). The reaction products were subjected to SDSpolyacrylamide gel electrophoresis. The gels were fixed, dried, and autoradiographed.
Phosphoamino Acid Analysis-Radioactive bands excited from dried gels were hydrolyzed in 6 N HCl for 1 h at 110°C. The supernatant was lyophilized and dissolved in pH 1.9 buffer containing cold phosphoamino acids as markers. The phosphoamino acids were resolved electrophoretically in two dimensions using a thin layer cellulose (TLC) plate with two pH systems (pH 1.9 and 3.5). The markers were visualized by staining with ninhydrin (21).

RESULTS
Cloning and Sequencing of lok-To identify protein kinases expressed in lymphocyte precursor cells, degenerate oligonucleotide primers corresponding to conserved regions of subdomains VI and IX in the catalytic domain of protein kinases were used to amplify cDNA from a mouse precursor B lymphocyte line NFS5.3. The sequence determination of randomly chosen 21 clones of the amplified products revealed that 6 of them had an identical sequence that could encode a novel kinase, most likely a serine/threonine kinase. We named this molecule lymphocyte-oriented kinase (LOK) according to its expression pattern in tissues as shown below.
Since Northern blot analysis using the lok cDNA fragment as a probe detected the expression of ϳ5 kilobases of mRNA in NFS5.3 cell line as well as in thymus and spleen (data not shown), a cDNA library was prepared from mouse thymus and The kinase domain is located at the N terminus of the protein and contains all 11 subdomains of serine/threonine kinases ( Fig. 1, boxed, and Fig. 3B) (23). Within the kinase domain, LOK displays the highest homology to members of the STE20 family such as PAK and GCK with 38 -46% identity (Fig. 2). Moreover, the subdomain VIII of LOK contains the sequence GTPY/FWMAPEV characteristic for the STE20 family, indicating that LOK is a new member of the family. However, the C-terminal non-catalytic domain of LOK does not show any homology to known members of the family. Homology search of the non-catalytic domain of LOK failed to find any homologous proteins except rat AT1-46 carrying no kinase domain (see "Discussion"). The C-terminal region of LOK is predominately ␣-helical and supposed to form a coiled-coil structure (Fig. 1, shaded, and Fig. 3B), as predicted by the Pepcoil algorithm (Fig. 3A). Between the catalytic domain and the coiled-coil region, there is a proline-rich region containing putative SH3binding motifs, PXXP (Fig. 1, thickly underlined, and Fig. 3B).
LOK Is Expressed Predominantly in Lymphocytes-When FLAG epitope-tagged cDNA of lok was transfected into COS7 cells, the gene product was detected as a 130-kDa protein by Western blotting with anti-FLAG antibody (data not shown).
The polyclonal antibody generated against LOK-GST fusion proteins could detect the same protein in lok cDNA-transfected COS7 cells (Fig. 4C). The expression of LOK in various mouse tissues was then examined by Western blotting with this antibody. Surprisingly, LOK expression was found not ubiquitous and rather restricted to lymphoid organs such as spleen, thymus and bone marrow (Fig. 4, A and B). Both T and B lymphocytes in spleen expressed LOK (Fig. 4B). Analysis of LOK in a panel of in vitro cell lines confirmed the predominant expression of the kinase in lymphocytes (Fig. 4C, not all data shown). Eventually this pattern of expression led us to designate this kinase lymphocyte-oriented kinase, LOK.
LOK Possesses the Activity of Serine/Threonine Kinase-Since the nucleotide sequence predicted LOK as a novel serine/ threonine kinase, we next analyzed whether the LOK protein was enzymatically active (Fig. 5). FLAG epitope-tagged LOK immunoprecipitated from COS7 transfectants autophosphorylated itself and phosphorylated substrates such as myelin basic protein and histone IIA when analyzed by the in vitro kinase assay. Furthermore, native LOK immunoprecipitated from the B cell line WEHI 231 also showed the kinase activity (Fig. 5A,  lane 10). The phosphoamino acid analysis revealed that serine and threonine residues but not tyrosine residues were phosphorylated in LOK and histone IIA (Fig. 5B). The substitution of lysine at position 65 in the ATP-binding site of the catalytic domain with arginine (KR mutant) abolished the kinase activity of LOK (Fig. 5A, lane 9), indicating that the observed kinase activity was directly from LOK and not from other molecules LOK Activates None of the Known MAP Kinase Cascades-Many of the STE20 family members have been shown to activate the JNK/SAPK MAP kinase cascade and/or the p38 MAP kinase cascade (24 -30). Therefore, we asked a question which cascade of the known MAP kinases (ERK, JNK/SAPK, and p38) could be activated by LOK. The lok cDNA tagged with FLAG was transfected to COS7 cells together with HA epitope-tagged cDNAs encoding either ERK, JNK/SAPK, or p38. The activation of each MAP kinase was analyzed by the immune complex kinase assay using c-Jun, ATF-2, and myelin basic protein as substrates, respectively (Fig. 6). Even though slight elevation (1.2ϳ1.9-fold increase) of the activity was observed in all three MAP kinases coexpressed with wild-type LOK as compared with controls coexpressed with empty vectors, the coexpression of the kinase-dead KR mutant of LOK gave comparable increase of the MAP kinase activity. In contrast, the coexpression of MAP kinase kinases (MAPKK, SEK1/MKK4, and MKK6) greatly enhanced the activity of the corresponding MAP kinases. Therefore, we concluded that LOK activated none of the known MAP kinase cascades at least in COS7 cells. DISCUSSION The present study has revealed that LOK is a new member of the STE20 family and has unique features among the family. First of all, LOK was found to be expressed predominantly in lymphocytes. GCK (germinal center kinase) is expressed ubiquitously, despite its original naming, like many of other family members (31). A few members show the expression pattern relatively restricted to a certain tissue: PAK3/PAK␤ in brain (32), and HPK1 in hematopoietic cells (25,26). Those tissuespecific kinases including LOK may play important roles in response to a certain external stimulus in a tissue-specific manner. LOK is expressed in both B and T lymphocytes throughout their life, from early stage of their development in bone marrow and thymus till the differentiated effector phase in the periphery. Since recent studies have clarified that pre-cursor cells of both B and T lymphocytes use the same or very similar mechanisms to regulate their differentiation (3,4), LOK may be involved in such regulations.
Second, LOK has a unique non-catalytic domain, which does not have any similarity to that of other members of the STE20 family. The family can be divided into three subfamilies based on the overall structure (6): (a) the archetypal PAK subfamily, containing an N-terminal p21-binding domain and a C-terminal kinase domain; (b) the pleckstrin homology-PAK subfamily, having a structure similar to the archetypal PAK subfamily, except they contain a pleckstrin homology domain N-terminal to the p21-binding domain (this subfamily has been found so far only in yeast but not in higher eukaryotes); (c) the GCK subfamily, having a kinase domain at the N terminus and lacking a recognizable p21-binding domain. Among this subfamily GCK (31), KHS (27), HPK1 (25,26), and NIK (28) have extensive homology with each other in their non-catalytic domain to make one subgroup, whereas Mst1 (33) and Mst2 (34) makes another subgroup. Although the overall structure of LOK indicates that LOK belongs to the GCK subfamily, the C-terminal non-catalytic domain displayed no resemblance to that of other members of the GCK subfamily including SOK1 (35) and Sps1 (36). Thus, LOK appears to be a distinct member of the subfamily. Homology search of the nucleotide sequence of LOK non-catalytic domain identified rat AT1-46 cDNA with 85% identity, and the C-terminal half of LOK shows 96% amino acid identity to AT1-46 protein (37). This gene was originally cloned from rat brain and predicted to encode a 472-amino acid protein with no kinase domain, which corresponds to the coiledcoil region of LOK. Even though the authors claimed to have obtained a full-length AT1-46 cDNA of 4320 base pairs, their Northern blot analysis revealed the presence of a single band of 5.2-kilobase transcripts hybridized with the cDNA. Moreover, the expression of the transcripts was highest in thymus and spleen and lowest in brain. Therefore, AT1-46 appears to be a part of the cDNA encoding rat homologue of mouse LOK.
Outside of the kinase domain, LOK possesses two intriguing regions that have potential to interact with other molecules. The proline-rich region with PXXP motifs found near the middle of the protein is a good candidate for the binding site of SH3-carrying molecules (38,39). PAK and NIK among the STE20 family members have been shown to associate through their proline-rich motifs with the adaptor protein Nck, which is composed of three SH3 domains and one SH2 domain and a common target for a variety of growth factor receptors (28, 40 -42). It remains to be determined what kind of a SH3carrying molecule(s) is bound to the proline-rich region of LOK. The C-terminal half of LOK is predicted to form a ϳ400-amino acid coiled-coil structure. This region exhibits a 24% amino acid sequence identity with the intermediate filament-associated protein, trichohyalin, which associates with keratin intermediate filaments (43). Thus, the coiled-coil structure of LOK is indicative of its interaction with itself (homodimerization) or with other coiled-coil proteins (heterodimerization). LOK activated none of the known MAP kinase cascades, ERK, JNK/SAPK, and p38 (44), when expressed in COS7 cells together with the MAP kinases. In contrast, the archetypal PAK subfamily members and many of GCK subfamily members have been shown to activate the JNK/SAPK MAP kinase cascade, most likely via MEKK1 (or MLK3) and MKK4/SEK (24 -30). At least two possibilities can be considered to explain why LOK failed to activate the known MAP kinase cascades. Ample of evidence indicates that the STE20 family resides near the top of the MAP kinase cascades and functions as a MAP kinase kinase kinase kinase (6). Since the expression of LOK is mostly restricted to lymphocytes, it may not be surprising that a putative MAPKKK and/or MAPKK downstream of LOK is also expressed predominantly in lymphocytes. If this is the case, LOK could not activate the MAP kinases in the ectopic system with COS7 cells in the absence of these MAPKKK and MAPKK. Alternatively, LOK may be involved in an as yet unidentified MAP kinase cascade(s) working in lymphocytes. In this sense it is interesting to note that several "orphan" MAP kinase cascade modules such as ERK5 and its activator MEK5 have been described, in which upstream activators and down-  4 and 5). The amounts of transfected wild type or mutant LOK plasmids in lanes 3 and 5 were 10 times that in lanes 2 and 4. As a positive control, pSR␣-MAPKK (A), pSR␣-SEK1 (B), or pSR␣-MKK6 (C) were co-transfected in place of the LOK plasmids (lane 6). HA-MAPK, HA-SAPK, or HA-p38 was immunoprecipitated from lysates of the COS7 transfectants by anti-HA antibody. Their kinase activity was measured by the in vitro kinase assay using MBP, c-Jun, and ATF-2 as a substrate, respectively. Phosphorylation of each substrate was quantified by Fuji BAS 2000. stream targets remain to be determined (45). Since at least five MAP kinase cascades have been identified in yeast (46), it would be reasonable to speculate the existence of more distinct MAP kinase cascades in mammals in addition to the known ones.
Some members of the STE20 family have been shown to become activated by stress such as inflammatory cytokines and H 2 O 2 (9,35). Therefore, we examined whether such stress can activate LOK in lymphocytes. The stimulation of lymphocytes with tumor necrosis factor ␣, H 2 O 2 , and mitogens such as lipopolysaccharide and anti-CD3 antibody failed to increase LOK kinase activity significantly, as determined by the immune complex kinase assay (unpublished observation). The problem in this in vitro kinase assay was that the basal activity of LOK isolated from unstimulated lymphocytes was already considerably high and might have obscured the activation of LOK. This high basal activity seems a common feature of the GCK subfamily in this assay (25,26,31,(33)(34)(35). It is not clear yet that this high basal activity is due to autophosphorylation and autoactivation of LOK in the presence of ATP in vitro or indeed reflects the constitutive activation of LOK in vivo. Since NIK (NFB-inducing kinase), a newly identified MAPKKK, has been shown to be involved in activation of NFB (47), we explored the possibility of involvement of LOK in this activation. However, LOK did not activate NFB, and the KR mutant of LOK did not interfere the activation of NFB by tumor necrosis factor ␣, at least when analyzed in L929 fibroblast. 2 Thus, possible elements upstream and downstream of LOK remain to be identified. Nevertheless, the predominant expression of LOK in lymphocytes and its distinctive non-catalytic domain make LOK very attractive among the STE20 family members. The expression of LOK not only in mature lymphocytes but also in their precursors in bone marrow and thymus may suggest an intriguing potential involvement in regulation of differentiation, proliferation, and activation of lymphocytes. To explore this possibility, we are in the process of establishing mice deficient for LOK. Furthermore, identification of molecules bound to the proline-rich motif and the coiled-coil structure of LOK should help us to understand the pathway of signal transduction through LOK.