Identification and Characterization of a Novel Protein Kinase, TESK1, Specifically Expressed in Testicular Germ Cells*

We have isolated cDNA clones encoding the rat and human forms of a novel protein kinase, termed TESK1 (testis-specific protein kinase 1). Sequence analysis in- dicates that rat TESK1 contains 628 amino acid residues, composed of an N-terminal protein kinase consen- sus sequence followed by a C-terminal proline-rich region. Human TESK1 contains 626 amino acids, sharing 92% amino acid identity with its rat counterpart. The protein kinase domain of TESK1 is structurally similar to those of LIMK (LIM motif-containing protein ki-nase)-1 and LIMK2, with 49–50% sequence identity. Phy- logenetic analysis of the protein kinase domains revealed that TESK1 is most closely related to a LIMK subfamily. Chromosomal localization of human TESK1 gene was assigned to 9p13. Anti-TESK1 antibody raised against the C-terminal peptide of TESK1 recognized two polypeptides of 68 and 80 kDa in cell lysates of COS cells transfected with human TESK1 cDNA expression plasmid. TESK1 protein expressed in COS cells exhibited serine/threonine kinase activity, when (0.05 mg/ml, Sigma) (19). The samples were mixed with Laemmli’s sample buffer and then resolved by 15% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes, and the 32 P-labeled proteins were visualized by autoradiography. Phosphoamino Acid Analysis— Phosphoamino acid analysis was per- formed as described (20). The region of the membrane containing the radioactive MBP band was excised and incubated with 6 N HCl at 105 °C for 2 h. After removal of the membrane, the hydrolysates were separated by two-dimensional electrophoresis. The 32 P-labeled phos- phoamino acids were detected by autoradiography, and a comparison was made with the ninhydrin-stained phosphoamino acid standards. Fluorescence in Situ Hybridization— Direct R-banding fluorescence in situ hybridization was carried out, using the biotinylated human TESK1 cDNA probe, as described elsewhere (21).

Protein kinases play a pivotal role in intracellular signal transduction systems involved in the regulation of cell proliferation, differentiation, metabolism, and other activities. There are two broad categories of protein kinases; one is tyrosine kinase with substrate specificity toward the tyrosine residue, and the other is the serine/threonine kinase with specificity toward serine and threonine residues, although the latter includes proteins with dual specificity toward both serine/threonine and tyrosine residues. The protein kinases in each category are further divided into many subfamilies, based on sequence similarities of the kinase catalytic domain and the extracatalytic domain (1)(2)(3).
An increasing number of protein kinase genes have been isolated by cDNA cloning. Some of the newly identified protein kinases contain catalytic and extracatalytic domains not related to any known members of the protein kinase family. It is assumed that these unclassified protein kinases have specific functions in signal transduction pathways not previously established and are regulated in a different manner. For example, a JAK family of protein kinases was identified as a solitary class of protein kinases structurally not related to other known kinases, and later specific roles in the signal transduction system linked to receptors for interferons and other cytokines were discovered (4). In view of the central role of protein kinases in diverse cell activities, further searches for a novel class of protein kinases and specification of their roles in various systems are important to better understand signal-transduction mechanisms.
We recently identified a novel class of protein kinases, termed LIM kinases (LIMKs), 1 composed of LIMK1 and LIMK2 (5,6). Both of these two closely related kinases contain characteristic structural features, consisting of the N-terminal two LIM motifs and the C-terminal unusual protein kinase domain. The LIM motif, a structural motif composed of two adjacent zinc fingers separated by a 2-amino acid linker (7), may be involved in the regulation of the kinase activity of LIMKs. The C-terminal kinase domains of LIMKs contain a consensus sequence of protein kinases, but are unique in that they have an unusual sequence motif (DLNSHN) in the kinase catalytic loop in subdomain VIB and a highly basic insert sequence between subdomain VII and VIII (5,6). Although the functional roles of LIMK family kinases have yet to be determined, the unique structural features of these kinases suggest specific roles for these kinases in previously uncharacterized signaling pathways.
During the search for LIMK-related protein kinases, we isolated the cDNA encoding a novel protein kinase. As the mRNA transcripts of this kinase are expressed almost exclusively in the testis of rat and mouse, we termed this novel protein kinase TESK1 for "testis-specific protein kinase." The protein kinase domain of TESK1 is most closely related to those of LIMKs, but the overall domain structure is totally different. In contrast to LIMKs, TESK1 has no N-terminal extension including LIM motifs but does have an N-terminal protein kinase domain, followed by a long C-terminal extension rich in proline resi-dues. Here we report the molecular cloning and sequences of rat and human TESK1 cDNAs and the serine/threonine-specific kinase activity of TESK1 protein transiently expressed in COS cells. The predominant expression of the TESK1 mRNA in testicular germ cells and developmental changes in the expression of TESK1 mRNA in mouse testis indicate an important role of TESK1 at and after the meiotic phase of spermatogenesis.

EXPERIMENTAL PROCEDURES
Isolation and Sequencing of Rat and Human TESK1 cDNAs-Using as a probe the 32 P-labeled 1.9-kb PstI fragment of human LIMK1 cDNA (5), a random-primed rat brain cDNA library in ZAPII (approximately 3 ϫ 10 5 independent phage plaques) was screened, under conditions of low stringency hybridization, as described previously (8). One of the 16 positive clones, rLK-6, was isolated and subcloned into pBluescript (Stratagene), and the cDNA insert was subjected to nucleotide sequence analysis. To obtain full-length cDNA clones, an oligo(dT)-primed rat testis cDNA library in ZAPII (3 ϫ 10 5 independent plaques) was screened with the 32 P-labeled rLK-6 cDNA probe. Of the 24 positive clones detected, 7 positive ones were isolated, subcloned into pBluescript, and subjected to the nucleotide sequence analysis. To isolate the cDNAs encoding human TESK1, an oligo(dT)-primed ZAPII cDNA library of human HepG2 hepatoma cells (5 ϫ 10 5 phage plaques) was screened, using the rLK-6 probe. Two positive clones (hLK3-1 and hLK3-2) were isolated and used for sequencing.
DNA Sequencing-Nucleotide sequences were determined on both strands by the dideoxy chain termination method (9), using Taq polymerase and a dye terminator cycle sequencing kit with a model 370A DNA sequencer (Applied Biosystems). Overlapping cDNA fragments were obtained by sequential exonuclease III digestion (10) or by priming with sequence-specific oligonucleotides.
Preparation of Spermatogenic Cells-Dissociated germ cells were prepared from rat and mouse testes, as described previously (11,12). Briefly, the decapsulated testicles were incubated in bicarbonatebuffered RPMI 1640 (Life Technologies, Inc.) containing 1 mg/ml collagenase at 34°C for 15 min with constant agitation. The dispersed seminiferous tubules were washed three times with RPMI 1640 and treated with 0.25 mg/ml trypsin and 5 g/ml deoxyribonuclease I in RPMI 1640 at 34°C for 15 min. The cell aggregates were washed with RPMI 1640, gently pipetted, filtered through a 74-m nylon mesh, and then centrifuged to obtain an enriched suspension of germ cells. The germ cell suspension of mouse testicles was further purified by unit gravity sedimentation (13), using a 2-4% bovine serum albumin(w/v) gradient, as described (12). The purities of the cell populations of pachytene spermatocytes and round spermatids exceeded 90% in all experiments.
Northern Hybridization-Total RNA was extracted from various tissues or cells by the guanidine thiocyanate procedure (14) followed by CsCl centrifugation, or by the acid guanidine thiocyanate/phenol/chloroform extraction method (15). Poly(A) ϩ RNA was purified by two cycles of Oligotex dT-30 (Rosch) adsorption, according to the manufacturer's instruction. The RNA samples were denatured with glyoxal or formaldehyde, electrophoresed on 1.2% agarose gels, and transferred onto Hybond-N nylon membranes (Amersham Corp.). The blots were probed by 32 P-labeled 1.2-kb rLK-6 or by a 2.0-kb KpnI fragment of rat TESK1 cDNA and analyzed by a BAS2000 Bio-Image Analyzer (Fuji Film), as described previously (5).
Preparation of Antibody-A peptide TK-11 (TPSLQLPGARS) (5 mg), corresponding to the common C-terminal sequence of human and rat TESK1, was coupled to keyhole limpet hemocyanin (10 mg, Calbiochem) by glutaraldehyde. After dialyzing against phosphate-buffered saline (PBS), the resultant conjugate was mixed with an equal volume of Freund's complete adjuvant and inoculated subcutaneously into two rabbits (1 mg each) (16). The rabbits were given a booster every 2 weeks and were bled 1 week later. The IgG fraction was purified from collected antisera with a Protein A-Sepharose (Pharmacia Biotech Inc.) column, and an anti-TESK1 antibody was further purified on a column of Tresyl-activated Sepharose 4B (Pharmacia) coupled with the antigenic TK-11 peptide.
Phosphoamino Acid Analysis-Phosphoamino acid analysis was performed as described (20). The region of the membrane containing the radioactive MBP band was excised and incubated with 6 N HCl at 105°C for 2 h. After removal of the membrane, the hydrolysates were separated by two-dimensional electrophoresis. The 32 P-labeled phosphoamino acids were detected by autoradiography, and a comparison was made with the ninhydrin-stained phosphoamino acid standards.
Fluorescence in Situ Hybridization-Direct R-banding fluorescence in situ hybridization was carried out, using the biotinylated human TESK1 cDNA probe, as described elsewhere (21).

Isolation and Sequences of cDNAs Encoding
Rat and Human TESK1-To search for possible novel members of LIMK family protein kinases, a rat brain cDNA library was screened under conditions of low stringency hybridization, using as a probe the 32 P-labeled cDNA fragment of human LIMK1 (5). Of 16 positive clones, 6 were previously identified to encode rat LIMK1 and LIMK2. 2 Another positive clone, rLK-6, contained the 1.2-kb insert, which encoded a previously unpublished amino acid sequence and was partially homologous to the sequences of LIMK1 and LIMK2. Northern blot analysis revealed that the mRNA probed with rLK-6 insert was preferentially expressed in the rat testis (see Fig. 4A). We therefore screened a rat testis cDNA library, using the rLK-6 cDNA fragment as a probe, and identified 24 positive clones. Nucleotide sequence analysis of several overlapping cDNA clones yielded a 3600-base pair sequence, which has a single long open reading frame encoding a novel protein (termed TESK1) of 628 amino acid residues (Fig.  1A). The initiation ATG codon at nucleotides 1131-1133 resides in the Kozak consensus sequence (22) and is preceded by an ϳ1.1-kb 5Ј-noncoding region, which contains several in-frame stop codons. An ϳ0.6-kb 3Ј-noncoding sequence includes a poly(A) tail and two polyadenylation signals (AATAAA). Some of the isolated clones had a shorter 0.3-kb 3Ј-noncoding sequence with a poly(A) tail 30 -34-bases downstream of a 5Ј-side AATAAA signal. The size of the combined cDNA sequence (3.6 kb) coincides with the size of mRNA, measured by Northern analysis (see Fig. 4A); hence, the cloned cDNAs probably cover a practically full-length sequence of rat TESK1 mRNA.
The overlapping cDNA clones encoding human TESK1 were isolated from a human HepG2 hepatoma cDNA library, using the rat TESK1 cDNA fragment as a probe. Fig. 1B shows the combined 2,452-base pair nucleotide sequence and the predicted 626-amino acid sequence of human TESK1. Compared with the rat cDNA, human TESK1 cDNA contained a relatively shorter 5Ј-noncoding sequence.
Structural Characteristics of TESK1 Protein-The predicted amino acid sequences of rat and human TESK1 share characteristic structural features, composed of an N-terminal protein kinase consensus sequence and a C-terminal extracatalytic sequence highly rich in proline residues. The overall amino acid sequence identity of rat and human TESK1 is 92%, and identities within the protein kinase domain and the C-terminal proline-rich domain are 97% and 90%, respectively. A hydropathy plot analysis of TESK1 showed no hydrophobic segment indicative of a signal sequence or a transmembrane domain, suggesting that TESK1 functions as an intracellular protein. Alignment of the amino acid sequence of the protein kinase domain of TESK1 with known protein kinases revealed that TESK1 shared 17 out of 21 highly conserved residues throughout the protein kinase superfamily (3) (Fig. 2A). In the protein kinase domain, TESK1 is most similar to LIMK1 (49% identity) and LIMK2 (50% identity), while identities to other protein kinases are at most 32%. Phylogenetic analysis of the kinase domains also revealed that TESK1 forms an obvious cluster with LIMK1 and LIMK2 (bootstrap probability; 100%), and they are clearly separated from other known protein kinases on the tree (Fig. 3). Thus, although the overall domain structure of TESK1 is entirely different from those of LIMKs (LIMK1 and LIMK2) (Fig. 2B), TESK1 can be grouped as a distant relative of a LIMK subfamily. In addition, TESK1 and LIMKs are located on the phylogenetic tree adjacent to the activin receptor and Daf-1 within a serine/threonine kinase family. This suggests that the LIMK/TESK1 subfamily belongs to the serine/ threonine kinase subfamily and may be phylogenetically related to the activin receptor/Daf-1 subfamily.
The protein kinase domain is subdivided into 12 subdomains (2, 3). The short sequence motif in subdomain VIB is highly conserved within serine/threonine kinases and within tyrosine kinases. In this region, TESK1 contains an unusual short sequence motif DLTSKN, which does not match the consensus sequence for either serine/threonine kinases (DLKXXN) or tyrosine kinases (DLRAAN or DLAARN). This is also the case of LIMK1 and LIMK2, both of which possess a sequence motif DLNSHN in this region (5,6). Thus, we could not predict the substrate specificity of TESK1 from the sequence in this motif. TESK1 has no basic kinase insert sequence, which is observed between subdomain VII and VIII in LIMK1 and LIMK2 (5,6).
Another notable structural feature of TESK1 is the presence of a proline-rich region at the C terminus. Within the C-terminal 315 and 308 amino acid residues of rat and human TESK1, there are 64 and 63 proline residues, respectively, which correspond to 20% of residues within the C-terminal half. Additionally, the C-terminal half of TESK1 is also rich in Ser, Thr, and Glu residues and might be the region for the rapid protein degradation signal known as a PEST sequence (23).
Expression of TESK1 mRNA in Rat and Mouse Tissues and Human Cell -The tissue distribution of TESK1 gene expression was examined by Northern blot analysis of poly(A) ϩ RNAs from various tissues of adult rat, using as a probe the rat TESK1 cDNA fragment. A major band of TESK1 mRNA, with a size of ϳ3.6 kb, was detected almost exclusively in the testis (Fig. 4A). The relatively broad band of the mRNA may be due to the presence of transcripts derived by alternative use of two polyadenylation signals. A faint band of 2.5 kb was barely detectable in all tissues examined, after longer exposure (data not shown). It remains to be determined whether this 2.5-kb mRNA represents an alternatively spliced transcript of TESK1 or a cross-hybridized one from a closely related gene. The 3.6-kb mRNA of TESK1 was clearly detected in the total cellular RNA of rat spermatogenic cells, as in the case of mRNA of acrosin (12), a serine protease localizing in the sperm acrosome (Fig. 4B). Thus, the TESK1 mRNA was found to be mainly expressed in the testicular germ cells of the adult rat.
A similar pattern of TESK1 gene expression was observed in adult mouse tissues (Fig. 5A). Of 10 different mouse tissues examined, expression of TESK1 mRNA with the size of about 4.0 kb was detected only in the testis. No significant hybridization signal was detectable in other tissues, including the ovary. Expression of TESK1 mRNA was also observed in testicular RNAs from other species, including monkey, dog, guinea pig, and hamster, when the rat TESK1 cDNA fragment was used as a hybridization probe (data not shown). In human cell lines, a faint band of 2.5-kb mRNA, which hybridized to human TESK1 cDNA fragment, was detected in HepG2 hepatoma, A431 epidermoid carcinoma, and Lu99 lung carcinoma cells, while KB epidermoid carcinoma, HL-60 promyelocytic leukemic cells, and THP-1 acute monocytic leukemia cells gave no apparent mRNA hybridization signal (data not shown).
Developmental Change and Distribution of TESK1 mRNA Expression in Mouse Testis-To examine the developmental pattern of TESK1 mRNA accumulation in mouse testis, the

FIG. 4. Expression of TESK1 mRNA in rat tissues and testicular germ cells.
A, tissue distribution of TESK1 mRNA in rat. Poly(A) ϩ RNAs (2 g each) from various tissues of adult rats were subjected to Northern analysis using the rat TESK1 cDNA probe (rLK-6), as described under "Experimental Procedures." B, expression of TESK1 mRNA in rat testicular germ cells. Total RNA (10 g each) from rat whole testis or germ cell-enriched preparations from testis were analyzed using rat TESK1 cDNA probe (left panel) or mouse acrosin cDNA probe (12) (right panel), as described under "Experimental Procedures." Positions of molecular weight markers are indicated on the left. TESK1, Testis-specific Protein Kinase testicular total RNAs from prepuberal (14 -24-day-old) and sexually mature (70-day-old) mice were analyzed by Northern blotting, using rat TESK1 cDNA as a probe. About 4.0-kb TESK1 mRNA was first detectable in the testis at the 18th day of postnatal development. The level of TESK1 mRNA expression increased progressively with postnatal development (Fig.  5B). This expression pattern of the TESK1 gene was apparently consistent with the temporal appearance of haploid round spermatids in the seminiferous epithelium of mouse; approximately 1, 4, and 10% of total spermatogenic cells differentiate into the round spermatids at the 18th, 20th, and 22nd postnatal day, respectively (24). To verify whether or not the TESK1 gene is specifically expressed in haploid male germ cells after meiosis, total RNAs from purified populations of pachytene spermatocytes, round spermatids, and a mixture of elongating spermatids and residual bodies (a residual body fraction) were subjected to Northern blot analysis (Fig. 5C). The TESK1 gene was most abundantly expressed in round spermatids, but a lower level of TESK1 mRNA was present in pachytene spermatocytes and a residual body fraction. Although a very small amount of multinucleate spermatids is usually contaminated in the purified population of the pachytene spermatocytes, it is most likely that expression of the TESK1 gene initiates during meiosis prior to the haploid phase of spermatogenesis and continues through the early stages of spermiogenesis.
Expression and Serine/Threonine Kinase Activity of TESK1 Protein-In view of the unusual sequence of the protein kinase domain of TESK1, it is particularly important to verify the kinase activity and substrate specificity of this protein. The expression plasmid containing the full-length coding region of human TESK1 cDNA was transfected into COS cells, and the expression of TESK1 protein was analyzed by immunoblotting, using anti-TESK1 polyclonal antibodies raised against the synthetic C-terminal 11-amino acid peptide of TESK1. As shown in Fig. 6A, two major immunoreactive bands with estimated molecular masses of about 68 and 80 kDa were detected in COS cells transfected with TESK1 cDNA (COS/TESK1 cells), while no such band was detected in the lysates of COS cells mocktransfected with the vector DNA alone (COS/mock cells). Thus, these bands are thought to be products derived from the ectopically expressed human TESK1 cDNA. A slowly migrating 80-kDa protein, although its apparent molecular mass is significantly higher than the molecular mass predicted from the analysis of TESK1 cDNA, appears to be a primary translation product with a full-length amino acid sequence of TESK1. The high proline content of TESK1 seems to be responsible for the unusually slow migration of the protein on SDS-PAGE, which has been noted for other proline-rich proteins, such as zyxin and Kruppel (38). A rapidly migrating protein with an apparent molecular mass of 68 kDa may be a proteolytically processed product. When lysates of COS/TESK1 cells were immunoprecipitated with an anti-TESK1 antibody, separated on SDS-PAGE, and immunoblotted with the same antibody, protein bands corresponding to 68-and 80-kDa TESK1 proteins appeared (Fig. 6B, lane 1). These bands were not detectable when the lysates were immunoprecipitated with preimmune serum (Fig. 6B, lane 3) or with the anti-TESK1 antibody preincubated with excess amounts of antigenic peptide (Fig. 6B,  lane 4). These findings show the potency and specificity of the anti-TESK1 antibody as a tool for immunoprecipitation.
To assess the kinase activity of TESK1, the anti-TESK1 immunoprecipitates from COS/TESK1 cells were incubated with [␥-32 P]ATP in the presence or absence of substrate protein. No radioactive protein was detectable when the kinase reaction was carried out in the absence of substrate protein, thereby indicating that TESK1 has no autophosphorylating activity (data not shown). On the other hand, MBP and histone, but not enolase, were radiophosphorylated by in vitro kinase reaction (Fig. 7A). Radiophosphorylation of MBP was detected in immunoprecipitates of COS/TESK1 cells with anti-TESK1 antibody, but not in immunoprecipitates of COS/mock cells with anti-TESK1 antibody, or in those of COS/TESK1 cells with preimmune serum (Fig. 7B). Phosphoamino acid analysis of the radiolabeled MBP obtained by in vitro kinase reaction revealed that most of the radioactivity was incorporated into phosphoserine and phosphothreonine, but not into phosphotyrosine (Fig. 7C). Taken together, these results suggest that TESK1 has serine/threonine-specific kinase activity.
Chromosomal Localization of the Human TESK1 Gene-The chromosomal localization of human TESK1 gene was determined by fluorescence in situ hybridization analysis. Metaphase chromosomes with replication bands were hybridized with a biotinylated human TESK1 cDNA probe. The hybridization signals were located on human chromosome band 9p13, and no other hybridization site was observed (Fig. 8). DISCUSSION We isolated cDNAs encoding the rat and human forms of TESK1, a novel non-receptor type protein kinase, which is specifically expressed in the testis. TESK1 contains unique structural features composed of the N-terminal unusual protein kinase domain and the C-terminal proline-rich region. In the protein kinase domain, TESK1 has significant homology with LIMK1 and LIMK2, two related members of a LIM motifcontaining protein kinase subfamily which we recently identified (5,6). Phylogenetic analysis by comparing the kinase domains of TESK1 and other protein kinases revealed that TESK1 is most closely related to LIMKs. Despite the sequence similarity in the protein kinase domains, the extracatalytic domain of TESK1 is not related to those of LIMKs. TESK1 contains the C-terminal proline-rich domain, whereas LIMKs contain the N-terminal two LIM motifs and a Dlg homology region domain (35). Thus, it is likely that the genes for TESK1 and LIMKs evolved by duplication of a common ancestral protein kinase gene, followed by combining with genes encoding respective extracatalytic regions. Diverse extracatalytic domain structures between TESK1 and LIMKs suggest that the physiological functions and regulation mechanisms of TESK1 are distinct from those of LIMKs.
The short sequence motif in subdomain VIB is highly conserved within a serine/threonine kinase family and within a tyrosine kinase family, respectively, and thus is usually used to predict whether the newly identified protein kinase belongs to a serine/threonine-specific kinase or a tyrosine-specific kinase (2, 3). The diagnostic sequence motif for serine/threonine ki-nases is DLKXXN. For tyrosine kinases, the Src family tyrosine kinases contain DLRAAN, while other tyrosine kinases, including receptor-type tyrosine kinases (epidermal growth factor receptor, insulin receptor, etc.) and non-receptor-type tyrosine kinases (Abl, Csk, Syk, etc.) contain DLAARN in subdomain VIB. As we have noted previously (5,6), the protein kinase domains of LIMKs contain an unusual sequence motif, DLN-SHN, in subdomain VIB; hence, we could not predict the substrate specificity of LIMKs from the sequence data. TESK1 has sequence motifs DLTSKN in this region. As TESK1 and LIMKs 3 are now known to have serine/threonine kinase activity, as determined by in vitro kinase assay, DLXX(K/H)N motif in subdomain VIB can be defined as another acceptable sequence motif for serine/threonine kinases. Thus, the basic residue in this motif may significantly contribute to substrate specificity of the protein kinases. While it is known that protein kinases that contain the arginine residue at the third or fifth position of this motif (DLRAAN or DLAARN) have substrate specificity toward tyrosine residues, protein kinases that contain the lysine or histidine residue at the third or fifth position of this motif (DLKXXN or DLXX(K/H)N) might belong to a serine/threonine kinase family. However, the accumulation of data on the sequences and substrate specificity of the protein kinases is needed to support this thesis.
Another notable structural feature of TESK1 is the presence of a large C-terminal proline-rich region. The short stretches of proline-rich sequences (Pro-Pro-X-Pro) are recognized by Src homology 3 (SH3) domains of various signaling proteins, such as Src, Abl, Grb2, p85 subunit of phosphatidylinositol 3-kinase, and phospholipase C␥ (25). As the proline-rich region of TESK1 contains several Pro-Pro-X-Pro motifs, TESK1 may interact with these SH3-containing proteins through the C-terminal proline-rich regions. By analogy to other protein kinases, such as cyclin-dependent kinases, protein kinase activity of TESK1 may be regulated by such protein-protein interactions. The proline-rich domain is also found in the C-terminal region of the protein kinases, SPRK (also called PTK1) and MST (26,27). While these protein kinases have the extracatalytic domain composed of an SH3 domain and two leucine zipper domains in addition to a proline-rich domain, TESK1 has neither the SH3 domain nor the leucine zipper domain. Thus, the intramolecular interaction between a proline-rich domain and an SH3 domain, as suggested for SPRK and MST, is unlikely in the case of TESK1. In addition, the C-terminal half of TESK1 is rich in Ser, Thr, and Glu residues, indicating that this region may function as a PEST protein degradation signal for rapid turnover (23).
Of the rat and mouse tissues examined, the TESK1 mRNA was detected almost exclusively in the testis. Although very faint bands of 2.5-kb mRNA were detectable in other tissues including brain, from which the TESK1 cDNA fragment was originally isolated, it is not known at present whether the 2.5-kb mRNA is the alternatively spliced transcript of TESK1 gene or the transcript derived from a TESK1-related gene. Even if the 2.5-kb mRNA encodes TESK1, the level of TESK1 mRNA expression in the testis was incomparably higher than levels in other tissues. Thus, it is probable that the physiologically important function of TESK1 is the one in the testis. The finding that the TESK1 gene is mainly expressed in testicular germ cells means that the TESK1 probably has a role in spermatogenesis.
Spermatogenesis is a complex process, consisting of spermatogonial proliferation, meiotic division of spermatocytes into spermatids, and spermiogenesis, a morphogenic change of 3 I. Okano and K. Mizuno, unpublished data. TESK1, Testis-specific Protein Kinase spermatids into highly differentiated sperm. These events are synchronized in each seminiferous tubule and are thought to be regulated by numerous gene products at each stage of germ cell differentiation. The molecular mechanisms regulating these events are not well understood. In the present study, we showed the developmentally regulated pattern of expression of the TESK1 mRNA in mouse testis. As the mouse spermatocytes begin to undergo meiosis and to differentiate into haploid round spermatids on the 18th postnatal day, the expression of TESK1 mRNA in the mouse testis during development almost paralleled the appearance of haploid round spermatids. Cell fractionation studies of mouse spermatogenic cells revealed that the TESK1 mRNA is present mainly in round spermatids and at a lesser but significant level in the pachytene spermatocytes and residual body fractions. Thus, it is likely that the expression of TESK1 gene initiates in spermatocytes at the meiotic phase of spermatogenesis and the mRNA accumulates in the round spermatids. Taken together these findings suggest that the TESK1 gene product probably plays an important role in meiotic stages and/or the early stages of spermiogenesis. To precisely define the expression of TESK1 mRNA and the protein in testicular cells at each specific stage of spermatogenesis, in situ localization and immunohistochemical studies are ongoing in our laboratory.
Several novel protein kinases or isomeric forms of known protein kinases are specifically expressed in the testis (28 -34). Mak (male germ cell-associated protein kinase) distantly related to Cdc2 kinase is expressed exclusively in testicular germ cells at and after meiosis (28,29). TSK-1 is expressed exclusively in the testis and has sequence similarity to human Rac protein kinase-␤ and yeast SNF-1, Nim-1, KIN-1, and KIN-2 (30). MAST205 (205-kDa microtubule-associated serine/threonine kinase) was identified as the testis-specific protein kinase most closely related to members of cAMP-dependent protein kinase and protein kinase C subfamily, and is associated with microtubules in vitro and co-localized with the spermatid manchette (31). Expression of testis-specific isoforms of casein kinase I, c-Abl, and Fer have been reported (32)(33)(34). Structure of TESK1 is not related to any of these kinases, except for similarity (25-30% identity) in the kinase catalytic domain. These protein kinases are thought to be involved in the regulation of the certain stage of spermatogenesis, based on their testis-specific and stage-specific expression, but the physiological functions of these protein kinases have yet to be clearly defined.
In summary, TESK1 is a novel serine/threonine kinase that belongs to a second subgroup of a LIMK/TESK family of protein kinases. Since TESK1 has unique structural features not related to known protein kinases, it may be involved in previously uncharacterized signaling pathways. The testicular germ cell-specific expression and the developmental pattern of ex-pression of TESK1 gene suggest that TESK1 probably has an important role in spermatogenesis.