NORMAL GENOMIC

• Sato T, Katagiri K, Kojima K, Komeya M, Yao M, Ogawa T
• In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.
• PLoS One. 2015; 10: 130171-130171
• Display abstract

• Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

• Sato S, Maeda C, Hattori N, Yagi S, Tanaka S, Shiota K
• DNA methylation-dependent modulator of Gsg2/Haspin gene expression.
• J Reprod Dev. 2011; 57: 526-33
• Display abstract

• The Gsg2 (Haspin) gene encodes a serine/threonine protein kinase and is predominantly expressed in haploid germ cells. In proliferating somatic cells, Gsg2 is shown to be expressed weakly but plays an essential role in mitosis. Although the Gsg2 minimal promoter recognized by the spermatogenic cell-specific nuclear factor(s) has been found, to date, the molecular mechanism that differentially controls Gsg2 expression levels in germ and somatic cells remains to be sufficiently clarified. In this study, we analyzed the DNA methylation status of the upstream region containing the Gsg2 promoter. We found a tissue-dependent and differentially methylated region (T-DMR) upstream (-641 to -517) of the authentic promoter that is hypomethylated in germ cells but hypermethylated in other somatic tissues. Profiling of Gsg2 expression and DNA methylation status at the T-DMR in spermatogenic cells indicated that the hypomethylation of the T-DMR is maintained during spermatogenesis. Using the reporter assay, we also demonstrated that DNA methylation at the T-DMR of Gsg2 reduced the promoter activity by 60-80%, but did not fully suppress it. Therefore, the T-DMR functions as a modulator in a DNA methylation-dependent manner. In conclusion, Gsg2 is under epigenetic control.

• Cuny GD et al.
• Structure-activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors.
• Bioorg Med Chem Lett. 2010; 20: 3491-4
• Display abstract

• Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure-activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC50 <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC50 <400 nM) with a 5.4-fold selectivity over haspin was also identified.

• Kupinski AP, Muller-Reichert T, Eckmann CR
• The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation.
• Dev Biol. 2010; 344: 758-71
• Display abstract

• Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.

• Farkas R et al.
• Exploring some of the physico-chemical properties of the LAMMER protein kinase DOA of Drosophila.
• Fly (Austin). 2009; 3: 130-42
• Display abstract

• Members of the highly conserved LAMMER family of protein kinases have been described in all eukaryotes. LAMMER kinases possess markedly similar peptide motifs in their kinase catalytic subdomains that are responsible for phosphotransfer and substrate interaction, suggesting that family members serve similar functions in widely diverged species. This hypothesis is supported by their phosphorylation of SR and SR-related proteins in diverged species. Here we describe a 3-dimensional homology model of the catalytic domain of DOA, a representative LAMMER kinase, encoded by the Drosophila locus Darkener of apricot (Doa). Homology modeling of DOA based on a Sky1p template revealed a highly conserved structural framework within conserved core regions. These adopt typical kinase folding like that of other protein kinases. However, in contrast to Sky1p, some structural features, such as those in helix alphaC suggest that the DOA kinase is not a constitutively active enzyme but requires activation. This may occur by phosphorylation within an activation loop that forms a broad turn and in which interactions between the side chains occur across the loop. The fold of the activation loop is stabilized through interactions with residues in the C-terminal tail, which is not part of the conserved kinase core and is variable among protein kinases. Immediately following the activation loop in the segment between the beta9 sheet and helix alphaF is a P + 1 loop. The electrostatic surface potential of the DOA substrate-binding groove is largely negative, as it is in other known SR protein kinases, suggesting that DOA substrates must be basic. All differences between D. melanogaster and other Drosophila species are single amino acid changes situated in regions outside of any alpha-helices or beta-sheets, and after modeling these had absolutely no visible effect on protein structure. The absence of evolved amino acid changes among 12 Drosophila species that would cause at least predictable changes in DOA structure indicate that evolution has already selected evolved mutations for having minimal effect on kinase structure.

• Nakajima Y, Tyers RG, Wong CC, Yates JR 3rd, Drubin DG, Barnes G
• Nbl1p: a Borealin/Dasra/CSC-1-like protein essential for Aurora/Ipl1 complex function and integrity in Saccharomyces cerevisiae.
• Mol Biol Cell. 2009; 20: 1772-84
• Display abstract

• The Aurora kinase complex, also called the chromosomal passenger complex (CPC), is essential for faithful chromosome segregation and completion of cell division. In Fungi and Animalia, this complex consists of the kinase Aurora B/AIR-2/Ipl1p, INCENP/ICP-1/Sli15p, and Survivin/BIR-1/Bir1p. A fourth subunit, Borealin/Dasra/CSC-1, is required for CPC targeting to centromeres and central spindles and has only been found in Animalia. Here we identified a new core component of the CPC in budding yeast, Nbl1p. NBL1 is essential for viability and nbl1 mutations cause chromosome missegregation and lagging chromosomes. Nbl1p colocalizes and copurifies with the CPC, and it is essential for CPC localization, stability, integrity, and function. Nbl1p is related to the N-terminus of Borealin/Dasra/CSC-1 and is similarly involved in connecting the other CPC subunits. Distant homology searching identified nearly 200, mostly unannotated, Borealin/Dasra/CSC-1-related proteins from nearly 150 species within Fungi and Animalia. Analysis of the sequence of these proteins, combined with comparative protein structure modeling of Bir1p-Nbl1p-Sli15p using the crystal structure of the human Survivin-Borealin-INCENP complex, revealed a striking structural conservation across a broad range of species. Our biological and computational analyses therefore establish that the fundamental design of the CPC is conserved from Fungi to Animalia.

• Patnaik D, Jun Xian, Glicksman MA, Cuny GD, Stein RL, Higgins JM
• Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay.
• J Biomol Screen. 2008; 13: 1025-34
• Display abstract

• Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.

• Goldberg JM et al.
• The dictyostelium kinome--analysis of the protein kinases from a simple model organism.
• PLoS Genet. 2006; 2: 38-38
• Display abstract

• Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase-like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system.

• Maduzia LL et al.
• C. elegans serine-threonine kinase KIN-29 modulates TGFbeta signaling and regulates body size formation.
• BMC Dev Biol. 2005; 5: 8-8
• Display abstract

• BACKGROUND: In C. elegans there are two well-defined TGFbeta-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFbeta signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. RESULTS: kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. CONCLUSION: kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFbeta outputs, such as tail morphogenesis.

• Dai J, Sultan S, Taylor SS, Higgins JM
• The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment.
• Genes Dev. 2005; 19: 472-88
• Display abstract

• Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Furthermore we find that haspin, a member of a distinctive group of protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis. Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays progression through early mitosis. This work reveals a new kinase involved in composing the histone code and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function during mitosis and meiosis.

• Takahashi T et al.
• Rosbin: a novel homeobox-like protein gene expressed exclusively in round spermatids.
• Biol Reprod. 2004; 70: 1485-92
• Display abstract

• Mammalian spermiogenesis is a complex process occurring in a highly coordinated fashion within the seminiferous tubules. To elucidate the molecular mechanisms controlling haploid germ cell differentiation, we have isolated haploid germ cell- specific cDNA clones from a subtracted cDNA library of mouse testis. One of these cDNAs, Rosbin, is 3.2 kilobases (kb) long and has an open reading frame of 2385 nucleotides encoding a putative protein of 795 amino acid residues. A computer-mediated homology search revealed that it contained a domain similar to that of homeobox genes. Northern blot analysis revealed a 3.2-kb mRNA expressed exclusively in male germ cells. Transcription of the Rosbin gene was not observed in prepubertal testis but became detectable after Day 23. By Western blot analysis the protein encoded by this gene had a molecular mass of 89 kDa, expressing specifically in the testis and localized to the nucleus of stages IV-VIII haploid round spermatids, predominantly at stages VII-VIII of spermatogenesis. ROSBIN is associated with and is most likely phosphorylated by protein kinase A. We suggest that it plays an important role in transcriptional regulation in haploid germ cells.

• Wang Y et al.
• TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1.
• Biochem Biophys Res Commun. 2004; 323: 9-16
• Display abstract

• The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted alpha-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway.

• de Graaf K et al.
• Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain: phosphorylation by DYRK1A and colocalization with splicing factors.
• J Biol Chem. 2004; 279: 4612-24
• Display abstract

• A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

• Pike BL, Tenis N, Heierhorst J
• Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.
• J Biol Chem. 2004; 279: 39636-44
• Display abstract

• Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

• Parker N, Porter AC
• Identification of a novel gene family that includes the interferon-inducible human genes 6-16 and ISG12.
• BMC Genomics. 2004; 5: 8-8
• Display abstract

• BACKGROUND: The human 6-16 and ISG12 genes are transcriptionally upregulated in a variety of cell types in response to type I interferon (IFN). The predicted products of these genes are small (12.9 and 11.5 kDa respectively), hydrophobic proteins that share 36% overall amino acid identity. Gene disruption and over-expression studies have so far failed to reveal any biochemical or cellular roles for these proteins. RESULTS: We have used in silico analyses to identify a novel family of genes (the ISG12 gene family) related to both the human 6-16 and ISG12 genes. Each ISG12 family member codes for a small hydrophobic protein containing a conserved ~80 amino-acid motif (the ISG12 motif). So far we have detected 46 family members in 25 organisms, ranging from unicellular eukaryotes to humans. Humans have four ISG12 genes: the 6-16 gene at chromosome 1p35 and three genes (ISG12(a), ISG12(b) and ISG12(c)) clustered at chromosome 14q32. Mice have three family members (ISG12(a), ISG12(b1) and ISG12(b2)) clustered at chromosome 12F1 (syntenic with human chromosome 14q32). There does not appear to be a murine 6-16 gene. On the basis of phylogenetic analyses, genomic organisation and intron-alignments we suggest that this family has arisen through divergent inter- and intra-chromosomal gene duplication events. The transcripts from human and mouse genes are detectable, all but two (human ISG12(b) and ISG12(c)) being upregulated in response to type I IFN in the cell lines tested. CONCLUSIONS: Members of the eukaryotic ISG12 gene family encode a small hydrophobic protein with at least one copy of a newly defined motif of approximately 80 amino-acids (the ISG12 motif). In higher eukaryotes, many of the genes have acquired a responsiveness to type I IFN during evolution suggesting that a role in resisting cellular or environmental stress may be a unifying property of all family members. Analysis of gene-function in higher eukaryotes is complicated by the possibility of functional redundancy between family-members. Genetic studies in organisms (e.g. Dictyostelium discoideum) with just one family member so far identified may be particularly helpful in this respect.

• Kuroda TS et al.
• A novel nucleolar protein, PAPA-1, induces growth arrest as a result of cell cycle arrest at the G1 phase.
• Gene. 2004; 340: 83-98
• Display abstract

• We have identified a novel nucleolar protein, PAP-1-associated protein-1 (PAPA-1), after screening the interacting proteins with Pim-1-associated protein-1 (PAP-1), a protein that is a phosphorylation target of Pim-1 kinase. PAPA-1 comprises 345 amino acids with a basic amino-acid cluster. PAPA-1 was found to be localized in the nucleolus in transfected HeLa cells, and the lysine/histidine cluster was essential for nucleolar localization of PAPA-1. PAPA-1 protein and mRNA expression decreased upon serum restimulation of starvation-synchronized cells, which displayed maximum level of PAPA-1 expression at G0 and early G1 phase of the cell cycle. Ectopic expression of PAPA-1 induced growth suppression of cells, and the effect was dependent on its nucleolar localization in established HeLa cell lines that inducibly express PAPA-1 or its deletion mutant under the control of a tetracycline-inducible promoter. Furthermore, when PAPA-1-inducible HeLa cells were synchronized by thymidine, colcemid or mimosine, and then PAPA-1 was expressed, the proportion of cells at the G1 phase was obviously increased. These results suggest that PAPA-1 induces growth and cell cycle arrests at the G1 phase of the cell cycle.

• Madec E et al.
• Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis.
• J Mol Biol. 2003; 330: 459-72
• Display abstract

• We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.

• Tanaka H, Iguchi N, Egydio de Carvalho C, Tadokoro Y, Yomogida K, Nishimune Y
• Novel actin-like proteins T-ACTIN 1 and T-ACTIN 2 are differentially expressed in the cytoplasm and nucleus of mouse haploid germ cells.
• Biol Reprod. 2003; 69: 475-82
• Display abstract

• We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and Mr 43.1 x 10(3) and Mr 47.2 x 10(3), respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.

• Cziferszky A, Seiboth B, Kubicek CP
• The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1.
• Fungal Genet Biol. 2003; 40: 166-75
• Display abstract

• In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.

• Ramos MA, Sanchez-Lopez R, Olvera F, Alagon A
• Entamoeba histolytica genomic organization: identification, structure, and phylogenetic relationship of two serine-threonine protein kinases.
• Exp Parasitol. 2002; 100: 135-9

• Yan W, Burns KH, Ma L, Matzuk MM
• Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein.
• Mech Dev. 2002; 118: 233-9
• Display abstract

• Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) which were unique to ovary, testis, and egg libraries. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA encodes a novel protein of 341 amino acids with a nuclear localization signal. The carboxyl-terminus of the protein contains three C2H2 zinc fingers, and the NH(2)-terminus is proline and serine-rich. Based on the conserved zinc finger motifs, we have termed this novel protein as zinc finger protein 393 (ZFP393). Northern blot and RT-PCR analyses revealed that Zfp393 mRNA was exclusively expressed in testis and ovary. The expression sites were further localized by in situ hybridization to step 3-8 spermatids in testis and growing oocytes in ovary. The Zfp393 gene consists of three exons spanning approximately 8 kb on the distal part of mouse chromosome 4. The carboxyl-terminal zinc finger region is highly homologous to several zinc finger-containing proteins, but no proteins were found to share sequence similarity with the NH(2)-terminal region of ZFP393. Genomic database mining and Southern blot analysis indicate that Zfp393 is a single copy gene. We hypothesize that ZFP393 functions as a germ cell-specific transcription factor that plays important roles in spermatid differentiation and oocyte development.

• Egydio de Carvalho C, Tanaka H, Iguchi N, Ventela S, Nojima H, Nishimune Y
• Molecular cloning and characterization of a complementary DNA encoding sperm tail protein SHIPPO 1.
• Biol Reprod. 2002; 66: 785-95
• Display abstract

• Formation of the tail in developing sperm is a complex process involving the organization of the axoneme, transport of periaxonemal proteins from the cytoplasm to the tail, and assembly of the outer dense fibers and fibrous sheath. Although detailed morphological descriptions of these events are available, the molecular mechanisms remain to be fully elucidated. We have isolated a new gene, named shippo 1, from a haploid germ cell-specific cDNA library of mouse testis, and also its human orthologue (h-shippo 1). The isolated cDNA is 1.2 kilobases long, carrying a 762-base pair open reading frame that encodes SHIPPO 1, a sperm protein predicted to consist of 254 amino acids. The amino acid sequence includes 6 Pro-Gly-Pro repeats, which are also present in the human orthologue protein (hSHIPPO 1) as well as in 2 other newly reported proteins of Drosophila melanogaster. Transcription of shippo 1 is exclusively observed in haploid germ cells. Antibody raised against SHIPPO 1 identified a testis-specific M(r) 32 x 10(-3) band in Western blot analysis. The protein was further localized in the flagella of the elongated spermatids and along the entire length of the tail in mature sperm. SHIPPO 1 in sperm is resistant to treatment with nonionic detergents and coextracted with the cytoskeletal core proteins of the mouse sperm tail.

• Ostvold AC, Norum JH, Mathiesen S, Wanvik B, Sefland I, Grundt K
• Molecular cloning of a mammalian nuclear phosphoprotein NUCKS, which serves as a substrate for Cdk1 in vivo.
• Eur J Biochem. 2001; 268: 2430-40
• Display abstract

• We have isolated and characterized a cDNA encoding a mammalian nuclear phosphoprotein NUCKS, previously designated P1. Molecular analyses of several overlapping and full-length cDNAs from HeLa cells and rat brain revealed a protein with an apparent molecular mass of 27 kDa in both species. The deduced amino-acid sequences are highly conserved between human and rodents, but show no homology with primary structures in protein databases or with translated sequences of cDNAs in cDNA databanks. Although the protein has some features in common with the high mobility group proteins HMGI/Y, attempts to find a putative protein family by database query using both sequence alignment methods and amino-acid composition have failed. Northern blot analyses revealed that human and rat tissues contain three NUCKS transcripts varying in size from 1.5 to 6.5 kb. All human and rat tissues express the gene, but the level of transcripts varies among different tissues. Circular dichroism analysis and secondary structure predictions based on the amino-acid sequence indicate a low level of alpha helical content and substantial amounts of beta turn structures. The protein is phosphorylated in all phases of the cell cycle and exhibits mitosis-specific phosphorylation of threonine residues. Phosphopeptide mapping and back-phosphorylation experiments employing NUCKS from HeLa interphase and metaphase cells show that the protein is phosphorylated by Cdk1 during mitosis of the cell cycle.

• Tan M, Liang A, Brunen-Nieweler C, Heckmann K
• Programmed translational frameshifting is likely required for expressions of genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus.
• J Eukaryot Microbiol. 2001; 48: 575-82
• Display abstract

• Three macronuclear genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus syngen 1 were isolated and sequenced. All three deduced gene products share significant properties with a group of recently identified nuclear serine/threonine protein kinases named Ndr. The three predicted proteins contain the twelve conserved catalytic subdomains of protein kinases and 22 near universally-conserved amino acids residues that are characteristic of serine/threonine protein kinases. In addition, there is an approximately 30 amino acid-peptide insertion between subdomains VII and VIII that contains a potential nuclear localization signal. Sequence analysis suggests that expression of the Eondr2 gene requires a + 1 programmed translational frameshift for its translation. Comparison of the deduced EoNdr2 with other known Ndr protein kinases implies that a + 1 ribosomal frameshift occurs at the motif AAATAA.

• Orstavik S et al.
• Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95.
• Biol Cell. 2000; 92: 27-37
• Display abstract

• Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.

• Westbrook VA et al.
• Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa.
• Biol Reprod. 2000; 63: 469-81
• Display abstract

• Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

• Johnston AM, Naselli G, Gonez LJ, Martin RM, Harrison LC, DeAizpurua HJ
• SPAK, a STE20/SPS1-related kinase that activates the p38 pathway.
• Oncogene. 2000; 19: 4290-7
• Display abstract

• We have cloned a member of the STE20/SPS1 protein kinase family from a transformed rat pancreatic beta cell line. SPAK (STE20/SPS1-related, proline alanine-rich kinase) belongs to the SPS1 subfamily of STE20 kinases and is highly conserved between species. SPAK is expressed ubiquitously, although preferentially in brain and pancreas. Biochemical characterization of SPAK catalytic activity demonstrates that is a serine/threonine kinase that can phosphorylate itself and an exogenous substrate in vitro. SPAK is immunoprecipitated from transfected mammalian cells as a complex with another, as yet uncharacterized, serine/threonine kinase which is capable of phosphorylating catalytically-inactive SPAK and myelin basic protein in an in vitro kinase assay. SPAK specifically activates the p38 pathway in cotransfection assays. Like MST1 and MST2, SPAK contains a putative caspase cleavage site at the junction of the catalytic domain and the C-terminal region. Full-length SPAK is expressed in the cytoplasm in transfected cells, while a mutant corresponding to caspase-cleaved SPAK is expressed predominantly in the nucleus. The similarity of SPAK to other SPS1 family members, its ability to activate the p38 pathway, in addition to its putative caspase cleavage site, provide evidence that SPAK may act as a novel mediator of stress-activated signals. Oncogene (2000) 19, 4290 - 4297

• Inouye S et al.
• A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium.
• Microb Comp Genomics. 2000; 5: 103-20
• Display abstract

• Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

• Sillje HH, Takahashi K, Tanaka K, Van Houwe G, Nigg EA
• Mammalian homologues of the plant Tousled gene code for cell-cycle-regulated kinases with maximal activities linked to ongoing DNA replication.
• EMBO J. 1999; 18: 5691-702
• Display abstract

• The Tousled (TSL) gene of the plant Arabidopsis thaliana encodes a serine/threonine kinase that is essential for proper flower development. Here we report the cloning and characterization of two human putative homologues of the Arabidopsis TSL gene, termed TLK1 and TLK2 (Tousled-like kinase). At the protein level, the two human Tlks share 84% sequence similarity with each other and almost 50% with Arabidopsis Tsl. Furthermore, nuclear localization signals and predicted coiled-coil regions are conserved in the N-terminal domains of all three kinases. The mammalian Tlks share several functional properties with plant Tsl, including a broad expression, a propensity to dimerize and autophosphorylate, and a preference for similar substrates. Most interestingly, human Tlks are cell-cycle-regulated enzymes, displaying maximal activities during S phase. Whereas protein levels are virtually constant throughout the cell cycle, both Tlks appear to be regulated by cell-cycle-dependent phosphorylation. Drug-induced inhibition of DNA replication causes a rapid loss of Tlk activity, indicating that Tlk function is tightly linked to ongoing DNA replication. These findings provide the first biochemical clues as to the possible molecular functions of Tlks, a highly conserved family of kinases implicated in the development of multicellular organisms.

• Rosok O, Pedeutour F, Ree AH, Aasheim HC
• Identification and characterization of TESK2, a novel member of the LIMK/TESK family of protein kinases, predominantly expressed in testis.
• Genomics. 1999; 61: 44-54
• Display abstract

• In this study we present the cDNA sequence of a novel putative protein kinase, denoted TESK2. The open reading frame of TESK2 encodes a putative 555-amino-acid protein, including a protein kinase consensus sequence in the N-terminal half. The protein kinase domain of TESK2 is structurally similar to the kinase domain of the protein serine/threonine kinase TESK1 (64% identity) and to those of the LIMK1 and LIMK2 kinases (42 and 39% identity, respectively). TESK2, together with TESK1, constitutes a second subgroup of the LIMK/TESK family of protein kinases, as revealed by phylogenetic analysis of the protein kinase domains. Chromosomal localization of human TESK2 was assigned to 1p32. Expression analysis of human TESK2 revealed a single mRNA species of 3.0 kb predominantly expressed in testis and prostate and low expression in most other tissues examined. Rat testicles expressed a single species of TESK2 mRNA of approximately 3.5 kb. However, the transcript was first detectable in rat testis after day 30 of postnatal development and was predominantly expressed in round spermatids. These observations suggest that TESK2 plays an important role in spermatogenesis.

• Tanaka K, Nigg EA
• Cloning and characterization of the murine Nek3 protein kinase, a novel member of the NIMA family of putative cell cycle regulators.
• J Biol Chem. 1999; 274: 13491-7
• Display abstract

• We have cloned and characterized murine Nek3 (NIMA-related kinase 3), a novel mammalian gene product structurally related to the cell cycle-regulatory kinase NIMA of Aspergillus nidulans. By RNase protection, low levels of Nek3 expression could be detected in all organs examined, regardless of proliferative index. In contrast to Nek1 and Nek2, Nek3 levels were not particularly elevated in either the male or the female germ line. Nek3 levels showed at most marginal variations through the cell cycle, but they were elevated in G0-arrested, quiescent fibroblasts. Furthermore, no cell cycle-dependent changes in Nek3 activity could be detected, and no effects upon cell cycle progression could be observed upon antibody microinjection or overexpression of either wild-type or catalytically inactive Nek3. Finally, Nek3 was found to be a predominantly cytoplasmic enzyme. These data indicate that Nek3 differs from previously characterized Neks with regard to all parameters investigated, including organ specificity of expression, cell cycle dependence of expression and activity, and subcellular localization. Hence, the structural similarity between mammalian Neks may not necessarily be indicative of a common function, and it is possible that some members of this kinase family may perform functions that are not directly related to cell cycle control.

• Morales CR, Wu XQ, Hecht NB
• The DNA/RNA-binding protein, TB-RBP, moves from the nucleus to the cytoplasm and through intercellular bridges in male germ cells.
• Dev Biol. 1998; 201: 113-23
• Display abstract

• The genetically haploid spermatids are functionally diploid as a result of the sharing of gene products through their intercellular bridges. This movement of molecules among haploid cells is crucial for the production of spermatozoa since numerous essential proteins are encoded on sex chromosomes. Testis-brain RNA-binding protein (TB-RBP) is a single-stranded DNA- and RNA-binding protein prominent in the nuclei and cytoplasm of specific stages of differentiating male germ cells. Here we present evidence that TB-RBP moves from the nucleus to the cytoplasm and through intercellular bridges of male germ cells. Based on its RNA-binding capabilities, we propose a role for TB-RBP in the distribution of equal amounts of mRNAs in haploid male germ cells.

• Halford NG, Hardie DG
• SNF1-related protein kinases: global regulators of carbon metabolism in plants?
• Plant Mol Biol. 1998; 37: 735-48

• Ogawa K, Wakayama A, Kunisada T, Orii H, Watanabe K, Agata K
• Identification of a receptor tyrosine kinase involved in germ cell differentiation in planarians.
• Biochem Biophys Res Commun. 1998; 248: 204-9
• Display abstract

• To investigate external signals involved in germ cell differentiation from somatic stem cells, we have tried to identify protein kinases whose expression is regulated during the process of sexualization of asexual-state planarians. It is known that in planarians germ cells differentiate from totipotent somatic stem cells called "neoblasts" during sexualization. As a first step, we have isolated twelve protein kinase genes from cDNAs of sexual-state planarians, including three non-receptor tyrosine kinases, three receptor-tyrosine kinases and three non-receptor serine/threonine kinases, and then analyzed their expression patterns during sexualization. One of them, the DjPTK1 gene, is specifically expressed in germ cells of sexual-state planarians. DjPTK1-positive cells were also detected in the mesenchymal space during the process of sexualization, and it appears that these cells migrate to the dorsal side and then differentiate into spermatogonia/spermatocytes in testis. Sequence analysis indicated that the DjPTK1 gene encodes a receptor protein tyrosine kinase belonging to the FGFR/PDGF family. These results suggest that a receptor tyrosine kinase system may be involved both at an early stage of germ cell differentiation and in a step of germ cell maturation in planarians.

• Zelko I, Kobayashi R, Honkakoski P, Negishi M
• Molecular cloning and characterization of a novel nuclear protein kinase in mice.
• Arch Biochem Biophys. 1998; 352: 31-6
• Display abstract

• We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers. The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase. The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s). This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25. The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei. Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver. These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily.

• Osada H et al.
• Subcellular localization and protein interaction of the human LIMK2 gene expressing alternative transcripts with tissue-specific regulation.
• Biochem Biophys Res Commun. 1996; 229: 582-9
• Display abstract

• In our efforts to explore possible roles of proteins with a LIM domain, which is a cysteine-rich Zinc-binding motif, in differentiation and oncogenesis in the lung, we have cloned a human LIMK2 gene and identified two alternative transcripts, LIMK2a and LIMK2b, which are probably due to variation in transcriptional initiation. The former encodes a protein containing two LIM domains, a PDZ domain, and a kinase domain, while the latter has only one and half LIM domains. The predominance of the two transcripts appears to be regulated in a tissue-specific manner. Alteration of the regulation is also observed in some cancer cell lines. Transfection studies have shown an association of 63-kDa and 58-kDa proteins with the LIMK2a and LIMK2b protein; the former is distributed in the cytoplasm and nucleus and the latter occurs mainly in the cytoplasm and is scarcely translocated to the nucleus. In contrast, a truncated LIMK2-Kinase has a nuclear location, not showing the protein association.

• Walden PD, Cowan NJ
• A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.
• Mol Cell Biol. 1993; 13: 7625-35
• Display abstract

• To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.