Differential expression pattern of the novel serine⁄ threonine kinase, STK33, in mice and men Alejandro O. Mujica 1 *, Bastienne Brauksiepe 1 *, Sigrid Saaler-Reinhardt 2 , Stefan Reuss 3 and Erwin R. Schmidt 1 1 Institute of Molecular Genetics, Johannes Gutenberg-University, Mainz, Germany 2 Institute of Genetics, Johannes Gutenberg-University, Mainz, Germany 3 Department of Anatomy and Cell Biology, Johannes Gutenberg-University, Mainz, Germany The serine ⁄ threonine kinase 33 gene (STK33 ⁄ Stk33) was identified by comparative sequencing of human chromosome region 11p15.3 and its syntenic region in mouse chromosome 7 [1,2]. Chromosome 11p15.3 is a gene-rich region of clinical importance because several human diseases, including predisposition for some types of cancer, have been mapped there [3,4]. It has also been associated with several defects and malig- nancies, such as the Beckwith–Wiedemann syndrome, haemoglobinopathies, Long QT syndrome (Ward– Romano Syndrome), insulin-dependent diabetes mellitus I, Usher syndrome 1C, T-cell leukaemia, hypoparathy- roidism and Nieman–Pick disease type A and B (reviewed in [5]) as well as different types of cancer in urinary bladder, ovary, testis, breast and lung [6,7]. By phylogenetic analysis, the STK33 ⁄ Stk33 protein was classified as a member of the Ca 2+ ⁄ calmodulin dependent kinases family (CAMK) which was subse- quently confirmed by the human and mouse kinome catalogues [1,8–10]. Related members of the CAMK family of serine ⁄ threonine kinases have been associated with a variety of biological functions through regula- tion of transcription by, for example, phosphorylating cAMP response element-binding transcription factor [11–13]. While CAMK1 and CAMK2 are expressed ubiquitously [14], CAMK4 is differentially expressed in certain neural tissues, T cells and testis [13]. In mice, Camk2 and Camk4 expression has been associated with functions as diverse as spermatogenesis, memory formation and cardiac hypertrophy and heart failure Keywords serine ⁄ threonine kinase, spermatogenesis, STK33 antibody, STK33 expression Correspondence E. R. Schmidt, Institute of Molecular Genetics, Johannes Gutenberg-University, J J. Becherweg 32, D-55099 Mainz, Germany Fax: +49 6131 3925346 Tel: +49 6131 3925748 E-mail: eschmidt@uni-mainz.de *These authors contributed equally to the paper (Received 6 May 2005; revised 2 August 2005, accepted 3 August 2005) doi:10.1111/j.1742-4658.2005.04900.x Serine ⁄ threonine kinase 33 (STK33 ⁄ Stk33) is a recently discovered gene whose inferred amino acid sequence translation displays characters typical for a calcium ⁄ calmodulin dependent kinase (CAMK). In this study we ana- lysed the STK33 ⁄ Stk33 RNA and protein distribution and the localization of the protein. The STK33 ⁄ Stk33 expression pattern resembles those of some related members of the CAMK group. STK33 ⁄ Stk33 displays a non- ubiquitous and, in most tissues, low level of expression. It is highly expressed in testis, particularly in cells from the spermatogenic epithelia. Moreover, significant expression is detected in lung epithelia, alveolar macrophages, horizontal cells in the retina and in embryonic organs such as heart, brain and spinal cord. A possible role of STK33 ⁄ Stk33 in spermato- genesis and organ ontogenesis is discussed. Abbreviations STK33 ⁄ Stk33, human and mouse serine ⁄ threonine kinase proteins; STK33 ⁄ Stk33: human and mouse serine ⁄ threonine kinase genes; CAMK: Ca 2+ ⁄ calmodulin dependent kinases group; CAMK1 ⁄ Camk1, CAMK2 ⁄ Camk2, CAMK4 ⁄ Camk4: genes for Ca 2+ ⁄ calmodulin dependent kinases I, II and IV from human (in capitals) and mouse; FCS, fetal calf serum; dpp, days postpartum; MTE, multiple tissue expression; ISH, in situ hybridization; LSM, laser scanning microscopy; Hsa, Homo sapiens; Mmu, Mus musculus. 4884 FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS [15–21]. Certain alternative splicing variants of CAMK2 were found to be expressed preferentially in tumour cells [22] and the Camk2d isoform is downreg- ulated in both human and mouse tumour cells [23]. CAMK4 expression has also been associated with epi- thelial ovarian cancer [24]. Alternative splicing may also explain apparently contradicting results from Camk4 knockout experiments: mice void of Camk4, as a consequence of disrupted promoter structure and exons I and II, exhibit impaired follicular development and ovulation [25] and impaired spermiogenesis in males [26]. On the other hand, mice defective in Camk4 generated by disrupting exon III and hence still able to produce the shorter alternative splice transcript for calspermin, have shown neither spermatogenesis dysfunction nor infertility [27]. The PhKgT gene (phos- phorylase kinase, testis ⁄ liver, gamma-2) which displays a close relationship with STK33 in phylogenetic analy- ses, originally found to be expressed mainly in testis [28], has been shown to be associated with hepatic disorders [29,30]. DAP-kinases are serine ⁄ threonine kinases involved in apoptosis that phosphorylate myo- sin light chains in a calmodulin-dependent way and are associated with the cytoskeleton [31]. The sequence of STK33 ⁄ Stk33 is divergent enough from other kinase genes so that corresponding EST entries are unequivocally identified [1] and examination of the human and mouse genomes suggests that STK33 and Stk33 are single-copy genes [1,8,10]. The human chromosome 19 harbours a nonexpressed STK33 pseudogene [8] but no Stk33 pseudogene is detected in the mouse genome [10]. The NCBI’s Uni- Gene [32] human build no. 184 contains 106 EST ent- ries for the STK33 cluster, and the murine build no. 174 contains 50 for Stk33. This reflects significantly lower total expression than, for example, housekeeping genes such as GAPDH (16014 human EST ⁄ 1128 mouse EST) or b-actin (15776 ⁄ 4559). STK33 ⁄ Stk33 EST counts are similar to other CAMKs (CAMK1: 133 ⁄ 148, CAMK2A: 141 ⁄ 129, CAMK4:83⁄ 177) and their distribution suggests a nonubiquitous expression (Table 1 shows a human ⁄ mouse comparison together Table 1. Comparison of human and mouse STK33 ⁄ Stk33 expression levels between our data and expression databases. Organ ⁄ tissue Hsa STK33 Mmu Stk33 Hsa STK33 Mmu Stk33 Hsa STK33 Mmu Stk33 Hsa STK33 Mmu Stk33 This paper Positive experiments UniGene a count [32] Number ⁄ % of clones EST profile viewer a [32] Transcripts per million SOURCE Gene Report b [34] % Normalized Testis +++ +++ 17 ⁄ 16.0 34 ⁄ 68.0 124 329 10.89 71.57 Lung + + ++ 22 ⁄ 20.7 0 76 0 6.97 – Fetal lung + – 0 0 – – – – Heart – – 0 1 ⁄ 2 0 18 – 4.96 Fetal heart + + 0 0 – – – – Heart’s interv septum + n.a. 0 0 – – – – Brain – – 2 ⁄ 1.8 4 ⁄ 8.0 4 8 – 1.50 Fetal brain – + 0 0 – – – – Pituitary gland + n.a. 0 2 ⁄ 4.0 45 11.29 Ovary – – 3 ⁄ 2.8 0 31 0 2.11 – Kidney + – 2 ⁄ 1.8 0 14 0 – – Stomach – n.a. 1 ⁄ 0.9 0 9 0 – – Pancreas + n.a. 5 ⁄ 4.7 0 25 0 – – Trachea + n.a. 0 0 – – – – Thyroid gland + n.a. 1 ⁄ 0.9 0 – – – – Prostate – n.a. 2 ⁄ 1.8 0 14 – 0.76 – Uterus – n.a. 7 ⁄ 6.6 0 38 0 3.40 – Cervix n.a. n.a. 1 ⁄ 0.9 0 24 – 2.33 – Eye – + + 2 ⁄ 1.8 2 ⁄ 4.0 11 11 – 3.40 Lymph node – n.a. 2 ⁄ 1.8 0 15 0 1.33 – Mammary gland – n.a. 0 1 ⁄ 2 0 2 – 0.73 Embryonic stem cells n.a. n.a. 2 ⁄ 1.8 0 – – – – Neuroblastoma n.a. n.a. 1 ⁄ 0.9 0 – – 64.34 – Bone, intestine, liver, skin, spleen, muscle, amongst others –– 000––– a UniGene Hs.501833 STK33 and Mm.79075 Stk33. Consulted on 15.06.2005. ‘Other’ or ‘mixed’ entries not included. b H. sapiens UniGene Build no. 184, and Mus musculus UniGene Build no. 147, released on 2005-06-09. ‘Other’ or ‘mixed’ entries not included. A. O. Mujica et al. STK33 expression pattern FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS 4885 with the expression pattern obtained in this paper). ESTs from human lung and testis are most frequently represented (20.7% and 16.0%, respectively). Embry- onic and fetal tissues as well as diverse tumour tissues and cancer cell lines are also represented with several entries each. Some entries are present from tissues of the nervous system as well as from auditory and ocular systems. Prostate and uterus also show single STK33 entries. The EST coverage for Stk33 in the mouse seems to be more limited. Only testis is very well repre- sented with 68% of the entries, and interestingly there are no entries from the lung. In addition to testis, there are single ESTs from retina, pituitary gland and cir- cumventricular organs of the brain such as subfornical organ and area postrema. In this first survey, we have addressed the expression of STK33 ⁄ Stk33 focussing on RNA and protein distri- bution with emphasis on the mouse as an animal model. Manning and colleagues [8] explained the fail- ure to detect some novel kinases, despite their similar- ity to members of the superfamily, with the limited expression of these proteins. The results presented here suggest that this may be the case for STK33 and that its expression pattern also resembles that of members of the CAMK family of protein kinases. As a step towards understanding their function, we have analysed the distribution of STK33 ⁄ Stk33 RNA and protein as well as the subcellular localization of the protein. Results Presence of STK33 ⁄ Stk33 RNA in mouse and human tissues Northern blot experiments were performed with 4–20 lg immobilized poly(A) + RNA from various murine organs. Simultaneously, hybridization with a cDNA fragment of ribosomal protein L19 housekeep- ing gene was performed as normalization probe. L19 gene shows a ubiquitous expression and, in particular, high expression in testis, according to the Gene Expression Atlas [33]. The L19 probe hybridizes to an RNA with a significantly lower length (full length mRNA 673 bp) and hence yields a signal in a clearly different position than Stk33. Results (Fig. 1A) show a strong hybridization signal with Stk33 in RNA from testis corresponding to an RNA of  3000 bp. In addi- tion, there is a weak signal corresponding to a shorter RNA which could be the result of a second Stk33- RNA variant, possibly generated by alternative spli- cing. In this analysis, no signals were detected in heart, intestine, brain, kidney, ovary and lung. A B C Fig. 1. STK33 ⁄ Stk33 levels of expression in human and mouse tissues. (A) Northern blot of immobilized poly(A) + RNA from adult mouse organs with a Stk33-specific probe. The following amounts of poly(A) + RNA were loaded (lg): heart, 4; intestine, 20; brain, 20; kidney, 6; ovary, 6; testis, 12; lung, 15. Normaliza- tion was performed by simultaneous hybridization with a probe from mouse ribosomal protein gene L19. The arrow shows a possible shorter alternative transcript present in testis. (B) cDNA dot-blot (MTE, Clontech) hybridization with an STK33 specific probe, with samples from different regions of nervous system (NS), heart (He), digestive system (DS), several organs (SO), can- cer cell lines (Ca), fetal organs (FO) and diverse controls (Co). The first column with practically no signal corresponds to several regions of brain (see Fig. S1 for the identity of each dot). (C) Quantification of the signal obtained in the cDNA dot-blot normal- ized to the maximal signal in testis. Only results are shown from tissues with signals higher than the highest value for the negat- ive controls. STK33 expression pattern A. O. Mujica et al. 4886 FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS The expression pattern of STK33 in human was investigated by cDNA dot-blot hybridization using the human multiple tissue expression (MTE, Clontech, Palo Alto, CA) and a STK33 specific probe. The cDNA dot-blot contains 76 normalized dots of immo- bilized cDNA, 61 from adult normal tissues, eight can- cer cell lines and seven fetal tissues (see supplementary Fig. S1). In two hybridization experiments weak but reproducible STK33-specific signals were produced in a small number of tissues. Significant hybridization sig- nals were obtained in cDNA from testis, fetal lung, fetal heart, pituitary gland and kidney. Weak but still above background signals were found in interventricu- lar septum of the heart, pancreas, trachea, and thyroid gland (Fig. 1B,C). Hybridization signals were consid- ered nonsignificant if they were below the highest pro- duced by the negative controls (yeast total RNA, yeast tRNA, Escherichia coli rRNA, E. coli DNA, poly r(A), human Cot-1 DNA, human genomic DNA). Such ‘nonsignificant’ signals were found in cDNAs from a large number of tissues. The reason may be low or very low amount of Stk33 RNA in those tissues or unspecific binding of the hybridization probe. To localize Stk33 transcripts at the cellular level, mRNA in situ hybridization (ISH) on tissue sections of various adult murine organs were performed. Controls with sense RNA show negative results (Fig. S2) com- pared with the antisense RNA probes. As expected from the results from the northern analysis, strong hybridiza- tion signals were found in mouse testis sections. Stk33- specific signal appeared to be restricted to the cells of the spermatid differentiation process, from the sperma- togonia to the early spermatides with a remarkable maximum of signal in the spermatocytes (Fig. 2A,B). In all cases the signal was perinuclear, strongly supporting its association to germinal cells. No signal was observed in periluminar areas of the germinal epithelium or in spermatozoa, in cells from the interstitial tissue, Leydig cells, vascular cells or myoid cells in the lamina propria. Stk33-specific ISH signal were also detected in lung tissue sections, particularly in the epithelium of the bronchi (Fig. 2D,E). Strong signals were also observed in single alveolar cells (Fig. 2D,F), which, as revealed by nuclear staining, probably are alveolar macro- phages. In situ RNA hybridization in mouse retina showed a signal in the outer plexiform layer (Fig. 2G). Protein distribution To investigate protein distribution, a polyclonal anti- body against a Stk33-specific synthetic peptide was generated. The specificity of the anti-Stk33 antibody was determined by competition tests with synthetic Stk33-specific peptide, anti-Stk33 signal disappeared in testis sections, when antibody is preabsorbed with 12.5 ngÆlL )1 synthetic peptide (Fig. 3, C3). Immuno- staining on tissue sections with the Stk33 antibody (Fig. 4) was observed in the same regions as the mRNA in situ-hybridization (Fig. 2). In testis an intensive staining was observed in only few cells per tubulus, which may be classified as secondary sperma- tocytes according to nucleus morphology and localiza- tion (Figs 4 and 5). Round spermatides showed signals of lower intensity compared to the spermatocytes. Moreover, Stk33 positive and negative spermatides were often seen in groups restricted to distinct tubular profiles (Fig. 4A). Immunostaining signal was also vis- ible in Sertoli cells, concentrated in the perinuclear space. In all cases, the protein localization appeared to be cytoplasmic. In lung, immunostaining with anti-Stk33 antibody produced strong signals in epithelial cells and suppo- sedly alveolar macrophages. Laser scanning micro- scopy (LSM) as well as comparison of immunostaining with nuclear staining showed cytosplasmic localization of Stk33 in the putative alveolar macrophages (Fig. 4D, E, F and J). In the retina, a strong cytoplas- matic immunostaining signal was found in horizontal cells (Fig. 4K–M). Immunostaining with Stk33 antibody was also per- formed with 15-day mouse embryos (Fig. 6A). Stk33 specific signals were found in some areas of the ner- vous system, in particular in the intermediate zone of the cerebral hemisphere and between cerebellum prim- ordium and medulla oblongata (Fig. 6, A1 and A2). Also border regions between metencephalon and pons with the third ventricle (arrows in Fig. 6, A1 and A2) showed an augmentation of the signal. The spinal cord showed a strong signal with a maximum in its lumbar part, including neuronal processes (Fig. 6, A3). A clear signal was observed in heart ventricles but not in the atria (Fig. 6B). The signal is augmented in the endo- cardium (Fig. 6, B1 to B3). Signals were also observed in the trigeminal ganglion, rhinencephalon and tongue (see Fig. S3B). All immunostaining negative controls with no primary antibody reproducibly showed no staining (see Figs S2 and S3). Spermatogenesis specific signal To confirm spermatogenesis-specific Stk33 signal, Western blots with protein extracts from testis of mice of different ages were performed. The Western blot analysis showed signals in 20 and 30 days postpartum (dpp) mice, whereas 10 dpp were Stk33 negative (see Fig. 3D). Although the Western blot results were not A. O. Mujica et al. STK33 expression pattern FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS 4887 evaluated quantitatively, it seems obvious that the sig- nal is stronger in older mice. Discussion Here we report the first survey of the distribution of the recently discovered serine ⁄ threonine kinase 33 in mouse and human tissues. Our results are well in accordance with the STK33 ⁄ Stk33 expression pattern derived from the expressed sequence databases (compiled in Table 1). The predominant expression of STK33 ⁄ Stk33 in testis is also observable in UniGene EST database [32] (http://www.ncbi.nlm.nih.gov/UniGene), Gene Expres- sion Atlas [33] (http://symatlas.gnf.org/SymAtlas/) and Testis A B C Lung Retina D G E F Fig. 2. Distribution of Stk33 mRNA in testis, lung and retina demonstrated by in situ hybridization (ISH). (A,B) ISH in testis. Strong Stk33 signal is detected in spermatocytes (spc), spermatides (sd) and possibly in spermatogonia (sg). No signal is detected around Sertoli cell nucleus (sen) or in spermatozoa (sz), neither in Leydig cells nor any kind of cell in the interstitial space (is). (C) Nuclear staining with DAPI was used for char- acterization of the nuclei in all tissues and is shown here exemplary for testis. (D,E,F) ISH in lung. Stk33 signal was detected in epithelium (ep) and alveolar macrophages (am). No signal was found in cartilage (ca), smooth muscle (sm), connective tissue (ct), bronchioli (bri), aleveolar duct (avd), alveoli (av), artery (ar) and bronchus (bru). (G) ISH in mouse retina. Stk33 signal was visible in the outer plexiform layer (opl). No sig- nal was detected in ganglio cells (gc), inner plexiform layer (ipl), inner nuclear layer (inl), outer nuclear layer (onl), choroid (ch) and sclera (sc). Dark staining in the pigmented epithelium (pe) was also observable in negative controls (data not shown) and hence disregarded as signal. STK33 expression pattern A. O. Mujica et al. 4888 FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS Stanford’s SOURCE database [34] (http://source. stanford.edu). The dataset of the GeneCards from the Weizmann Institute of Science (http://bioinformatics. weizmann.ac.il/cards), which is restricted to human genes and does not provide results from testis, shows expression of STK33 in lung and heart. However, GeneCards expression data for STK33 are close to the background level, which is fitting to the low level expression in tissues other than testis. The only discrep- ancy of the UniGene data, is the fact that we have detected significant Stk33 expression in mouse lung in two of three experiments. All expression data are sum- marized in Table 1. Clearly, the expression of Stk33 is only high enough in testis to be detected by northern blot and cDNA dot-blot experiments. More sensitive methods, such as RNA in situ hybridization and immunostaining, reveal expression in a broader spectrum of tissues but restric- tion to particular cell types and populations. The absence of signal in regions of the nervous system in the human cDNA dot-blot analysis and mouse nor- thern blot is remarkable, as several CAMK genes are expressed there in high rates [35]. However, cells from the nervous system in adult mice retina, probably hori- zontal cells, display Stk33 expression as observed by ISH and immunostaining. Additionally, Stk33 RNA ⁄ protein is expressed in some regions of the ner- vous system in fetal mice (Fig. 6), and protein distribu- tion in the adult brain shows a distinct distribution that will be presented in a separate paper. Human fetal lung yielded the second highest signal in our cDNA dot-blot hybridization, whereas there was remarkably weaker or no signal in human adult lung samples. Northern blot analysis with mRNA also showed no signal in adult mice lung, even though more mRNA was immobilized from lung than from testis. On the other hand, both mRNA in situ hybridization and immunostaining experiments on tissue slices of mouse adult lung showed a reproducible signal in bron- chial epithelium and putative alveolar macrophages. It seems evident that low expression of Stk33 is difficult to examine by hybridization methods such as northern blot and cDNA dot-blots using whole organs in all tissues but testis. We were not able to detect Stk33 protein in mouse fetal lung. As we tested embryos only at 15 days postcoitus, it is conceivable that expres- sion in fetal lung occurs at different developmental stages. According to their predicted general biochemical features, STK33 and Stk33 are probably soluble pro- teins. psort analysis [36] found no notable known sig- nal in STK33 ⁄ Stk33 primary structure except from conserved C-terminal di-lysine motifs (511-Thr-Lys- Lys-Lys-514 in human and 488-Gly-Lys-Lys-Arg-491 in mouse), which are recognized as putative endoplas- matic reticulum membrane retention signals [37]. The highest psort scores for subcellular localization based on amino acid composition correspond to cytoplasm and nucleus, weakly favouring nuclear localization. The results of our immunostaining shown here (Figs 4–6) demonstrate a predominantly cytoplasmic localization of Stk33 protein. STK33 could be involved in the normal development of heart and other organs in embryonic and fetal stages. The third highest signal in the cDNA dot-blot corres- ponds to fetal heart and the sixth highest to the inter- ventricular septum of the heart; this is also in line with the strong immunostaining signal in mouse embryo A C D B Fig. 3. Stk33 recombinant protein, antibody and spermatogenesis specific signal in mice. Western blot of recombinant biotin–Stk33 fusion protein, affinity purified, detected with ExtrAvidinÒ-AP (A) and with anti-Stk33 IgG (B). The 22.5-kDa band corresponds to the E. coli native protein biotin carboxyl carrier protein (BCCP). Track 1 in (B) corresponds to the purge of the column and shows a trace of antigen. Track 2 and 8 contain 572 ng and 0.425 ng of purified protein, respectively. (C) Preabsorption of anti-Stk33 IgG in testis slides with the following concentrations of synthetic peptide: 0ngÆlL )1 (C1), 1.25 ngÆlL )1 (C2) and 12.5 ngÆlL )1 (C3). C2 and C3 were photographed with longer exposure times and accordingly exhibit the light-coloured regions in the interstitial space also seen in negative control (C4) with no primary antibody. (D) Stk33 sper- matogenesis specific expression demonstrated by anti-Stk33 anti- body staining. Immunoreactivity is observable in recombinant protein (Rec), protein extracts from testis of mice at 20 and 30 dpp but absent in 10 dpp. Coomassie blue staining of total protein shows the homogeneous presence of 75 lg protein extract in each track. Recombinant protein is not visible in the Coomassie blue stained control track (Rec), as the amount loaded (80 ng) is below the detection threshold [48]. A. O. Mujica et al. STK33 expression pattern FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS 4889 heart (Fig. 6B) and the lack of signal in adult mouse heart in our northern blot analysis (Fig. 1A). A study on human heart malformations using DNA micro- arrays [38] revealed STK33 among the most downregu- lated genes in patients with tetralogy of Fallot, a nonfatal congenital cardiovascular malformation in which ventricles are not fully separated by the septum and the pulmonary artery valve is narrowed, causing a partial mixing of venal and arterial blood and a decrease of blood flow to the lungs. On the other hand, data from the source database [34] suggests a higher expression of STK33 in neuroblastoma than in any Testis AB C Lung Retina DG EH F KL M I J Fig. 4. Stk33 protein localization in testis, lung and retina. (A,B) Localization in testis. High signals in spermatocytes (spc) followed by round spermatids (rsd) and around the Sertoli cell nucleous (sen). Stk33-positive and negative round spermatides (+ rsp, –rsp) exhibit a tubulus-spe- cific distribution. (C) LSM image demonstrating cytoplasmic localization of the spermatocytes signal (anti-Stk33 in red and anti-a-tubulin in green). (D–I) Immunostaining combined with nuclear staining in mouse lung preparations (anti-Stk33 in red and nuclear staining in blue). (D,E,F) Alveolar macrophages showing cytoplasmic Stk33-signal, confirmed in (J) by LSM (anti-Stk33 in red and anti-a-tubulin in green). (G,H,I) Lung epithelial cells with Stk33 specific signal. (K,L,M) Stk33 localization in mouse retina, in particular in horizontal cells. Immunostain- ing with anti-Stk33 antibody showing strong signal in horizontal cells (hc) in the outer plexiform layer. STK33 expression pattern A. O. Mujica et al. 4890 FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS normal tissue (Table 1). Both neuroblastoma and tetralogy of Fallot (more generally congenital cardio- vascular malformations) are childhood diseases which may be associated because of their common neural crest origin [39,40]. The fact that STK33 may be downregu- lated in tetralogy of Fallot but upregulated in neuro- blastoma is intriguing and supports the idea of a role in early organ development. More cells in testis were labelled by ISH than by immunostaining preparations, which may be due to a high turnover of the protein. Moreover, the observa- tion of a high immunostaining signal in spermatocytes and grouped round spermatides with differential expression pattern may reflect a cell division stage-spe- cific synchrony of Stk33 expression during spermato- genesis. This observation is strongly supported by the Western blot results showing no signal in 10 dpp mice and positive signal first by 20 dpp. The absence of sig- nal at 10 dpp excludes involvement of Stk33 in Sertoli cell or spermatogonia proliferation, which extends to 12 dpp [41]; but the signals at 20 and 30 dpp suggest a meiosis and ⁄ or spermiogenesis specific function which first occur at the periods of 8–18 dpp and 18–30 dpp [41]. It has been hypothesized that novel cell division regulatory checkpoints probably exist in the germ line of higher eukaryotic organisms which are not necessar- ily present in the basic ones already described for yeast [42]. On the other hand, STK33 orthologs are present in the genomes of several chordate organisms (Fig. 7), but not found in yeast, fly or nematode genomes. This is not too surprising as humans possess 74 members of the CAMK group of protein kinases whereas only 21 are detectable in yeast, 32 in fly and 46 in worm [8]. We propose that STK33 ⁄ Stk33 expression occurs only within a narrow time window during the spermatogen- esis. It remains to be established whether this ‘pulse like’ expression is directly associated with some germ cell development checkpoint, which based on our data seems suggestive, or rather reflects synchrony to other events of the spermatogenesis. For instance, the mech- anisms unleashing the pass of the haploid germ cells through the tight junctions from the adluminal to the luminal region are still unknown. It has been argued that the germ cells themselves may signal the Sertoli cells their entry to meiosis, triggering their transport through the blood–testis barrier [43]. On the other hand, apoptotic germ cells in wild-type rats, show a similar frequency and distribution pattern as we show here for Stk33 [44]. The molecular basis of these phe- nomena is not yet fully established and a role for Stk33 in some of these pathways is feasible. An involvement of STK33 in human spermatogenesis may also be postulated. STK33 is clearly related to the canonical kinases from the CAMK group and, as we demonstrate here, shows similar expression pattern. Hence, similar func- tions may be proposed. In particular, the maximum expression signals of Stk33 in certain stages of sperma- togenesis, in some embryonic fetal organs and its involvement in child diseases, let us to postulate a role in gametogenesis and organ ontogenesis that strongly deserves to be further investigated. Experimental procedures Radioactive DNA labelling Fifty to 500 ng of purified PCR-generated STK33 ⁄ Stk33- DNA was used as template for radioactive probes. In vitro random primed DNA labelling was carried out according to manufacturer’s protocols (Roche Diagnostics, Mann- heim, Germany). The labelling reaction was performed overnight at room temperature, or for 2 h at 37 °C. Typically, blot hybridizations were carried out overnight at stringent temperatures depending on the G + C con- tent of the probe (usually 64 °C for Southern blots A B C D E G H Fig. 5. Stk33 in mouse secondary spermatocytes. Double staining with anti-Stk33 IgG (red) and nuclear staining with DAPI (green). (A) Detail of a section of two seminiferous tubules showing a single Stk33-positive cell. Note the dashed line marking the borders between two different tubuli sections. The DAPI staining reveals the different nuclear morphologies of: sertoli cell nucleous (sen), spermatogonia (sg), primary spermatocytes (sc1), secondary sper- matocytes (sc2), round spermatides (rsd) and spermatozoa (sz). According to their chromatine condensation, Stk33-positive sperma- tocytes were found in metaphase II (A), prophase II (B,C,D) and anaphase II (E,F,G, chromosome segregation indicated by arrows). A. O. Mujica et al. STK33 expression pattern FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS 4891 and 42 °C +50% v ⁄ v formamide for northern blots). After several washes with 2 · NaCl ⁄ Cit and 0.1 NaCl ⁄ Cit at room temperature membranes were exposed to X-ray film with or without intensifying screens at )70 °C. Exposure time varied from several hours to sev- eral days. Fig. 6. Localization of Stk33 in mouse embryo, day 15 postcoitius. (A) Overview of the mouse embryo immunostaining analysed with non- confocal laser scanning. Regions with particular strong signal (a1–a3 and b) were photographed with the fluorescence microscope. Signal is detected in some regions in the head, for instance between pons and medulla oblongata (A1), intermediate zone of mesencephalon (A2) and medulla (A3). Strong signal is observed in heart ventricle (B) in particular in endocardium (B1–B3), here supported by nuclear staining in green. STK33 expression pattern A. O. Mujica et al. 4892 FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS DNA probes To avoid cross-hybridization, probes were designed exclu- ding the region encoding the evolutionary conserved kinase domain. The probes were checked for uniqueness by blast searches against human and mouse DNA sequences in the databases, in particular with the EST subset and whole gen- ome assembly, and for repeat sequences by Repeatmasker (Washington University, http://repeatmasker.org). The human DNA probe was amplified by PCR from uterus cDNA as described [1] with the forward primer 5¢-AA GCAATTTCCTGCAACC-3¢ and the reverse primer 5¢-CATGTGAATGACTGAAGC-3¢, yielding a 676-bp DNA fragment. For the murine Stk33 a 570-bp probe was amplified from lung cDNA spanning the last two exons with the forward primer 5¢-AACCCAGAAAGTGATGAG- 3¢ and the reverse primer 5¢-TAGAACTAAGCGAG CATG-3¢. For normalization purposes in hybridization experiments with mouse tissues, a ribosomal protein gene L19-specific probe was amplified by PCR from the IMAGE:2648593 (kindly provided by GENterprise GmbH, Mainz, Germany) using the standard primers T7 and SP6. All PCR products were purified, checked by electro- phoresis, quantified and sequenced before use as probes for hybridization. Northern blot Total RNA isolation from mouse tissues was performed by guanidinium thiocyanate ⁄ phenol ⁄ chloroform extraction [45]. mRNA was isolated from total RNA with the Nucleo- Trap Ò isolation kit (Macherey-Nagel, Du ¨ ren, Germany), following the manufacturer’s instructions. mRNA was quantified spectrophotometrically, and separated by electro- phoresis on a 1.2% agarose gel under denaturing conditions (5.5% formaldehyde), transferred to Nylon membranes (Roche, Mannheim, Germany) and UV-cross linked. The blot was hybridized with a DNA Stk33-specific probe and a probe from the L19 housekeeping gene as normalization control. BioMax MS autoradiography films were exposed to the radioactive blot with intensifying screen at )70 °C. Exposure time was determined empirically. Fig. 7. Amino acid sequence alignment of Stk33 kinase domain in some chordates. Proteins sequences from human (Hsa) and mouse (Mmu) as described in [1]. Sequences from Rattus norvegicus (Rno), Fugu rubripes (Fru), Danio rerio (Dre) and Ciona intestinales (Cin), were inferred from their respective genome projects already available [49–51]. Partial Stk33 EST sequences are detected in Xenopus laevis and X. tropicalis, but they still do not extend the whole kinase domain and were in con- sequence excluded from the analysis. The putative ATP-binding subdomain, serine ⁄ threonine phosphorylation signature and the Mg 2+ chelating Asp-Phe-Gly are shown, by similarity with known kinase structures [52–54]. Alignment accession number: ALIGN_000866. A. O. Mujica et al. STK33 expression pattern FEBS Journal 272 (2005) 4884–4898 ª 2005 FEBS 4893 [...]... GTCACAGTAGG-3¢ and reverse 5¢-TGCAGATGGAC TGTAGAC-3¢ primers The 1006-base-long Stk33 cDNA fragment (database accession number AM056057) spans the complete coding sequence for the kinase domain including the peptide used for immunization and is in frame with the start and stop codons of the PinPointä Xa)1T (Promega, Madison, WI, USA) plasmid This inducible expression vector contains a biotin coding region... Regulation of the mitotic and meiotic cell cycles in the male germ line Recent Prog Horm Res 57, 75–101 Mruk DD & Cheng CY (2004) Sertoli-Sertoli and Sertoli–germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis Endocrinol Rev 25, 747–806 Jeyaraj DA, Grossman G & Petrusz P (2003) Dynamics of testicular germ cell apoptosis in normal mice and. .. merged images in the double-staining experiments Acknowledgements The authors thank Tilmann Laufs for kindly providing the mouse embryo slides, Christof Rickert for assistance with the LSM and Oliver Bitz for assistance with the Laser Scanner Financial assistance was obtained 4896 A O Mujica et al from the Rheinland-Pfalz Stiftung fur Innovation and ¨ through funding by the German Human Genome Project... phenylenediamine in 50% v ⁄ v phosphate-buffered glycerol Control incubations were carried out essentially as the immunostaining reactions but omitting primary antibodies The specificity of the immunostaining was demonstrated performing preabsorption tests of the Stk33-antibody with synthetic peptide Before applying it to the testis cryostat sections, the antibody was diluted 1 : 40 in NaCl ⁄ Pi, 10% FCS, incubated... start codon and upstream from the cloning site Ligation of the Stk33-PCR product with the vector was carried out according to the manufacturer’s instructions The recombinant DNA was isolated using the E.Z.N.A Plasmid-Miniprep-Kit (PeqLab, Erlangen, Germany) The insert was checked for proper orientation by restriction analysis and confirmed by sequencing with standard vector primers Transformation of E coli... Hunter T & Manning G (2004) The mouse kinome: Discovery and comparative genomics of all mouse protein kinases Proc Natl Acad Sci USA 101, 11707–11712 11 Corcoran EE & Means AR (2001) Defining Ca2+ ⁄ calmodulin-dependent protein kinase cascades in transcriptional regulation J Biol Chem 276, 2975–2978 12 Ikura M, Osawa M & Ames JB (2002) The role of calcium-binding proteins in the control of transcription:... independently of thickness, flatness or placement of the object due a 500-lm depth range Pictures were taken with a percentage point of gain about 25–30% (250–300 V) for preparations with anti-Stk33 and about 50% for the negative controls The resolution varied between 1 and 5 lmÆpixel)1 depending on the size of the section of the image The Adobe photoshop program was used to produce merged images in the double-staining... Genome sequence of the Brown Norway rat yields insights into mammalian evolution Nature 428, 493–521 52 Hanks SK & Hunter T (1995) Protein kinases 6 The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification FASEB J 9, 576– 596 53 Hanks SK, Quinn AM & Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains Science... domains Science 241, 42–52 54 Taylor SS, Radzio-Andzelm E & Hunter T (1995) How do protein kinases discriminate between serine ⁄ threonine and tyrosine? Structural insights from insulin receptor protein-tyrosine kinase FASEB J 9, 1255–1266 Supplementary material The following material is available for this article online: Fig S1 Distribution of cDNAs in Clontech’s MTE array, catalog # 7775–1, Protocol... for 15 min at 4 °C to remove cellular debris The biotinylated Stk33-fusion protein was affinity-purified using Soft LinkTM Soft Release Avidin Resin (Promega) following the manufacturer’s instructions Western blotting Recombinant Stk33-Protein was separated on 12% SDS ⁄ polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using 48 . Differential expression pattern of the novel serine⁄ threonine kinase, STK33, in mice and men Alejandro O. Mujica 1 *, Bastienne. 68% of the entries, and interestingly there are no entries from the lung. In addition to testis, there are single ESTs from retina, pituitary gland and