Abbreviations STK33 ⁄ Stk33, human and mouse serine ⁄ threonine kinase proteins; STK33 ⁄ Stk33: human and mouse serine ⁄ threonine kinase genes; CAMK: Ca2+⁄ calmodulin dependent kinases
Trang 1serine ⁄threonine kinase, STK33, in mice and men
Alejandro O Mujica1*, Bastienne Brauksiepe1*, Sigrid Saaler-Reinhardt2, Stefan Reuss3and
Erwin R Schmidt1
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 Ca2+⁄ 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 transcripregula-tion 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 ontospermato-genesis is discussed
Abbreviations
STK33 ⁄ Stk33, human and mouse serine ⁄ threonine kinase proteins; STK33 ⁄ Stk33: human and mouse serine ⁄ threonine kinase genes; CAMK:
Ca2+⁄ 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.
Trang 2[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]
CAMK4expression 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
Bone, intestine, liver, skin,
spleen, muscle, amongst
others
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.
Trang 3with 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 hybridizaNormaliza-tion 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.
Trang 4The 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 sechybridiza-tions
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
Trang 5evaluated 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
Lung
Retina
D
G
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.
Trang 6Stanford’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 popularestric-tions 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 distribudistribu-tion 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 ExtrAvidin-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:
0 ngÆlL)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 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].
Trang 7heart (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
Lung
Retina
F
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 showImmunostain-ing strong signal in horizontal cells (hc) in the outer plexiform layer.
Trang 8normal 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 upregudownregu-lated 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 37C Typically, blot hybridizations were carried out overnight
at stringent temperatures depending on the G + C con-tent of the probe (usually 64C for Southern blots
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).
Trang 9and 42C +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.
Trang 10DNA probes
To avoid cross-hybridization, probes were designed
exclu-ding the region encoexclu-ding 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
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 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.