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J Biol Chem, Vol. 274, Issue 26, 18597-18604, June 25, 1999
From the Gladys P. Stahlman Cardiovascular Research Laboratory,
Vanderbilt University Medical Center,
Nashville, Tennessee 37212-6300
We report the cloning of a novel murine cDNA,
LEK1, that is related to human CENP-F and mitosin and more distantly to
chicken CMF1. The proteins from these three organisms have significant homology, yet differ in their temporal, spatial, and subcellular localizations. The human proteins bind the kinetochore in mitotic cells, whereas the chicken protein is found only in skeletal and cardiac muscle and is developmentally regulated. Mouse LEK1 is a single
copy gene that codes for two developmentally regulated transcripts. The
LEK1 protein is expressed early and ubiquitously in mouse development
and is generally down-regulated as development proceeds in a manner
that correlates to a cessation of mitosis. In adult tissues, the LEK1
protein is detected exclusively in the pronucleus of the oocyte and was
not observed in other actively dividing tissues. Subcellular
localization revealed that the LEK1 protein in mitotic cells does not
bind the kinetochore. From these data, we hypothesize that chicken
CMF1, human CENP-F, mitosin, and mouse LEK1 are members of an emerging
family of genes that have important and functionally distinct roles in
development and cell division.
CMF1 is a recently described gene whose product has been
implicated in early chicken heart myogenesis. The possible function of
the CMF1 gene product in the differentiation of cardiac muscle was
demonstrated by disruption of CMF1 RNA function (1). These experiments
revealed that cardiomyocytes infected with CMF1 antisense-containing retrovirus failed to express stage-appropriate markers of
differentiation, whereas their uninfected or vector only-infected
counterparts expressed these proteins. Data base searches for CMF1-like
sequences found significant homology with
CENP-F1 and mitosin that were
cloned by independent groups from human HeLa cell cDNA libraries
(2, 3). The full-length CENP-F and mitosin cDNA sequences are
nearly identical. Comparisons between these human cDNAs and chicken
CMF1 found them 56% identical at the nucleotide level and 65% similar
at the amino acid level.
CENP-F was first isolated as an immunoreactive protein using antisera
obtained from a patient with an autoimmune disease (4) and was
subsequently cloned from a HeLa cDNA expression library (2). The
complete transcript of CENP-F codes for a large, 367-kDa protein.
Antisera directed against CENP-F localize to the outer plate of the
kinetochore during mitosis in dividing HeLa cells. The CENP-F protein
is up-regulated in late S-phase of cell division and during prophase
and metaphase localizes to paired foci near the centromere of each
chromosome. During anaphase, CENP-F was found in the intercellular
bridge and later, during telophase, was seen as two narrow bands on
either side of the midbody. Pulse-chase experiments showed the CENP-F
protein is rapidly turned over during mitosis (2). Recently, specific
regions of the CENP-F protein have been shown to interact with another
kinetochore protein, the kinesin-like motor protein, CENP-E (5).
Mitosin was originally cloned by its ability to bind the tumor
suppressor protein retinoblastoma (Rb) (3). This and subsequent investigations showed mitosin to be a nuclear protein that not only
binds the Rb protein but also binds microtubules, and similar to
CENP-F, binds the outer plate of the kinetochore in dividing tissue
culture cells (3, 6). The intracellular localization of mitosin is
nearly identical to that of CENP-F, particularly during mitosis. Recent
studies have shown that mitosin is capable of binding a putative
cytoplasmic retention protein, BRAP2 (7). CENP-F and mitosin have both
been associated with a number of human disorders such as chronic graft
versus host disease and various neoplasias (8-12). Taken
together, these studies led to the hypothesis that CENP-F/mitosin has
an essential role in mitotic cell division.
Previous data demonstrate the complexity of this emerging gene family.
Several basic questions concerning the structure, expression, and
function of the CENP/mitosin proteins remain unanswered. In an effort
to understand the role of this gene family in development, we sought to
clone gene products related to CENP/mitosin and CMF1 in mice,
characterize the expression of their mRNAs and proteins, and
initiate analysis of function. We have isolated a novel gene product,
LEK1, that has many structural elements shared with CENP-F/mitosin and
CMF1. Still, analysis of LEK1 expression and subcellular distribution in vivo suggests that it has a distinct role in mitosis and
cell differentiation. In addition, overexpression of a
dominant-negative form of LEK1 accelerated differentiation of a
myogenic cell line, suggesting a novel function for LEK1 among
CENP-F/mitosin-related proteins. Taken together, our data suggest that
LEK1 is a novel member of this emerging gene family.
Cloning, Sequencing, and Sequence Analysis of LEK1--
The
Access RT-PCR system (Promega) was used to amplify cDNAs from 100 ng of 9.5-dpc total ICR (Harlan Sprague-Dawley) mouse heart RNA using
two sets of degenerate primers (5'-GGCTNCCAGAAGTNGTTAAA-3' and
5'-CTTTTGTGATNTGCTGCCACC-3', 5'-TTAYATGAWCAGCACTGT-3', and 5'-TTCCTYAKTTTTCATAWTCYCTTG-3') from two homologous regions between chicken CMF1 and CENP-F/mitosin. These cDNA fragments were then cloned into the T-easy vector (Promega) and sequenced using an ABI
Prism Genetic Analyzer (Perkin-Elmer). Inserts from these clones were
then used as probes to screen a whole embryo 8.5-dpc mouse cDNA
library (courtesy of the laboratory of Dr. B. L. M. Hogan).
Overlapping clones were obtained by using standard protocols for
cDNA library screening, RT-PCR cloning, 5'-rapid amplification of
cDNA ends, and Genewalking (13) and sequenced as stated above.
Southern Blot Analysis--
Using standard protocols (13), 10 µg of mouse, human, and chicken genomic DNA were digested with the
indicated (Fig. 2) restriction endonuclease overnight at 37 °C as
per the manufacturer's instructions (Promega). This DNA was processed
for Southern blot analysis. The blot was then prehybridized in RapidHyb
(Amersham Pharmacia Biotech) for 1 h at 65 °C. A probe
corresponding to 7017-7222 nucleotides of the LEK1 transcript random
prime-labeled with 50 µCi of [ Northern Blot Analysis--
Staged embryonic and adult tissues
were collected, and the total RNA was isolated using the Trizol reagent
as per the manufacturer's instructions (Life Technologies, Inc.). 10 µgs of total RNA was electrophoresed on a denaturing 1% agarose, 2.2 M formaldehyde gel and visualized with ethidium bromide as
a loading control. The RNA was transferred to a nylon membrane via
capillary action overnight and UV-cross-linked. This blot was probed
with the same region of the mouse LEK1 cDNA as the mouse-probed
Southern above, using the same hybridization conditions. The blot was
then washed three times at 65 °C in 2× SSC, 0.1% SDS for 1 h/wash
and exposed overnight using a Biomax intensifying screen and Biomax
film (Kodak).
In Vivo Immunofluoresence in Mice--
Adult and staged
embryonic tissues were processed for cryo-sectioning using a Jung CM
3000 cryostat (Leica) in 10-µm increments and collected on
gelatin-coated slides. The slides were then fixed with 70% methanol
for 10 min, permeabilized in 0.25% Triton X-100 for 10 min, and
blocked in 2% bovine serum albumin overnight at 4 °C.
Affinity-purified anti-LEK1 (Biosynthesis) was incubated with these
samples for 1 h at room temperature using a 1:800 dilution. After
extensive washing in phosphate-buffered saline, slides were incubated
in donkey anti-rabbit Cy3 (The Jackson Laboratory) and DAPI (Roche
Molecular Biochemicals) counterstain at room temperature for 1 h
and again washed extensively in phosphate-buffered saline. These
samples were visualized using fluorescence microscopy (Olympus). Controls for these experiments included no primary antibody and peptide
competition. In both cases, staining was completely absent. The 9.5-dpc
limb bud in Fig. 4 was obtained using a confocal laser-scanning LSM410
microscope (Zeiss) on tissues prepared in a similar fashion as stated
above. The nuclear marker YoPro-1 (Molecular Probes) was used in these
experiments because of incapability of DAPI with this microscope.
In Vitro Immunofluoresence of C2C12 Cells--
C2C12 cells
(ATCC) were passaged once in 20% fetal bovine serum in DMEM. 5 × 106 cells were placed into single-well chamber slides.
Myotubes were induced to form by growing cells in 4% heat-inactivated
horse serum in DMEM for 4 days. Mitotically active C2C12 cells were maintained in 20% fetal bovine serum in DMEM for this same time period. These slides were processed and analyzed in an identical manner
to the mouse tissues above.
Transient Transfection of C2C12 Cells--
A dominant-negative
LEK1 (dnLEK1) construct was produced by cloning 4.49 kilobases of the
3'-most cDNA sequence of the LEK1 into the mammalian expression
vector pCI Neo (Promega). This protein is missing the N-terminal half
of LEK1. A similar dominant-negative of mitosin produced marked
phenotypic changes when transfected into human fibroblasts (3).
Preliminary analysis demonstrated that this construct produces the
expected dnLEK1 protein of the predicted size with the same subcellular
distribution as the native protein.2 For transfection,
C2C12 cells were grown to approximately 75% confluence in 20% fetal
bovine serum in DMEM. These cells were transfected with either 5 µg
of the dnLEK1 and 200 ng of p Isolation of LEK1 cDNA Clones--
Standard low stringency
hybridization techniques were initially employed to isolate the murine
CMF1 gene (13). A number of libraries (genomic and cDNA) and
hybridization conditions were assayed, none of which produced clones.
Consequently, an RT-PCR cloning strategy was developed using two
homologous regions between the chicken CMF1 and human CENP-F/mitosin
proteins to design degenerate primers (see "Experimental
Procedures"). These degenerate primers were used to amplify cDNAs
from heart RNA of 9.5-dpc mouse embryos. To obtain larger regions of
this mouse transcript, the RT-PCR clones were then used as probes to
screen an 8.5-dpc whole embryo library (courtesy of Dr. B. L. M. Hogan). The resulting cDNAs were subsequently cloned and
sequenced and found to be approximately 75% identical to the human
CENP-F/mitosin cDNAs. A combination of RT-PCR, cDNA cloning,
genomic walking, and 5'-rapid amplification of cDNA ends techniques
were used to isolate overlapping clones such that the primary structure
of the LEK1 transcript could be determined. It should be noted that an
antiserum (described below) was developed against a sequence from one
of the cDNA clones. Subsequently, this antiserum was used to
reclone LEK1,3 demonstrating
the specificity of this antibody.
Sequence Analysis of LEK1--
Analysis of the nucleotide
sequences of chicken CMF1, human CENP-F/mitosin, and mouse LEK1 shows
that mouse LEK1 is more closely related to human CENP-F/mitosin than to
chicken CMF1. CMF1, CENP-F/mitosin, and LEK1 proteins share a
significant amount of homology at the primary, secondary, and tertiary
levels. A prominent feature of these proteins is a preponderance of
leucine (L), glutamic acid (E), and lysine (K) amino acid residues in
these proteins, approximately 40% of the amino acid composition. Thus,
we have named the mouse cDNA LEK1 and tentatively refer to these
three proteins as the LEK family of genes.
Like CMF1 (1) and CENP-F/mitosin (2, 3), computer analysis (14)
predicts that the LEK1 protein is largely composed of
Mouse LEK1 protein shares a number of other protein motifs with
CENP-F/mitosin and CMF1. As would be expected with such large proteins,
there are a number of conserved, potential sites for post-translational
modifications, such as glycosylation, phosphorylation, myristylation,
and amidation. The most conserved region among the human, mouse, and
chicken proteins is the C terminus (Fig. 1.) In this region, the LEK1
protein contains a predicted helix-loop-helix dimerization domain (Fig.
1, asterisk), a bipartite nuclear localization signal (Fig.
1, yellow stripe and bracketed) (18), an atypical Rb binding site (Fig. 1, black stripe and
bracketed) (3), and a P-loop, which is an ATP/GTP binding
domain (Fig. 1, bracketed). These motifs are not only well
conserved between the three different proteins but are distributed in a
collinear fashion (Fig. 1). Though these proteins do share these
abundant similarities, it is important to note that comparisons between
them do have regions of significant divergence.
Genomic Analysis of LEK1--
A Southern blot analysis of mice,
human, and chicken genomic DNA was conducted to investigate the number
of potential LEK1-like genes in the genomes of other representative
vertebrates. Mouse, human, and chicken genomic DNAs were probed with
the well conserved 3' region of the LEK1 cDNA. At low stringency
conditions (see "Experimental Procedures"), numerous bands were
observed in all species.3 By gradually increasing the
stringency of the washes, the number and intensity of bands decreased
until at 65 °C and 2× SSC, 0.1% SDS, only a single
band/lane was observed (Fig.
2A). When this same blot was
stripped and reprobed with a corresponding region of the chicken CMF1
cDNA, single bands were also observed (Fig. 2B),
although their migration differed from those in the mouse-probed Southern blot (compare the arrows in Fig. 2, A
and B). Interestingly, bands with similar mobility were
observed in a low stringency wash of the mouse-probed
Southern.3 This is consistent with the hypothesis that two
homologous genes exist in these organisms, a CMF1-like and a LEK1-like
gene.
Expression of the LEK1 mRNA during Embryogenesis--
To
determine both the size and the temporal/spatial distribution of LEK1
transcripts in the murine tissues, Northern analysis experiments were
performed. RNA was extracted from staged mouse embryonic and adult
tissues. As seen in Fig. 3A,
two bands (approximately 10 kilobases) hybridized to the same well
conserved sequence used in the Southern analysis. These two transcripts
are most likely the result of alternative products from a single gene
as only one band was observed in the Southern blot experiments using
the same probe and high stringency conditions. The highest levels of
LEK1 expression were observed in the early stages of mouse development:
8.5-dpc whole embryo, 9.5-dpc head, and 9.5-dpc caudal regions
posterior to the heart (Fig. 3A). It is interesting to note
that the relative levels of the LEK1 message appeared to increase
during the course of embryonic murine heart and liver development. In
other tissues, such as the developing head and brain, the abundance of
the LEK1 transcripts decreased with the age of the embryo (Fig.
3A).
Because LEK1-like proteins such as CENP-F and mitosin have been
implicated in the mitotic process in vitro, we explored the expression of LEK1 in the developing heart, where the time course of
mitotic activity is well documented (19, 20). Using a Northern blot
kindly provided by the laboratory of Dr. Loren Field, we determined the
temporal expression of LEK1 in the developing mouse heart. High levels
of expression were found in 14-dpc embryonic mouse hearts (Fig.
3B) and continued through 4 days postpartum (Fig.
3B, N4). Little if any expression was observed in
the 7-day-old mouse, and a low yet detectable level of LEK1 expression
was observed in the adult mouse heart.
Localization of the LEK1 Protein In Vivo--
The previous RNA
data suggested that the LEK1 message is widely distributed in the early
embryo, but its expression drops as development proceeds (Fig.
3A). To determine the localization of LEK1 protein during
embryogenesis, we generated an affinity-purified antibody against LEK1.
As seen in Fig. 4, the vast majority of cells in this 9.5-dpc limb bud have LEK1-positive nuclei, although some
cells are negative (arrows in Fig. 4). The definitive
ectoderm and mesoderm in this tissue are examples of the extensive
expression pattern of LEK1 in early embryonic nuclei regardless of germ
layer origin. As tissues differentiated, we found a general decrease in
LEK1-positive nuclei.
The general pattern of LEK1 expression coincides with the loss of
mitotic activity in the early embryo. Because LEK1 mRNA expression
is down-regulated in the developing mouse heart at a time when
cardiomyocytes cease nuclear division (19, 20), we investigated the
distribution of the LEK1 protein in this organ using
immunohistochemical analysis. The majority of the nuclei in early
embryonic hearts were found to be LEK1-positive (compare LEK1 Ab and
DAPI counterstain in 16.5-dpc heart in Fig.
5). This pattern dramatically changed
between the N5 and N7 time points (Fig. 5). During this interval, the
number of LEK-positive nuclei dropped from 90% to less than 10%.
Thus, LEK1 is developmentally regulated at both the mRNA and
protein levels at this critical time in heart development. The low
level of LEK1-positive nuclei seen in N7 is maintained in N21 mouse
hearts and remains low in adult hearts.3 A survey of adult
organs and tissues was conducted to determine the pattern of LEK1
protein expression in differentiated tissues. Only rare cells were
observed to have LEK1-positive nuclei. Interestingly, LEK1 was not
observed in skin and intestinal epithelia, where cell renewal via
mitosis is common.3 In fact, in all of the adult tissues
examined thus far, LEK1 has only been found in the pronucleus of the
oocyte in the mouse ovary (Fig. 6).
However, no LEK1-positive pronuclei are observed in mature sperm cells
or any other cells in the seminiferous tubules of the testes (Fig.
6).
Subcellular Localization of LEK1 Protein during Mitosis and
Skeletal Myogenesis in Vitro--
CENP-F and mitosin have been
implicated in the mechanical/structural aspects mitosis (2-7, 21). Our
current study found a correlation between the loss of mitotic activity
during development and the down-regulation of the LEK1 protein (Fig.
5). However, this correlation was not maintained in differentiated
actively dividing cells of the adult mouse (testes in Fig. 6). To
investigate the localization of the LEK1 protein in a system where the
process of differentiation can be controlled, we employed the mouse
skeletal myogenic cell line C2C12. These cells can be maintained as
actively dividing cells in mitogen-rich medium or induced to form
mitotically inactive, multinucleated myotubes in differentiation medium
(22-25). As shown in Fig. 7, nuclei of
individual C2C12 cells were stained with anti-LEK1. Over 95% of
mononucleated C2C12 cells had LEK1-positive nuclei (compare red
fluorescence with DAPI counterstain, Fig. 7, B and
C). There is some minor LEK1 staining in the cytoplasm of
some cells. In mitotic cells, the LEK1 protein (Fig.
8) is present in all parts of the cell as
the nuclear envelope breaks down in prophase (Fig. 8, column
1). At metaphase and anaphase (Fig. 8, columns 2 and 3), the LEK1 protein remains fully cytoplasmic but is
absent in the area of the condensed chromosomes as determined by
counterstaining with DAPI (Fig. 8). At cytokinesis, the LEK1 protein is
localized to an area that is slightly greater than the DAPI-staining
region (Fig. 8, last column (Myotube)). With the
fusion of C2C12 into myotubes, DNA synthesis and mitosis abruptly stops, and the cells enter Go (25). As seen in Fig. 8,
there are LEK1-positive nuclei present in differentiated myotubes
(compare LEK1 Ab and DAPI in the myotube), and increased cytoplasmic
staining is observed. Later, however, LEK1 is absent in differentiated C2C12 myofibers.4 Thus, there
is no sharp boundary of LEK1 protein expression as these cells are
exiting the cell cycle. This pattern of LEK1 protein localization
contrasts with previously published reports for both CENP-F and mitosin
in mitotic cells (2-4).
Functional Analysis of LEK1 during Differentiation--
Data from
the previous sections suggest that the subcellular distribution of LEK1
is dynamic during mitosis and differentiation of skeletal myogenic
cells. In addition, our data suggest a general relationship between the
presence of LEK1 protein and maintenance of mitosis during
embryogenesis. LEK1 distribution during mitosis and differentiation is
significantly different from that reported for CENP-F, mitosin, and
CMF1, suggesting variation in function. Therefore, in an initial effort
to determine LEK1 function during cell differentiation, we sought to
overexpress a dominant-negative form of LEK1 in the C2C12 myogenic
system. Previous studies have demonstrated that a similar
dominant-negative lacking 5' sequences in mitosin leads to phenotypic
alterations in cultured fibroblasts (3). C2C12 cells were
co-transfected the dnLEK1 construct and a In the present study, we have cloned a novel mouse cDNA, LEK1,
that codes for transcripts that are related to human CENP-F/mitosin and
more distantly to chicken CMF1. The predicted LEK1 protein has
significant structural homology to these other proteins in their
primary sequence, the type and location of a number of protein motifs,
and their predicted overall protein structure. We present data that
show that LEK1 is a single copy gene that codes for two developmentally
regulated transcripts. These transcripts code for nuclear proteins that
are expressed ubiquitously at high levels early in development when
cells are most proliferative (26). Alteration of LEK1 function using a
dominant-negative form of the protein leads to phenotypic changes in
myogenic cells during differentiation. Thus, although LEK1 is
structurally related to the chicken and human genes, our data suggest
that it is a unique member of this gene family.
Conserved Motifs in the LEK family of Genes--
CMF1,
CENP-F/mitosin, and LEK1 share many structural characteristics.
Computer predictions of the LEK family of proteins show them to be
highly
The globular C terminus of LEK proteins contains a number of collinear,
conserved motifs. A bipartite nuclear localization signal (NLS) (Fig.
1) is a prominent feature in all three proteins (18). The present data
showing nuclear localization suggest that this NLS is functional in the
LEK1 protein. Just C-terminal to the NLS is an atypical Rb protein
binding domain (Fig. 1), that Lee and co-workers (3) have shown to bind
Rb. We have determined that the C terminus of CMF1 can bind E-proteins
that are helix-loop-helix transcription
proteins.5 Furthermore, LEK1
and CENP-F/mitosin have a computer-predicted helix-loop-helix
dimerization domain (Fig. 1, asterisk) (13). Thus, LEK
proteins may participate in the transcriptional activities via this
C-terminal domain.
Potential Differences among the LEK Proteins--
Independent
immunological observations have localized the CENP-F and mitosin
proteins to paired foci near the centromeres of chromosomes in the late
prophase and metaphase stages of mitosis (2, 3). These researchers have
gone further to show that the CENP-F and mitosin proteins bind the
outer plate of the kinetochore in dividing cells (4, 6). In the current
study, we found that the LEK1 protein is localized differently. As seen
in Fig. 8, LEK1 staining is observed throughout the entire cell during these stages of mitosis, with the exception of the condensed
chromosomes (columns 2 and 3, compare LEK1 Ab and
DAPI). Another notable difference between LEK1 and CENP-F/mitosin is
the localization of these proteins during cytokinesis.
Anti-CENP-F/mitosin antibodies brightly stained areas near the midbody
during cytokinesis (2, 3). The arrows in the last column
(Myotube) of Fig. 8 clearly show that anti-LEK1 does not
stain the midbody in actively dividing C2C12 cells. Additionally, CENP-F/mitosin has been shown to be regulated in a cell
cycle-coordinated manner, being up-regulated in late G1/S
and down-regulated in late telophase (2, 3), whereas LEK1 is present in
the nuclei of cells that are essentially at G0 (myotubes in
Fig. 8) (25). In addition, not just cells that are actively dividing
but the vast majority of early embryonic cells had nuclear staining
throughout the entire nucleus (Fig. 4). This in vivo
staining indicates that the LEK1 protein is not regulated like
CENP-F/mitosin is in HeLa cells. Thus, despite the similarities among
the primary, secondary, and tertiary structures of CENP-F/mitosin,
CMF1, and LEK1, these proteins differ significantly in their
subcellular localizations and temporal regulation in mitotic cells.
Evidence of a LEK Family of Genes--
As mentioned above, the
chicken, human, and mouse proteins have significant homology along
their entire lengths and at all levels of protein structure (Fig. 1).
Yet, the nature of the relationship of these molecules to each other is
confounded by the differences that we have observed among them. The
evolutionary distance between these molecules can be explained by two
hypotheses. 1) The three genes are the same gene in three different
species (i.e. orthologous genes) and have undergone
significant divergence since the avian (reptilian)/mammalian split, or
2) they are related members of a novel gene family (i.e.
paralogous genes). The observation that a CMF1 probe and a LEK1 probe
hybridize to two distinct bands in chicken genomic Southern blots
supports the latter hypothesis (arrows in Fig. 2).
Furthermore, the numerous bands that we have observed in low stringency
Southern analysis are indicative of a gene family, although some of
these genomic bands may correspond to pseudogenes. Additional support
for CMF1, CENP-F/mitosin, and LEK1 being paralogous members of a gene
family is provided by the subcellular localization of the three
proteins. CMF1 subcellularly localizes to the cytoplasm and is
restricted to developing cardiac and skeletal muscle,5
human CENP-F/mitosin binds the kinetochore of dividing cells and is
cell cycle-regulated, and mouse LEK1 does not bind the kinetochore, is
ubiquitously expressed, and is developmentally regulated. The potential
differences in protein localization and function may be explained by
multiple genes or by differential post-transcriptional processing of a
single gene product (hypothesis 1). We postulate that the LEK1 gene
produces two transcripts that may account for two proteins with variant
function and subcellular localization.
We found that mouse LEK1 is a developmentally regulated nuclear protein
that is down-regulated in the heart when mitotic activity of
cardiomyocytes ceases (19, 20). However, we do not see any evidence
that LEK1 is part of the general mitotic apparatus because it, unlike
CENP-F and mitosin, does not appear to bind the kinetochore, because it
is not associated with the condensed chromosomes and appears to be
present in all phases of mitosis. Furthermore, we do not see LEK1 in
actively dividing adult tissues such as the seminiferous tubules (Fig.
6) or crypts of the intestinal epithelium.3 The chicken
CMF1 protein has not been found to be part of the mitotic apparatus and
is tissue-restricted, being found only in the cytoplasm of heart and
skeletal muscle cells.5
From these data, we postulate that LEK1 function may be involved in
developmental mitosis but not in general cell reduplication. The human
CENP-F and mitosin proteins may also have developmental roles but are
deregulated in transformed cells. This interpretation is consistent
with these proteins having been found in a variety of human cancers and
autoimmune disorders (8-12), in which the normal expression pattern of
CENP-F and mitosin may well be perturbed. Chicken CMF1 is a
developmentally regulated, tissue-restricted protein that, like other
members of this proposed family, has protein motifs that allow it to
interact with other proteins. The LEK family of proteins may provide
cytoskeletal structural information in the differentiating cell to the
nucleus, where they could participate in transcriptional activities
and/or the cell cycle machinery.
Potential Functions for LEK1--
Our initial experiments using
dnLEK1 suggest that disruption of LEK1 function leads to phenotypic
changes in myogenic differentiation. Previous studies using similar dn
forms of mitosin also demonstrate phenotypic changes in mitotic
activity in cultured cells (3). Our data show that differentiation of
skeletal myoblasts transfected with dnLEK1 is accelerated or enhanced.
It is possible that disruption of LEK1 function alters the relationship
between cell division and differentiation in skeletal myogenesis. Many
groups have shown that decreasing mitotic activity enhances myogenic
potential (Ref. 28 and references within). Alternatively, disruption of
LEK1 function may directly promote myogenic differentiation. In either case, the present study suggests LEK1 has a potential role in regulating skeletal myogenesis and may even have a broader role in cell
differentiation in embryogenesis.
*
This work was supported by National Institutes of Health (NIH)
Grants HL37675 (to D. B.) and HL09916 (to R. L. G).
Experiments/analysis were performed in part through use of the VUMC
Cell Imaging Resource supported by NIH Grants CA68485 and DK20593.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 615-936-1976;
Fax: 615-936-3527; E-mail: david.bader{at}MCMAIL.vanderbilt.edu.
2
L. M. Pabón-Peña and D. Bader,
unpublished results.
3
R. L. Goodwin, L. M. Pabón-Peña, G. C. Foster, and D. Bader, data not shown.
4
M. E. Dees, R. L. Goodwin, and D. Bader, manuscript in preparation.
5
L. M. Pabón-Peña, R. L. Goodwin, and D. Bader, manuscript in review.
The abbreviations used are:
CENP, centromere
protein;
Rb, retinoblastoma;
DAPI, 4,6-diamidino-2-phenylindold;
NLS, nuclear localization signal;
DMEM, Dulbecco's modified eagle medium;
dpc, days post-coitum;
RT-PCR, reverse transcription-polymerase chain
reaction;
dn-, dominant-negative;
The Cloning and Analysis of LEK1 Identifies Variations in the
LEK/Centromere Protein F/Mitosin Gene Family*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32P]dCTP (Amersham
Pharmacia Biotech). This probe was then hybridized to the genomic blot
overnight at 65 °C. The blot was first washed three times in 2× SSC
(sodium chloride (150 mM) and sodium citrate (15 mM), pH 7.0), 0.1% SDS for 1 h/wash at 55 °C and
exposed overnight using Biomax intensifying screen and Biomax film
(Eastman Kodak Co.). This washing and exposure protocol was repeated at
60 and 65 °C. All counts were stripped from the blot with boiling
0.05% SDS. It was then reprobed with the corresponding of region of the chicken CMF1 transcript in a similar manner as noted above.
-Gal, 5 µgs of the control pCI Neo
vector, and 200 ngs of p-Gal or no DNA using Fugene (Roche Molecular
Biochemicals) as per the manufacturer's instructions. These plates
were grown in 20% fetal bovine serum DMEM for 24 h. The media was
then changed to differentiation media (4% heat-inactivated horse
serum) to promote myogenesis. Cells were maintained in this media for 3 days, at which time the plates were stained with
5-bromo-4-chloro-3-indolyl -D-galactopyranoside using
the manufacturer's instructions provided in the -Gal kit (Invitrogen). Blue-staining cells were counted and scored as single cells or differentiated myotubes in control and experimental groups. Transfection data was analyzed by two-sided 
2 methods
with a significance level set at p < 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices
separated by turns except for the proline-rich, globular C terminus. A
number of these -helices are perfect leucine zippers, whereas others
have aliphatic heptad repeats. These secondary structures have been
shown to be important mediators of both protein-DNA and protein-protein
interactions (15-17). The number and general positions of the leucine
zippers (Fig. 1, blue ovals)
are conserved among these proteins. Another shared feature of the three
proteins is their predicted tertiary structures. Using the Coiledcoil
program (14), the -helices of the LEK1 protein are predicted to fold into four coiled coils with intervening turns or loops.
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Fig. 1.
General structure of the LEK family of
proteins. A scheme of the conserved structures is shown in
A. The blue ovals represent leucine zippers. The
red rectangle denotes a spectrin repeat. The yellow
stripe symbolizes the bipartite NLS. The black stripe
depicts an atypical Rb binding domain (3). The asterisk
indicates the position of a myc-type basic helix-loop-helix
heterodimerizaton domain. In B, the amino acids of the well
conserved C termini of the mouse LEK1, the human CENP-F/mitosin, and
chicken CMF1 proteins are aligned (MacVector). The various motifs are
bracketed and labeled.
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Fig. 2.
Genomic Southern blot analysis of humans,
mice, and chickens with LEK1 and CMF1 sequences. 10 µgs of
genomic DNA from mice, humans, and chickens were cut with the denoted
restriction endonucleases and blotted. In A, the blot was
probed with the well conserved 3' region of the mouse LEK1 cDNA. In
B, the same blot was stripped and reprobed with the
corresponding region of the chicken CMF1 cDNA. The
arrows mark the migration of the LEK1-hybridizing band and
the CMF1-hybridizing band in chicken genomic DNA.
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Fig. 3.
Temporal and spatial Northern blot analysis
of the LEK1 transcript in the developing mouse. The same well
conserved 3' region of the mouse LEK1 cDNA used in the Southern
blot analysis was used to determine the expression of this transcript
in mouse development. In A, 10 µgs of total RNA from the
indicated embryonic and adult tissues were analyzed for the presence of
the LEK1 transcript. Two bands of approximately 10 kilobases each are
observed. The most abundant levels of the LEK1 message are seen in the
earlier stages of mouse development. Ethidium bromide staining of 28S
rRNA is shown as a loading control. In B, a Northern blot of
staged mouse heart RNA (kindly provided by the laboratory of Dr. Loren
Field) was used to determine temporal expression of the LEK1 transcript
in this organ. 28 S rRNA is shown as a loading control. A sharp drop in
LEK1 transcript levels was observed between 4 and 7 days after birth
(compare N4 and N7).
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Fig. 4.
Localization of the LEK1 protein in the limb
bud of a 9.5-dpc mouse embryo. The torso of a 9.5-dpc embryo was
cryo-sectioned and stained with affinity-purified anti-LEK1 and
counterstained with YoPro-1, a nuclear marker. The sections were
visualized via confocal microscopy. A shows the position of
all cells with the YoPro nuclear marker. B shows the
LEK1-positive nuclei. C is a composite of the two images.
The arrows show nuclei that are YoPro-positive and
LEK1-negative. The vast majority of the cells in the apical ectodermal
ridge and subjacent mesenchyme of the limb bud are LEK1-positive, as
shown by the number of yellow nuclei.
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Fig. 5.
Localization of the LEK1 protein in the
developing mouse heart. Staged embryonic and post-partum mouse
hearts were sectioned and stained with an affinity-purified anti-LEK1
antibody (red fluorescence in LEK1 Ab row) and
counterstained with DAPI (blue fluorescence in DAPI row).
The sections were also photographed under phase contrast light
microscopy (Phase row). The LEK1 protein is down-regulated
in between 5 and 7 days post-partum in a manner consistent with the
Northern blot analysis. All photographs are at 500×; the same exposure
was used for each row.
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Fig. 6.
Localization of the LEK1 protein in adult
gonads. Mouse adult gonads were cryo-sectioned and stained with
anti-LEK1 and counterstained with DAPI. The pronucleus of the oocyte
stained prominently, whereas the pronucleus of mature sperm were
LEK1-negative.
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Fig. 7.
Localization of the LEK1 protein in
mononucleated C2C12 cells. C2C12 cells maintained in high serum
are proliferative. All nuclei in these cells were observed to be
LEK1-positive with minor, punctuate staining observed in the cytoplasm
of some cells.
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Fig. 8.
Localization of the LEK1 protein in mitotic
C2C12 cells and differentiated C2C12 myotubes. Mitotic C2C12 cells
were observed in proliferating cultures and stained with anti-LEK1 and
counterstained with DAPI. The mitotic figures were staged by the
appearance of the chromosomes. Column 1 is a cell in late
prophase/early metaphase. Column 2 shows a cell in
metaphase. Column 3 shows a cell in late anaphase/early
telophase. Column 4 shows a cell in cytokinesis. During
metaphase, LEK1 staining is observed in all parts of the cell except
for the condensed chromosomes. The arrow shows the location
of the midbody, an area that stains with anti-CENP-F and anti-mitosin
antibodies (2, 3). Myotubes were induced to form by maintaining C2C12
cells in low serum conditions for 4 days. Nuclei in these myotubes are
LEK1-positive; there also appears to be punctuate staining in the
cytoplasm as well.
-Gal-containing plasmid,
control vector, and the -Gal-containing plasmid, or
mock-transfected. Cells were maintained in growth medium for 24 h,
switched to differentiation medium for 72 h, and processed for
-Gal staining. In transfected cultures, blue-stained cells were
scored as either mononucleated cells or differentiated myotubes in each
experiment. In control cultures, the ratio of transfected cells in
differentiated myotubes to mononucleated myoblasts was 0.17 (Fig.
9). In contrast, in dnLEK1-transfected cultures, the ratio of transfected cells in differentiated myotubes rose significantly to 0.46 (significance is p < 0.0001). The results of these experiments indicate that dnLEK1
accelerated or enhanced the differentiation of C2C12 myoblasts into
myotubes in culture.
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Fig. 9.
Transfection analysis of LEK1 function during
skeletal myogenesis. C2C12 cells are maintained, transfected with
control or dnLEK1 plasmids, and analyzed for myogenic differentiation
as described under "Experimental Procedures." Transfected cells
were visualized by 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside staining and scored as
mononucleated cells or differentiated myotubes. Examples of these two
phenotypes are shown in A. A.1 shows a
dnLEK1-transfected myotube, whereas A.2 shows a
mononucleated cell in of a control transfection. Fig. 9B
summarizes the transfection data as ratios of differentiated cells over
mononucleated cells in dnLEK (solid bar) and control
(striped bar) groups. The difference in the two groups was
analyzed using a two-sided 2 and found to be significant
to p < 0.0001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical with intervening turns. The conserved C termini of
these proteins are rich in proline and glycine (helix breakers) and
have no obvious computer-predicted secondary structure, although this
region does contain some other important motifs that are discussed
below. The highly -helical regions contain a number of leucine
zippers with many charged residues. Leucine zippers have been shown to
be important mediators of protein-protein interactions and protein-DNA
binding (15-17). Indeed, CENP-F and mitosin have been shown to
homodimerize and interact with a number of other proteins (3, 4-7).
The -helices of LEK1 are predicted to fold into four coiled coil
structures (13) again with intervening turns. Yen and co-workers (2)
have similarly reported that CENP-F is predicted to contain four coiled
coils and a globular C terminus that is proline-rich. In LEK1, one of
the coiled coils located near the middle of the protein contains a
spectrin repeat (red bar in Fig. 1) and may interact with
elements of the cytoskeleton such as spectrin, -actinin, dystrophin,
or utrophin (27).
FOOTNOTES
Supported by National Institutes of Health Training Grant HL07723.
ABBREVIATIONS
-Gal, -galactosidase.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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