(Received for publication, September 3, 1995)
From the
Genetic manipulation has proven valuable in identifying the role
of specific genes in cellular function. Genomic disruption of genes
that are expressed during embryonic development or in multiple tissue
types, however, complicates phenotypic analysis. We demonstrate that
targeted expression of an inhibitor peptide derived from myosin light
chain kinase can neutralize the function of calmodulin. We have shown
that elimination of the nuclear function of
Ca-calmodulin causes disruption of the nuclear
structure. Targeted expression of this calmodulin inhibitor gene in the
lung epithelium of transgenic mice leads to cellular death and
dysfunctional lung development. This approach is a strategy to modify
the activity of a targeted protein within a specific organelle in order
to evaluate its role in cellular and tissue function.
Calmodulin (CaM) ()mediates intracellular
Ca
by regulating cellular enzymes in a
Ca
-dependent manner. A variety of in vitro techniques in many biological systems have been used to implicate
CaM in nuclear activities. The nucleus contains several CaM-binding
proteins (1) including protein kinases and
phosphatases(2, 3) . Ca
-CaM is
capable of directly binding transcription factors containing basic
helix-loop-helix domains and can differentially inhibit in vitro transcription(4) . In vitro, CaM kinase II can
phosphorylate the cAMP response element-binding
protein(5, 6) . Attenuation of interleukin-2
transcription occurs in T-cells expressing a constitutively active CaM
kinase II(7) . Inhibitors of calcineurin, a
Ca
-CaM-dependent phosphatase, alter the in vitro transcription of many early response
genes(8, 9) .
Proteolytic mapping of a major CaM
target enzyme, skeletal muscle myosin light chain kinase (MLCK), was
used to identify a peptide that binds Ca-CaM with an
affinity (10
M) similar to that of the
native enzyme(10, 11) . This peptide is an effective,
specific competitive inhibitor of CaM-dependent enzymes. In the present
study, the MLCK CaM binding domain was used to design a synthetic
concatemer gene that would target and functionally neutralize CaM in
the nuclei of cultured cells and in lung epithelium of transgenic mice.
Affinity columns were
constructed using 5 mg of the free peptide dissolved in 125 mM borate (pH 8.4) containing 75 mM NaCl and 5 ml of
CNBr-activated Sepharose (Pharmacia Biotech Inc.) Immune sera were
centrifuged (10,000 g for 15 min) and 5 ml applied to
the affinity resin. The column was washed with 100 ml of 0.1 M
NaHCO
(pH 8.4) containing 0.5 M NaCl. The bound
IgG was eluted with 0.1 M glycine (pH 2.7) and immediately
neutralized to pH 7.4 with 1 M Tris (pH 9.0).
We used the rabbit skeletal muscle MLCK 19-amino acid
CaM-binding sequence to design a gene, which, when expressed, would
eliminate the cellular function of Ca-CaM by
competing with endogenous target proteins. In order to test whether the
synthetic gene product would inhibit CaM, the MLCK peptide gene was
expressed in bacteria as a fusion protein. The fusion protein, like the
chemically synthesized peptide, inhibits
Ca
/CaM-dependent protein kinase II in a
concentration-dependent manner (Fig. 1).
Figure 1:
Inhibition of
Ca/CaM-dependent kinase II activity by recombinant
and synthetic calmodulin binding peptides. The
and
subunits
of CaM kinase II and myelin basic protein were phosphorylated in the
presence of 100 µM Ca
and 0.5 µg of
CaM. The molar ratio of synthetic peptide to CaM was approximately 1:3,
1:1.5, and 1:0.75, and fusion protein to CaM was approximately 1:3,
1:2, and 1:1, respectively. The synthetic peptide, KRRWKKNFIAVSAANRFKK,
and the purified GST-MLCK peptide fusion protein inhibited CaM kinase
II activity in a concentration-dependent
manner.
CaM is composed of
two highly structured globular Ca-binding domains
connected by an
-helical peptide(14) . In the presence of
Ca
, the lobes engulf the target peptide like a
vise(15) . The inhibitor peptide gene for in vivo studies was designed to include two properties, high binding
capacity and intracellular targeting. Due to the fact that calmodulin
is abundant in cells, the monomer gene was self-ligated into four
tandem repeats in order to increase the calmodulin binding capacity of
each transcript and to target localization to the nucleus. The sequence
of the first two repeats of the concatemer is:
MKRRWKKNFIAVSAANRFKKLGMKRRWKKNFIAVSAANRFKKLG. This sequence contains
several basic amino acid clusters and the bipartite lysine-lysine
motif, KK . . . . . . . KK (underlined). These are potential nuclear
localization signals common in many karyophilic proteins such as the
SV40 T-antigen and transcription factors(16) . In order to
evaluate the cellular targeting of the CaM inhibitor gene and its
effects in intact cells, COS-7 cells were transiently transfected with
the concatemer gene. The cells were examined by indirect
immunofluorescence using affinity purified antibody produced against
the inhibitor peptide. As shown in Fig. 2a,
approximately 10% of the cells expressed the MLCK peptide concatemer,
which was concentrated in the nuclei of transfected cells (Fig. 2, c and d). During early stages of
expression, the peptide staining pattern was ``donut-like'' (Fig. 2e). By 72 h, the nuclei became extremely large
and multi-lobed, and contained condensed chromatin indicating
disruption of nuclear function (Fig. 2f). It did not
appear that the MLCK peptide-transfected cells entered mitosis. A
splicing isoform of CaM kinase II contains the nuclear targeting
sequence, KKRK, and is localized in the nucleus(3) .
Microinjection of a CaM kinase II inhibitory peptide into sea urchin
eggs causes a delay in nuclear envelope breakdown(17) .
Targeted overexpression of the MLCK peptide in the nucleus may inhibit
CaM kinase II activity preventing cells from entering mitosis. Double
immunostaining of transfected cells demonstrates coincident
localization of CaM and the MLCK peptide in the nucleus. CaM is also
present throughout the cytoplasm, indicating that only nuclear
functions of CaM were being neutralized (Fig. 2, g and h).
Figure 2: Immunolocalization of the MLCK concatemer peptide in transfected COS cells. COS-7 cells transiently transfected with the MLCK peptide gene (a) or pSVL vector alone (b) were immunostained with affinity-purified anti-MLCK peptide antibody 48 h following transfection (10x), c, transfected cells stained with anti-MLCK peptide; d, DNA counter-stained with 50 µg/ml propidium iodide (100x); e, ``donut'' staining pattern in nucleus following 24 h of transfection (100x); f, multi-lobed nucleus with intense nuclear focal staining after 72 h of transfection (100x). g, double immunostaining of cells 72 h after transfection with affinity-purified rabbit anti-MLCK peptide identified with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (100x); h, double immunostaining of cells 72 h after transfection with affinity-purified sheep anti-calmodulin visualized with CY3-conjugated rabbit anti-sheep IgG.
In order to evaluate the consequences of expression of the MLCK peptide during cell growth and differentiation, the CaM inhibitor gene was targeted to a specific cell type in transgenic mice. Due to the potent inhibitory effects of the peptide observed in the CaM kinase II assay and in cultured cells, the lung was selected as the target organ since the animals would not require respiratory function until birth. The 3.7-kb 5` promoter region of the human SP-C gene dictates specific expression to type II cells of the distal airway epithelium(18) . Activation of gene expression by the 3.7-kb SP-C promoter occurs approximately on day 11 of fetal life, when a definable lung epithelium first appears.
Our transgene was composed
of the SP-C promotor, the four-repeat MLCK peptide concatemer gene, and
SV40 RNA processing motifs (Fig. 3a). Offspring of
transgenic founder mice displayed varying degrees of lung pathology,
which corresponded to congenital cystic adenomatoid malformation. In
this condition, the terminal bronchioli continue to develop in the
absence of alveolar epithelial proliferation. At birth, trapped air
causes cystic dilation of the bronchioles. Alveoli that appeared normal
were found among the cysts in some transgenic animals. One founder, a
female, demonstrated no obvious physical symptoms. Her litters
contained two transgenic phenotypes. Some pups displayed cyanosis and
died within 15 min of birth (Fig. 3b). All of the
surviving transgenic animals were females. They demonstrated retarded
post-natal development as exhibited by a 10-20% reduction in body
size and displayed delayed hair growth, eye opening, and tooth eruption (Fig. 3c). Affinity-purified antibodies produced
against the MLCK peptide showed that expression of the transgene
product occurs by approximately day 13.5 of embryonic development (Fig. 3d). Cellular differentiation and bronchial
branching began to decrease. Dilated tubules appeared at day 15.0 (Fig. 3e). There was no anti-MLCK peptide staining in
the lung of day 16.5, 17.5, and post-natal male transgenic animals.
Histological examination of tissue sections revealed that the
transgenic male animals that died at birth had not developed a lung
epithelium or lower airways (Fig. 3f). These results
indicate that the type II cells that express the inhibitor peptide did
not survive early development of the lung epithelium. The trachea and
all major organs were histologically normal. Analysis of subsequent
F, F
, and F
generations confirmed
that all of the transgenic males died at birth and that all of the
surviving transgenic animals were female (Fig. 4).
Figure 3: Morphological analysis of mice expressing the MLCK peptide transgene. a, schematic representation of a transgene designed to target expression to the lung epithelium. The arrows designate the primer sites used for PCR analysis of the transgene using DNA obtained from tail-clips. b, transgenic mice (TG), which died within 15 min after birth and demonstrated cyanosis and little movement. The non-trangenic (NTG) littermates appeared normal. c, female transgenic (TG) mice demonstrated retarded physical development, including reduced body weight and hair growth when compared to their non-transgenic (NTG) littermates. Immunostaining of lung cross-sections from 13.5-day-old (d) and 15.0-day old (e) transgenic embryos using the affinity-purified anti-MLCK peptide antibodies (20x). f, dilated cysts in the lung of a newborn male transgenic mouse (10x). g, alveoli of the lung of a non-transgenic littermate (10x).
Figure 4:
Pedigree of transgenic mice Carrying the
X-linked dominant MLCK peptide concatemer gene. This pedigree shows
F, F
, and F
generations of X-linked
founder mouse (F
) in which
&cjs0604; =
transgenic males that died at birth;
&cjs0604; =
transgenic females that died at birth;
= surviving
transgenic females;
= non-transgenic male;
= non-transgenic female.
The transgene is lethal in males, suggesting an X-linked dominant trait. During morula development of females, there is random inactivation of one of the X-chromosomes, which form a Barr body. All of the cells in a given lineage inactivate the same X-chromosome throughout the life of the animal(19) . In the present study the ``Lyonized'' females develop a mosaic lung epithelium. In some proliferating type II cells, and their clonal descendants, the CaM inhibitor peptide gene would remain silent, enabling some of the heterozygotic transgenic females to survive. Most of the transgenic females lived to adulthood, reproduced, and had a normal lung morphology. These studies confirm that progenitor type II cells are important for normal lung development.
Genetic manipulation of organisms at all levels of complexity has proven valuable in identifying the role of specific genes in cellular function. Gene ``knock-outs'' by genomic homologous recombination in embryonic stem cells allows for the selective disruption of targeted genes(20) . Ablation of genes that are critical for the function of many cell types influences fetal development and can prove lethal. Indeed, disruption of fungal CaM genes demonstrates that CaM is essential for survival(21, 22, 23) . Our study demonstrates that binding peptides can be used to selectively neutralize the function of a targeted protein such as CaM in a specific organelle of a defined cell type. Our findings also suggest that the mosaic inactivation of the MLCK peptide gene in lung allowed the unaffected clonal type II cells to proliferate and compensate for early loss of the cells expressing the concatemer peptide. These findings demonstrate that dominant lethal genes can be sustained in the germ-line of animals by targeting transgene integration into the X-chromosome. Male progeny display the most severe phenotype, while females demonstrate a broad spectrum of phenotypes depending upon the ratio of X-chromosome inactivation of the transgene during early embryogenesis.
Organism organization is based upon recognition between individual biochemical components. Cell adhesion, assembly of organelles, and signal transduction pathways are each, for example, a complex series of molecular interactions. Properties of individual proteins are altered through covalent modifications and ligand binding. Stimuli initiate a cascade of protein conformational changes. These changes, in turn, modify the properties of other proteins in sequential order. These physical interactions transmit signal bifurcation and propagation throughout the cell, ultimately leading to a cellular response. Analysis of protein-protein and protein-nucleic acid associations has shown that the interactive domains are highly structured and are often composed of less than 25 amino acids(24, 25, 26, 27, 28, 29, 30) . These domains of recognition are attractive target sites for the design of peptides that modify specific cellular pathways. Identification and design of binding peptides that modify a targeted protein activity can be obtained by several techniques including biochemical fragmentation, three-dimensional structural analysis, mutagenesis of interacting species, two-hybrid transcriptional systems, and libraries of random peptides(27) . Once identified, the binding properties of a selected peptide can be further refined by mutagenesis and reselection. Due to the fact that cellular processes involve a cascade of protein-protein interactions, the interference of molecular recognition by the use of binding peptides should be applicable to modulate any targeted cellular function.