(Received for publication, October 27, 1995; and in revised form, November 13, 1995)
From the
Myotrophin is a soluble-12 kilodalton protein isolated from
hypertrophied spontaneously hypertensive rat and dilated
cardiomyopathic human hearts. We have recently cloned the gene coding
for myotrophin and expressed it in Escherichia coli. In the
present study, the expression of myotrophin gene was analyzed, and at
least seven transcripts have been detected in rat heart and in other
tissues. We have further analyzed the primary structure of myotrophin
protein and identified significant new structural and functional
domains. Our analysis revealed that one of the ankyrin repeats of
myotrophin is highly homologous specifically to those of
IB
/rel ankyrin repeats. In addition, putative
consensus phosphorylation sites for protein kinase C and casein kinase
II, which were observed in I
B
proteins, were identified in
myotrophin. To verify the significance of these homologies,
B gel
shift assays were performed with Jurkat T cell nuclear extract proteins
and the recombinant myotrophin. Results of these assays indicate that
the recombinant myotrophin has the ability to interact with
NF-
B/rel proteins as revealed by the formation of ternary
protein-DNA complexes. While myotrophin-specific antibodies inhibited
the formation of these complexes, rel-specific p50 and p65
antibodies supershifted these complexes. Thus, these results clearly
indicate that the myotrophin protein to be a unique rel/NF-
B interacting protein.
Cardiac myocyte cell hypertrophy has been used as an in
vitro model for studying cardiac hypertrophy. Cardiac myocytes
respond to hemodynamic overload by altering the expression of specific
set of genes, which are needed for hypertrophy. Our laboratory has been
studying the molecular basis of myocardial hypertrophy using
spontaneously hypertensive rat as an animal
model(1, 2, 3) . Earlier, Sen et al.(1, 2) isolated a novel 12-kilodalton protein,
which we named myotrophin, from the hypertrophied ventricles of
spontaneously hypertensive rat (1) and dilated cardiomyopathic
human hearts (2) based on its ability to stimulate protein
synthesis specifically in cardiac myocytes(1) . Recently, we
have isolated the cDNA clones encoding rat myotrophin (4) ()and found that the cardiac myotrophin is
identical to a previously reported rat brain v1 protein (7, 8) whose function is not determined at present in
brain. In addition, we have expressed the myotrophin protein in Escherichia coli and showed that the recombinant myotrophin
has the ability to stimulate protein synthesis in neonatal cardiac
myocytes (4) .
In the present study, analyzed the
expression of myotrophin in various tissues, identified new structural
and functional domains, and, using the recombinant myotrophin,
determined one of the key activities of myotrophin.
Figure 1: A, distribution of myotrophin mRNAs in various tissues. H, heart; B, brain; S, spleen; L, lung; Li, liver; Sk, skeletal muscle; K, kidney; T, testis. B, Northern analyses of myotrophin transcripts in 9-day-old WKY hearts. Lane a represents the no-salt buffer-eluted poly(A) RNA, and lane b represents RNA from low salt wash fraction.
Figure 2:
A,
ankyrin repeats and putative phosphorylation sites for protein kinase C
and casein kinase II are highlighted on the indirectly predicted amino
acid sequence of myotrophin. B, homology of myotrophin ankyrin
repeat 2 to IB
ankyrin repeats. It should be noted that in
addition to rel-associated pp40, the BLASTP analysis
identified other I
B
members (MAD3, RL/IF-1, and ECI-6) on the
same ankyrin repeats 2 and 4 with similar Poisson
values.
The ankyrin repeats of myotrophin span from amino acid residues 9 to 107 (Fig. 2A). Myotrophin possesses two full-length ankyrin repeats (repeat 2, 27-58 and repeat 3, 59-91) and two incomplete (half) repeats (repeat 1, 9-26 and repeat 4, 92-107). One incomplete repeat (9-26) is a carboxyl half of a typical ankyrin repeat, and the other (92-107) is an amino half (Fig. 2A). Ankyrin repeats are generally 33 amino acids in length and possess two regions: one region is highly conserved and the other is highly variable. A typical ankyrin repeat sequence is shown below, where X indicates a highly variable region compared with the rest of the conserved region: XGXTPLHXAXXLLXXGADXXXDX.
Since the ankyrin repeats are found in various classes of proteins
(cytoplasmic, nuclear, and cell surface), Hatada et al.(13) have attempted to classify these ankyrin repeats. Based on
the sequence homology in the variable region of these ankyrin repeats,
Hatada et al.(13) have proposed a unique subgroup of
ankyrin repeats for rel and related IB transcription
factors (9, 10, 11, 12) . The BLASTP
analysis of myotrophin revealed a significant homology between the
ankyrin repeat 2 (residues 27-58) of myotrophin and two ankyrin
repeats of I
B
proteins (Fig. 2B). The
myotrophin ankyrin repeat 2, in addition to the homology in the core
consensus sequence, possesses homologous residues in the variable
region similar to the I
B
ankyrin repeat (Fig. 2B). Since the ankyrin repeats are considered as
modular protein-interacting domains with sub-regions of these repeats
conferring unique specificity, the observed homology between myotrophin
ankyrin repeat 2 and the two ankyrin repeats of I
B
proteins
(9-13) is considered very significant (Fig. 2B).
It is well known that the IB proteins interact with
NF-
B/rel factors through its ankyrin repeats
(15-19). Specifically, it has been shown that I
B
ankyrin repeats bind to rel domains of NF-
B subunits (p50
and p65). In addition to the ankyrin repeats, putative consensus
phosphorylation sites for protein kinase C and casein kinase II, which
were observed in I
B
proteins (9-12), were also observed
in myotrophin (Fig. 2A). However, myotrophin is only a
12-kilodalton protein with fewer ankyrin repeats than other known
I
B
proteins. The observation of I
B
homologous
ankyrin repeats and putative consensus phosphorylation sites for
protein kinase C and casein kinase II in myotrophin suggested that
myotrophin may be a unique I
B-related protein.
Figure 3:
Electrophoretic mobility shift assays
analyzing the effect of recombinant myotrophin on the
NF-B/rel/
B DNA (A) and Oct-1/oct DNA (B) complexes. Phorbol ester-treated Jurkat T cell nuclear
extracts were used as source of NF-
B/rel and Oct factors.
Bacterially expressed recombinant myotrophin was added
(``+'' = 1 µl = 200 ng) to the binding
reactions, and its effect was analyzed on 4% PAGE. JNE-P,
phorbol ester-treated Jurkat T cell nuclear extract;
B,
radiolabeled
B DNA probe;
-myo, native
myotrophin-specific antibody IgG(5) ;
-p65,
antibody to p65 of NF-
B (supershifting); PI, preimmune
serum IgG; Oct, radiolabeled Oct DNA probe; SC,
myotrophin-shifted slower migrating complexes; FC,
myotrophin-shifted faster migrating complexes; NF-
B, rel-
B heterodimeric protein-DNA
complexes.
To further confirm the myotrophin interaction with NF-B/rel factors, EMSAs were performed in presence of p50 and p65
antibodies. The results are shown in Fig. 4. When incubated with
either p50 (lane 6) or p65 (lane 4) antibodies, the
myotrophin-shifted
B complexes were supershifted to slower
migrating ternary complexes (SSC-50 and SSC-65 in Fig. 4), and
the intensity of these complexes increased when more myotrophin was
present in the reaction. These results clearly show that
myotrophin-shifted protein complexes actually contain NF-
B/rel factors. It should also be noted that phorbol 12-myristate
13-acetate-induced Jurkat T cells probably contain a sufficient amount
of endogenous myotrophin since myotrophin-shifted
B complexes were
also detected at a lower level in the control experiments (lanes 1 and 2 in Fig. 4).
Figure 4:
Electrophoretic mobility shift assays
analyzing the effect of rel-specific p50 and p65 antibodies on
the myotrophin-shifted NF-B
rel-
B DNA
complexes. Phorbol ester-treated Jurkat T cell nuclear extracts were
used as a source of NF-
B/rel factors. rel-specific p50 and p65 antibodies were added to the
appropriate
B binding reactions. Bacterially expressed recombinant
myotrophin was added (``+'' = 1 µl =
200 ng) to the binding reactions along with appropriate antibodies, and
its effect was analyzed on 4% PAGE. JNE-P, phorbol
ester-treated Jurkat T cell nuclear extract;
B,
radiolabeled
B DNA probe; myo, recombinant myotrophin;
-p65, antibody to p65 of NF-
B complex;
-p50, antibody to p50 of NF-
B complex; SC,
myotrophin-shifted slower migrating complexes; NF-
B, rel-
B heterodimeric protein-DNA complexes; SSC-50, supershifted complex by
-p50; SSC-65,
supershifted complex by
-p65.
In the present study, we have shown that the myotrophin gene is expressed in various rat tissues and as much as seven myotrophin-specific transcripts have been detected in rat heart and in other tissues. These transcripts were most abundant in brain and least in skeletal muscle compared to other tissues. Based on its ubiquitous distribution, it appears that the myotrophin protein may be playing a very important role in the basic functions of various tissues.
Our
analysis on the primary structure of the myotrophin protein also
revealed the homology between one of the ankyrin repeats of myotrophin
and to those of IB
/rel ankyrin repeats. Furthermore,
our analysis showed putative consensus phosphorylation sites for
protein kinase C and casein kinase II in myotrophin protein, which were
also observed in I
B
proteins. The significance of these
homologies were experimentally confirmed with
B gel shift assays.
The results of these gel shift assays clearly show that the recombinant
myotrophin has the ability to interact with NF-
B/rel proteins in vitro. In vivo experiments are currently
being conducted to further confirm these results. Thus, these results
clearly indicate that the 12-kDa myotrophin protein is a unique rel/NF-
B interacting protein.
It has been very well
documented that upon exposure to a variety of external stimuli,
NF-B/rel proteins are involved in the rapid induction of
genes whose products play a central role in the immune responses,
inflammation, and cell
proliferation(15, 16, 17, 18, 19) .
The most obvious characteristic of NF-
B is its rapid translocation
from cytoplasm to nucleus in response to extracellular signals. They
are kept dormant in the cytoplasm by the members of the I
B family
of proteins. Many signals inactivate the inhibitor I
B, thereby
allowing the NF-
B to enter nuclei and rapidly induce coordinate
sets of defense-related genes. It is possible that upon exposure to
chronic hemodynamic overload signals, cardiac myocytes respond through
their NF-
B rapid response system to alter myocardial gene
expression. In the present preliminary study, we have shown by its
ability to interact with NF-
B in vitro that myotrophin is
probably a component of such a rapid response system, which might
influence the transcription of hypertrophy-specific genes. Based on the
present study, we speculate that myotrophin is probably involved in
regulating the expression of hypertrophy-specific genes in the
myocardium through rel factors and
B DNA sites. It should
be noted that no transcription regulatory factor has been reported so
far to be involved in cardiac hypertrophy. Further studies are in
progress to determine the exact mechanism of action of myotrophin.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U21661[GenBank].