(Received for publication, November 3, 1994; and in revised form, February 8, 1995)
From the
Dematin is an actin-bundling protein of the erythroid membrane
skeleton and is abundantly expressed in human brain, heart, skeletal
muscle, kidney, and lung. The 48-kDa subunit of dematin contains a
headpiece domain which was originally identified in villin, an
actin-binding protein of the brush-border cytoskeleton. The headpiece
domain of villin is essential for its morphogenic function in
vivo. Here we report the primary structure of 52-kDa subunit of
dematin which differs from the 48-kDa subunit by a 22-amino-acid
insertion within its headpiece domain. A unique feature of the
insertion sequence of the 52-kDa subunit is its homology to erythrocyte
protein 4.2. The insertion sequence also includes a cysteine residue
which may explain the formation of sulfhydryl-linked trimers of
dematin. Actin binding measurements using recombinant fusion proteins
revealed that each monomer of dematin contains two F-actin binding
sites: one in the headpiece domain and the other in the undefined
N-terminal domain. Although the actin bundling activity of intact
dematin was abolished by phosphorylation, no effect of phosphorylation
was observed on the actin binding activity of fusion proteins. Using
somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of
chromosome 8. The dematin locus, 8p21.1, is distal to the known locus
of human erythroid ankyrin (8p11.2) and may contribute to the etiology
of hemolytic anemia in a subset of patients with severe hereditary
spherocytosis.
Dematin is an actin-bundling protein that was originally
identified as a component of the human erythroid membrane skeleton (1, 2, 3) . Purified dematin consists of two
polypeptides of apparent molecular mass of 48 and 52 kDa,
respectively(1, 2, 3) . Recently we reported
the primary structure of the 48 kDa subunit of dematin that contains a
headpiece domain which was first defined in villin, an actin-binding
and -bundling protein(4) . The headpiece domain of villin is
essential for its actin bundling activity and is required for
villin's actin modulating function in the microvillar
cytoskeleton (5) . Dematin differs from villin in two important
respects. 1) In contrast to villin's restricted expression in
absorptive epithelia, dematin is expressed widely in many tissues of
distinct origin. 2) Unlike villin, the actin bundling activity of
dematin is regulated by phosphorylation of dematin by cAMP-dependent
protein kinase (2) . Based on these distinctive features, it
was suggested that dematin may substitute for villin in villin-negative
tissues to modulate the organization of filamentous actin in a
phosphorylation-dependent manner(4) .
In solution, dematin
exists as a trimer, and electron microscopy has shown that purified
dematin exhibits a trilobed
structure(1, 2, 3) . Each trimer of dematin
appears to contain two polypeptides of 48 kDa and one polypeptide of 52
kDa, as estimated by the elution of Coomassie Blue dye from bands after
gel electrophoresis(3) . Although the two subunits appear to be
structurally related, the origin of the 52-kDa subunit remained
unknown(3, 4) . The recent cloning of the 48-kDa
subunit showed that there is only a single cysteine residue in its
amino acid sequence(4) . This observation implied that there
must be an additional cysteine residue in the 52-kDa subunit to account
for the formation of sulfhydryl-linked trimers containing two subunits
of 48 kDa and one subunit of 52 kDa.
To resolve these issues, we
have determined the primary structure of the 52-kDa subunit of dematin.
Here we report the amino acid sequence of the 52-kDa subunit which was
deduced from the cDNA clones of dematin transcripts present in human
reticulocytes. We also show that each subunit of dematin contains two
actin binding sites: one in the headpiece domain and the other in an
N-terminal undefined domain. Phosphorylation assays demonstrate that
the actin binding activity of these domains is not regulated by
phosphorylation. Finally, the fluorescence in situ hybridization data show that the dematin gene is located distal to
the ankyrin locus on the short arm of chromosome 8.
Glutathione-Sepharose, TA Cloning
The following primers were used to produce the cDNA
constructs. 1) Villin headpiece, Ala-753 to Phe-826, sense:
5`GCTAACAGCAACCTC; antisense: 5`TCAAAATAGTCC. 2) Dematin headpiece
domain with and without the insert sequence, Ser-309 to Phe-383, sense:
5`TCAGGGAGTGAGACTGGAAGCCCA; antisense: 5`GAAGAGAGAGGCCTTCTTCTTGA. 3)
The truncated headpiece domain of dematin, Pro-362 to Phe-383, sense:
5`CCTGAAGAGTTTGGC; antisense, 5`GAAGAGAGAGGCCTT. 4) N-terminal
undefined domain of dematin, Met-1 to Pro-308, sense:
5`ATGGAACGGCTGCAG; antisense: 5`TGGGCTGAACTCCGT. 5) The truncated
N-terminal domain, Ser-103 to Pro-308, sense: 5`AGCCGGTCGCCTGGA;
antisense: 5`TGGGCTGAACTCCGT. Thirty five cycles of PCR amplification
were carried out 55 °C annealing for 1.0 min, 72 °C elongation
for 1.0 min, and 94 °C denaturation for 1.0 min.
Figure 1:
Two-dimensional
Figure 2:
Cloning and sequence alignment of the
52-kDa subunit. A, domain organization of the 52-kDa subunit
of dematin. The primary structure of the 52-kDa subunit is identical
with that of the 48-kDa subunit except for the insert sequence within
the headpiece domain. B, the primary structure of the insert
sequence encodes 22 amino acids. C, a comparison of the insert
sequence with protein 4.2. Note that the first three amino acids (GLQ)
are contributed from the headpiece domain which is common between the
48-kDa and 52-kDa subunits. The remaining eight amino acids are derived
from the insert sequence of the 52-kDa
subunit.
Figure 3:
Expression of recombinant fusion proteins.
Glutathione S-transferase (GST) fusion proteins containing
defined domains of dematin, and the headpiece domain of villin, were
expressed in bacteria as described under ``Experimental
Procedures.''
Figure 4:
Actin binding activity of recombinant
fusion proteins. Specified domains of dematin and villin were expressed
in bacteria as glutathione S-transferase fusion proteins. The
location of respective constructs is described in Fig. 3. Actin
binding activity of purified fusion proteins was measured by a
sedimentation assay (see ``Experimental Procedures''). Bovine
serum albumin (BSA) was added to reduce nonspecific binding.
Coomassie Blue-stained SDS-PAGE pellet (P) and supernatant (S). The abbreviations are: GST-VHP, villin
headpiece; GST-DHP, dematin headpiece; GST-DHPI,
dematin headpiece with insert; GST-THP, dematin truncated
headpiece; GST-TUD dematin truncated undefined
domain.
Figure 5:
Effect of phosphorylation on the actin
binding activity of fusion proteins. Purified fusion proteins were
phosphorylated by cAMP-dependent protein kinase (see
``Experimental Procedures''). The details of actin binding
and abbreviations of fusion proteins are mentioned in Fig. 4.
The autoradiograph shows the pellet (P) and supernatant (S) of the actin binding experiments. Note that the in
vitro phosphorylation had no effect on the binding of fusion
proteins to actin filaments. Villin headpiece is not shown because it
was not phosphorylated under these conditions. The undefined domain of
dematin (GST-UD) is included to highlight the extreme
sensitivity of this construct to
proteolysis.
Figure 6:
Localization of genomic dematin clone 1977
to human chromosome 8p21.1 by fluorescence in situ hybridization. A, both chromatids are hybridized on each
chromosome 8 (arrows). B, corresponding DAPI-banded
chromsomes. The hybridized chromatids (two for each chromosome) are
indicated. Note: the signals were not amplified with anti-avidin so as
to reduce nonspecific background
hybridization.
In this manuscript, we describe the complete primary
structure of the 52-kDa subunit of dematin, an actin-bundling
phosphoprotein. The amino acid sequence contains an N-terminal
undefined domain and a C-terminal headpiece domain, an organization
similar to that of the 48-kDa dematin subunit (Fig. 2, Table 3). However, the headpiece domain of the 52-kDa subunit
contains an additional sequence of 22 amino acids suggesting that this
isoform arises from the inclusion of an alternatively spliced exon that
is not present in the 48-kDa isoform. This inserted sequence starts
after glutamine 319 of the headpiece domain of the 52-kDa subunit (Fig. 2). The elucidation of the primary structure of the 52-kDa
subunit may explain the formation of trimeric dematin in solution. Each
trimer of dematin appears to consist of two polypeptides of 48 kDa and
one polypeptide of 52 kDa (3) . Because of the presence of only
one cysteine residue in the 48-kDa subunits(4) , it was
postulated that there must be an additional cysteine residue in the
52-kDa subunit to allow formation of disulfide-linked trimers. As
proposed in Fig. 7, the second cysteine in the insert sequence
of the 52-kDa subunit should allow formation of a disulfide-linked
trimer between the 48-kDa and 52-kDa subunits. This result is also
consistent with our previous observation that the 52-kDa subunit
exhibits decreased mobility in the absence of dithiothreitol, a
reducing agent, whereas the mobility of the 48-kDa subunit remained the
same regardless of whether dithiothreitol was present(3) . It
is relevant to note that the disulfide-linked proteins are often
resistant to reduction in vivo(16) , and, thus, the
presence of sulfhydryl-linked trimers of dematin may be of
physiological significance in intact cells.
Figure 7:
A proposed model for the formation of
trimeric dematin. The presence of a second cysteine residue in the
insert sequence of the headpiece domain is sufficient to form a
disulfide-linked trimer between a 52-kDa subunit and two subunits of 48
kDa. The chemical cross-linking experiments have previously shown that
both subunits of dematin are cross-linked via disulfide bonds in
dematin trimers(1, 3) .
The availability
of the primary structures of dematin subunits allowed us to map the
location of actin binding sites within respective domains of each
subunit (Fig. 3). Each dematin subunit contains two actin
binding sites: one in the headpiece domain and the other in the
undefined N-terminal domain. The actin binding site in the headpiece
domain is located within the C-terminal 22 amino acids (Fig. 3).
The presence of an insert sequence of 22 amino acids in the headpiece
domain of the 52-kDa subunit has no effect on the actin binding
activity of this isoform in vitro. The location of an actin
binding site in the headpiece domain is consistent with the
localization of an actin binding site in the villin headpiece domain (5) . An attempt to further reduce the primary sequence of the
actin binding site in the dematin headpiece domain was not successful,
perhaps because of insufficient folding of the synthetic peptide. A
similar observation has been made previously with the headpiece domain
of villin(5) .
A protein monomer must contain at least two
actin binding sites to function as an actin-bundling protein.
Alternatively, self-association of monomers containing a single actin
binding site can also provide multiple sites for actin binding. Our
results show that both subunits of dematin contain a second binding
site for F-actin in their N-terminal undefined domains and hence allow
a dematin trimer to hold six actin filaments (Fig. 3). Moreover,
it appears that the actin binding activity of fusion proteins
containing either the headpiece domain or undefined domain was not
influenced by the state of their phosphorylation (Table 1, Fig. 5). This result is consistent with our previous observation
that the phosphorylated dematin remains bound to actin filaments
although it cannot bundle actin filaments(2, 3) .
Since both dephospho- and phosphorylated forms of dematin exist as
trimeric particles(1, 2, 3) , we propose that
phosphorylated dematin fails to bundle actin filaments because it
cannot hold filaments in correct orientation due to
phosphorylation-induced conformational changes. If true, the proposed
model may also explain the rapidity with which dematin regains its
actin bundling activity upon dephosphorylation(2, 3) .
The experimental evidence in support of this model will come from
elucidation of dematin's three-dimensional structure in both
dephosphorylated and phosphorylated forms.
The results of somatic
cell hybrids suggest that there is a single copy of the dematin gene on
chromosome 8. Further analysis by fluorescence in situ hybridization revealed that the dematin gene is located on the
short arm of chromosome 8 (8p21.1), which is distal to the SPH2 locus
containing the ankyrin gene (8p11.2)(19) . Previously, several
groups have shown that the locus for hereditary spherocytosis resides
on the short arm of human chromosome 8(20, 21) . Lux et al.(19) have demonstrated that a single copy of
the ankyrin gene is missing in two children with severe hereditary
spherocytosis due to a heterozygous deletion (p11-p21.1) of chromosome
8. Since red cell membrane proteins spectrin, actin, protein 4.1,
protein 4.2, and band 3 are not located on chromosome 8, it was
suggested that the partial deficiency of ankyrin 1 leads to severe
hereditary spherocytosis(19) . Our finding showing the location
of dematin gene on the short arm of chromosome 8 raises the possibility
that the abnormalities in the dematin gene may also contribute to the
genesis of hereditary spherocytosis perhaps by destabilizing the actin
binding end of spectrin tetramers in mutant erythrocytes(22) .
Alternatively, the mutant dematin gene may functionally manifest in the
abnormalities of nonerythroid tissues. Interestingly, a subset of
hereditary spherocytosis patients with chromosome 8 deletions suffers
from multiple neurological defects(19, 20) , and the
abundant expression of dematin in human brain may be of physiological
consequence in this context.
While our search continues for a
physiological basis of dematin's actin bundling activity, it was
considered necessary to elucidate the primary structure of its two
subunits. This information may help to explain the assembly of dematin
subunits into trimers and sheds light on the mechanism by which
phosphorylation regulates dematin's actin bundling activity. We
have previously suggested that, in mature erythrocytes, dematin may
function to link spectrin-actin complexes to the plasma membrane, a
function akin to another adaptor protein 4.1(4, 22) .
To accomplish its function in mature erythrocytes where actin bundles
appear to be absent, the actin binding activity of dematin appears to
be sufficient whereas dematin's actin bundling activity may be
functionally more relevant during erythropoiesis. Dematin is expressed
in early erythroblasts
Dematin is a
member of the villin superfamily by virtue of a common C-terminal
headpiece domain(4) . It is noteworthy that other members of
the villin superfamily such as gelsolin, severin, and fragmin lack
villin's headpiece domain and therefore do not bundle actin
filaments (24) . The presence of a headpiece domain therefore
appears to be required for the actin bundling function. To date, three
proteins are known to contain the headpiece domain. In villin, the
headpiece domain is necessary for the actin bundling activity and is
essential for villin's morphogenic function in
vivo(5) . Recently, the Drosophila quail gene has
been shown to encode a villin-like protein with a headpiece
domain(25) . Drosophila villin-like protein is
germ-line-specific, and its null mutations result in female sterility
due to an abnormal transport of cytoplasm from nurse cells into the
oocyte(25) . The primary molecular defect appears to be the
failure of actin bundle assembly in mutant nurse cells, demonstrating
that the villin-like protein functions by bundling actin filaments in vivo(25) . Unlike villin and Drosophila villin-like protein, dematin is unique in its widespread tissue
distribution and is the only actin-bundling protein with a headpiece
domain whose activity is reversibly controlled by
phosphorylation(2, 3) . A dematin-like protein, RET52,
has been suggested to play a role in the regulation of disk membrane
assembly and synapse formation within photoreceptors(26) . Now
that the primary structure of the dematin subunits has been elucidated,
it will be possible to co-express a reconstituted trimer in a
heterologous expression system in order to assess the biological
function of dematin in vivo.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U28389[GenBank® Link].
We thank Lisa White for expert molecular cytogenetic
assistance and Dr. Monique Arpin of the Institut Pasteur, France, for
providing the cDNA of human villin.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
vector, and
cAMP-dependent protein kinase were purchased from Pharmacia,
Invitrogen, and Sigma, respectively.
Construction of Recombinant Fusion Proteins
Constructs
representing defined domains of dematin and villin were amplified from
the corresponding cDNA templates using the polymerase chain reaction
(PCR).(
)The PCR amplified products were
subcloned into pGEX-2T plasmid, and fusion proteins were expressed in
the Escherichia coli strains of DH5
and HB101. Fusion
proteins containing glutathione S-transferase (GST) were
purified by affinity chromatography as described(6) .
Proteolysis of fusion proteins was reduced by adding leupeptin and
phenylmethylsulfonyl fluoride in the solubilization and washing
buffers.
Actin Binding Assay
Actin was isolated from rabbit
skeletal muscle as described (7) and further purified by gel
filtration on a Sephacryl 300 column. The actin binding assay mixture
contained 240 µg/ml actin and an appropriate amount of the GST
fusion protein in the following binding buffer: 10 mM Tris-HCl, pH 7.5, 1.0 mM EDTA, 1.0 mM
dithiothreitol, 10 mM KCl, 75.0 mM NaCl. After
incubation for 2.0 h at room temperature, actin was sedimented by
centrifugation at 35,000 rpm in a Type 42.2 Ti rotor at 4 °C. The
supernatant was carefully separated from the pellet, and both fractions
were analyzed by SDS-polyacrylamide gel electrophoresis. The amount of
proteins bound to actin filaments was quantified by either densitometry
or the elution of Coomassie Blue by pyridine(8) .
Other Procedures
A control peptide was synthesized
containing the following sequence: biotin-T-S-P-P-P-S-P-E-V-W. Peptide,
biotin-N-E-L-K-K-K-A-S-L-F, corresponds to the C terminus of the
dematin headpiece domain. The sequence includes serine 381 which is a
consensus site for phosphorylation by cAMP-dependent protein kinase.
The two-dimensional peptide mapping was carried out as
described(9) . Dematin was purified after treating intact red
cells with a membrane-permeable protease inhibitor (MDL28170) as well
as a protease inhibitor set (Boehringer Mannheim) which was included
during subsequent steps of purification. These modifications in the
original purification protocol (1, 2) improved the
stoichiometry of dematin to 69.9% (48-kDa subunit) and 30.1% (52-kDa
subunit).
Phosphorylation Assay
Phosphorylation of GST
fusion proteins by the catalytic subunit of cAMP-dependent protein
kinase was carried out as described(2, 3) .
Phosphorylation of biotinylated peptides was measured after coupling
these peptides to streptavidin-agarose beads. To determine the effect
of phosphorylation on the actin binding activity of fusion proteins,
purified proteins were phosphorylated by the catalytic subunit of cAMP
kinase, and kinase was inactivated at 68 °C as described
before(2) .
Chromosome Mapping Using Somatic Cell Hybrids
Two
panels of somatic cell hybrids were used to localize dematin gene on
human chromosome. The first panel (A) contains nine human hamster
hybrid cell lines containing varying complements of human chromosomes
as described previously(10) . The second panel is NIGMS
monochromosomal somatic cell hybrid panel 2 (11) which was
screened as described(12) . Selected individual hybrid DNAs
were amplified by polymerase chain reaction to confirm assignments
based on the pooled DNAs. Twenty-eight to thirty cycles of PCR
amplification were carried out in an MJ Research PTC-100 thermal cycler
set for 60 °C annealing for 45 s; 72 °C elongation for 30 s;
and 94 °C denaturation for 30 s. PCR products were visualized on 1%
agarose gels stained with ethidium bromide.
Isolation of P1 Genomic Clones
The following
primers were used to amplify segments of the dematin gene from human
genomic DNA. 1) Sense: OL1, 5`ACAGGAGGAAGAAAGGGAGAG; antisense: OL13,
5`AAACGAGGCAAGTCATCCA. This pair of primers amplified a 120-bp fragment
from the 5`-untranslated region of dematin cDNA. 2) Sense: OL4,
5`ATGTCCCCTGAAGAGTTTGGC; antisense: OL6, AACTCTGTGTGCCAGAGCCCA. This
pair of primers amplified a 332-bp fragment from the 3`-untranslated
region of dematin cDNA. Using a PCR-based screen, the primer pair 4/6
was used to isolate three P1 clones from the DuPont Merck human
foreskin fibroblast P1 library (DMPC-HFF1) by Genome Systems, Inc., St.
Louis, MO.
Fluorescence in Situ Hybridization
Human metaphase
chromosomes were prepared from phytohemagglutinin-stimulated
lymphocytes of a normal male by standard techniques. Purified DNA from
the P1 clone 1977 was labeled with biotin-16-dUTP by nick translation.
Hybridization and detection were performed with 150 ng of labeled probe
as described previously(13) . Hybridization signals were
detected with fluorescein-labeled avidin (Vector Laboratories, 7.0
µg/ml). Chromosomes were counterstained with propidium iodide and
DAPI. Fluorescein and propidium iodide were visualized through a
fluorescein isothiocyanate/Texas red dual band pass filter set (Omega
Optical), and DAPI banding was viewed through a single band pass filter
set (Zeiss). Photomicrographs were taken with Ektar 1000 color film
(Kodak) or ektachrome 400 color slide film (Kodak).
Isolation of Human Reticulocyte cDNA Encoding the
52-kDa Subunit
Despite many similarities between the 48-kDa and
52-kDa subunits of dematin(3) , the 52-kDa subunit appears to
have subtle structural differences from the 48-kDa subunit based on the
following observations. First, the electrophoretic mobility of the
52-kDa subunit changes depending upon the presence and absence of
reducing agents, whereas no such mobility shift is observed with the
48-kDa subunit of dematin(3) . Secondly, the two-dimensional
peptide mapping analysis revealed that although the two subunits of
dematin shared many peptides, spots unique to both subunits were also
detected (Fig. 1). The respective positions of these unique
spots did not change even after an extensive in vitro dephosphorylation of purified dematin (not shown). These results
indicated that the primary structure of the 52-kDa subunit may be
similar to but not identical with that of the 48-kDa subunit of
dematin. Based on this assumption, a PCR strategy was designed to
amplify short segments of the 48-kDa subunit from total RNA isolated
from human reticulocytes. Primers flanking the headpiece domain of the
48-kDa subunit produced a cDNA product that appeared as a doublet on
agarose gels (not shown). Subsequent subcloning and sequencing of the
cDNA doublet revealed that two distinct cDNA fragments were present in
the band. One cDNA encoded the regular headpiece domain of the 48-kDa
subunit(4) , whereas the second cDNA fragment contained a novel
headpiece domain with an additional 66-base pair sequence (Fig. 2). This insert sequence encodes an extra 22 amino acids
in-frame, including a cysteine residue, in the headpiece sequence of
the 48-kDa subunit (Fig. 2).
I-tryptic
peptide maps. The 48-kDa and 52-kDa subunits of dematin were separated
by electrophoresis of dematin purified from human erythrocyte
membranes. The Coomassie-stained polypeptides were excised from the
gel, radioiodinated, and digested as described before(9) . The
spots shared between the 48-kDa and 52-kDa subunits have been
identified by arrowheads, and circles highlight the
position of unique spots.
We have previously reported that
the molecular mass of the 48- and 52-kDa subunits, as measured by the
laser desorption mass spectrometry, corresponds to 43,265 Da and 46,175
Da, respectively(4) . This difference of 2.91 kDa is in
agreement with the 2.2-kDa difference calculated from the insert
sequence of the 52-kDa subunit (Fig. 2). Using insert-specific
primers, cDNA clones encoding the full-length 52-kDa subunit were
isolated from human reticulocyte mRNA. The primary structure of the
52-kDa subunit is identical with that of the 48-kDa subunit except for
the presence of a 22-amino-acid sequence inserted in the headpiece
domain of the 52-kDa subunit (Fig. 2). The 52-kDa subunit cDNA
clones contained three additional nucleotide changes. The codon for
threonine 304 was changed from ACG to ACA without changing the amino
acid. The codon for valine 325 (GTG) was changed to methionine (ATG)
and serine 358 (TCT) to phenylalanine (TTT). The functional
significance of these amino acid substitutions is not currently known.
Alternatively, these changes may reflect polymorphisms in the 52-kDa
subunit. The observed changes were confirmed by four independent PCR
amplifications, and the original codons of the 48-kDa subunit, as
reported previously(4) , were confirmed.
The 52-kDa Subunit of Dematin Shows Homology to
Erythrocyte Protein 4.2
Alignment of the 22 amino acids of
insert sequence with the available data bank showed that the N-terminal
eight amino acids were identical with a motif found in human
erythrocyte protein 4.2 (Fig. 2). Additional alignment analysis
of the 52-kDa subunit containing the insert sequence revealed homology
with protein 4.2 that extended further and included three additional
amino acids derived from the headpiece domain just preceding the insert
sequence (Fig. 2). Therefore, 11 amino acids of the 52-Da
subunit of dematin are identical with a motif present in protein 4.2 (Fig. 2). This 11-amino-acid motif is also conserved in murine
protein 4.2 except that a histidine residue has replaced an arginine of
the human protein 4.2 as shown in Fig. 2. The functional
significance of this shared motif in dematin and protein 4.2 is not yet
known.
Each Monomer of Dematin Subunits Contains Two Actin
Binding Sites
In order to determine the number and location of
actin binding sites in dematin, segments of dematin cDNA were expressed
in bacteria as glutathione S-transferase (GST) fusion proteins (Fig. 3). As shown in Fig. 4and Table 1, the
headpiece domain of both 48-kDa and 52-kDa subunits sedimented with
actin filaments. The presence of a 22-amino-acid insert sequence did
not affect the actin binding activity of the headpiece domain (Fig. 4). To further map the actin binding site within the
headpiece domain, a GST fusion protein was made containing the
C-terminal 22 amino acids of the headpiece domain (Fig. 4). This
fusion protein containing a truncated headpiece domain also sedimented
with actin filaments showing that the actin binding site is located
within the C-terminal 22 amino acids of the headpiece domain. To
further narrow the actin binding site, a synthetic peptide containing
the last 10 amino acids of the headpiece domain was produced. This
biotin-conjugated peptide, N-L-E-L-K-K-K-A-S-L-F, did not sediment with
actin filaments (not shown) indicating that although the consensus
sequence for the actin binding site is located within the last 10 amino
acids of the headpiece domain (see ``Discussion''), the in vitro actin binding activity of the headpiece domain
requires participation of at least 22 amino acids (Fig. 3).
The primary structure of dematin contains an N-terminal undefined
domain which does not show significant sequence homology to any protein
in the GenBank data base(4) . In this respect,
dematin differs from villin which has an actin-binding N-terminal
domain that is homologous to gelsolin, severin, and
fragmin(14, 15) . The N-terminal undefined domain of
dematin contains a PEST sequence and a highly negatively charged motif
of 10 glutamic and aspartic acid residues(4) . In order to
determine whether this N-terminal domain of dematin contains a second
actin binding site, the respective fusion proteins were assayed for
actin binding activity. Because of the presence of the PEST sequence,
the GST fusion protein containing a full length N-terminal undefined
domain was proteolyzed during purification from bacterial lysates (not
shown). Nevertheless, this proteolyzed fusion protein containing 25-kDa
fragments sedimented with actin filaments (Table 1). To further
confirm this binding, a GST fusion protein was made which lacked the
N-terminal PEST sequence (Fig. 3). This fusion protein encoding
a truncated undefined domain did not undergo proteolysis and also
sedimented with actin filaments (Table 1). The fusion protein
containing only the PEST sequence was extensively proteolyzed and
therefore could not be tested for its actin binding activity (not
shown). In summary, the results of actin binding measurements show that
each subunit of dematin contains at least two actin binding sites: one
in the headpiece domain and the other in the N-terminal undefined
domain.
Phosphorylation of Serine 381 by cAMP-dependent Protein
Kinase
The headpiece domain of dematin contains a consensus
phosphorylation site for protein kinase A at serine 381(4) . In
contrast, the villin headpiece domain contains an alanine residue at
the corresponding position(13, 14) . Because the actin
bundling activity of dematin, but not of villin, is regulated by
phosphorylation (2, 3) , it was suspected that the
phosphorylation of serine 381 may regulate this effect. To test this
hypothesis, we first examined whether the headpiece domain of dematin
was phosphorylated by protein kinase A in vitro. The
glutathione S-transferase fusion proteins containing headpiece
domains of the 48-kDa and 52-kDa subunits were excellent substrates of
protein kinase A, whereas no phosphorylation was observed with the
headpiece domain of villin (Table 1). To further confirm that the
site of phosphorylation in the headpiece domain was in fact serine 381,
a synthetic peptide was produced that contained the C-terminal 10 amino
acids of the headpiece domain of dematin. This synthetic peptide was an
excellent substrate of cAMP-dependent protein kinase confirming that
serine 381 is indeed the site of phosphorylation (not shown). We then
examined whether the actin binding activity of the dematin headpiece
domain was regulated by phosphorylation. Actin binding by fusion
proteins containing headpiece domains of dematin remained unchanged
upon their phosphorylation by protein kinase A (Fig. 5, Table 1).
We also examined the possibility that the inhibitory
effect of phosphorylation on dematin's actin bundling activity
may be mediated via the phosphorylation of an N-terminal undefined
domain (Fig. 2). The fusion protein containing the truncated
undefined domain of dematin was also phosphorylated in vitro by protein kinase A, but again no effect of phosphorylation was
observed on its actin binding activity (Fig. 5, Table 1).
The above results show that although the actin bundling activity of
dematin is completely abolished by phosphorylation (2, 3) , the actin binding activities of respective
domains are not altered by phosphorylation. This result is consistent
with our previous observation that the phosphorylated dematin remains
associated with actin filaments even though it cannot hold filaments in
a bundled conformation(2, 3) .
Dematin Gene Is Located on Human Chromosome 8
The
chromosomal location of the dematin gene was determined using somatic
cell hybrid panels and dematin specific primers. The primer pairs
OL1/OL13 and OL4/OL6 were combined and used to detect the dematin gene
sequences in two somatic cell hybrid panels segregating human
chromosomes in a rodent background. These pooled primers did not
amplify the dematin gene in control rodent DNAs but segregated with
human chromosome 8 in hybrids and were discordant with the segregation
of all other human chromosomes (Table 2). This assignment was
confirmed by positive amplification of the dematin gene in the
monochromosomal hybrid GM/NA10156B containing only human chromosome 8
(not shown). These results indicate that the dematin gene is located on
human chromosome 8.
Regional localization of the dematin gene on
chromosome 8 was performed by fluorescence in situ hybridization with a genomic P1 clone of dematin. The primer pair
OL4/OL6 was used to isolate three P1 genomic clones specific for human
dematin from the DuPont Merck P1 library (see ``Experimental
Procedures''). The identity of these clones was established by
Southern blot analysis (not shown). The DNA from one of the three P1
clones, designated P1-1977, was biotinylated and hybridized to human
chromosomes. Fifteen metaphases with single or double chromatid
hybridizations were examined using the P1-1977 genomic probe. All
revealed hybridization on the short arm of chromosome 8. The dematin
gene was localized to 8p21.1 by simultaneous visualization of the
hybridization signals on DAPI-banded chromosomes and by fractional
length measurements (Fig. 6).
A unique feature of the
insert sequence in the 52-kDa subunit is its homology with erythrocyte
protein 4.2 (Fig. 2). The 11 amino acids of the 52-kDa subunit
are identical with a motif present in protein 4.2. Of these 11 amino
acids, 8 residues are contributed from the insert sequence of 52 kDa,
and 3 amino acids are derived from the original headpiece domain (Fig. 2). Although the function of this motif is not yet known,
it is highly conserved between human and murine protein 4.2 which are
considerably different in their primary structures(17) .
Interestingly, this conserved motif is not found in transglutaminases
which share significant sequence identity with human and murine protein
4.2(18) . These observations raise the possibility that the
role of this motif may be specific to the functions of dematin and
protein 4.2 and is currently under investigation.
(
)where actin bundles do
exist(23) . In fact, actin bundling events have been correlated
with the process of enucleation in murine erythroblasts (23) .
Whether dematin plays a role in such actin bundling events during
erythoid development is currently under investigation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.