From the Division of Reproductive Biology, Department
of Gynecology and Obstetrics, Stanford University School of Medicine,
Stanford, California 94305-5317 and the ¶ Department of
Immunology, Berlex Biosciences, Richmond, California 94804-0099
Received for publication, July 21, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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Subcellular targeting of the components of the
cAMP-dependent pathway is thought to be essential for
intracellular signaling. Here we have identified a novel protein, named
myomegalin, that interacts with the cyclic nucleotide phosphodiesterase
PDE4D, thereby targeting it to particulate structures. Myomegalin is a
large 2,324-amino acid protein mostly composed of Only in recent years has it been realized that the components of
the signal transduction machinery are organized in macromolecular complexes located in close proximity to the plasma membrane or in
discrete subcellular structures. These complexes are assembled by
scaffold proteins often devoid of enzymatic activity and for the sole
function of bringing together different signaling molecules (1, 2).
These signaling/scaffold protein complexes are necessary and often
indispensable for signaling, as disruption or blocking the formation of
these complexes decreases the efficiency of signaling or negates it altogether.
Even though cAMP is a diffusible second messenger, the enzymes involved
in this signaling pathway are targeted to different subcellular
compartments. With few exceptions, adenylyl cyclases are plasma
membrane-associated proteins, and protein kinase A (PKA),1 one of the effectors
of cAMP signaling, is anchored to discrete structures within the cell
via regulatory subunit interaction with A kinase anchoring proteins
(AKAPs) (3, 4). Several different AKAPs that function as scaffold
proteins have been characterized. For instance, AKAP79 is part of a
multicomplex, which, in addition to the PKA regulatory subunit RII,
includes protein kinase C and the phosphatase PP2B/calcineurin (5).
Disruption of the PKA interaction with AKAPs abolishes the
cAMP-dependent regulation of ion channels (6, 7). PKA
regulatory subunits have also been localized in the centrosome in
culture cells (8), and in cells of the central nervous system (9), thus
opening the possibility that microenvironments of cAMP signaling may be
necessary for intracellular trafficking and organelle movements during
cell replication.
Cyclic nucleotide phosphodiesterases, the enzymes that degrade and
inactivate cAMP, may play an important role in signaling compartmentalization by controlling cAMP diffusion to reach the PKA
isoenzymes anchored to different organelles. Of the wide array of PDE
isoforms present in a cell (10-13), several are recovered in the
particulate fraction of the homogenate and have been immunolocalized to
different cellular compartments. We and others have shown that variants
derived from the PDE4D gene are particulate or soluble, depending on
the amino terminus present in the protein (14-16), and are located in
different subcellular compartments as shown by immunofluorescence
studies. In FRTL-5 thyroid cells, two variants (PDE4D3 and PDE4D4) with
long amino termini are recovered in the particulate fraction, and are
localized in a perinuclear region corresponding to centrosomal
inner/Golgi structures, as well as to the membrane cortical region
(16). Conversely, a third variant (PDE4D2), the expression of which is
cAMP-dependent, is recovered mostly in the soluble fraction
and its induction by cAMP is associated with diffuse immunofluorescent
signals in these cells (16). Localization of PDE4D in discrete
subcellular structures has also been demonstrated in macrophages (17).
These findings open the possibility that the subcellular localization
of PDE4D is finely controlled and this localization may be dynamically
regulated during cAMP signaling.
To understand the mechanism of PDE4 subcellular targeting, we sought to
identify proteins that interact with these PDE4D variants and to anchor
them to different organelles. Here we describe the properties of a
novel protein named myomegalin, which is a major component of skeletal
and cardiac muscle but is expressed at low levels in all cells studied.
Although in nonmuscle cells it is targeted to the Golgi/centrosomal
region, myomegalin localizes in the sarcomeres in heart and skeletal
muscle. This protein colocalizes with PDE4D in the cells and tissue studied.
Yeast Strains and Growth Conditions--
The yeast strains of
Saccharomyces cerevisiae (Y190, HF7c, Y187), cloning
vectors, and the activating domain (AD) rat brain cDNA library used
for the yeast two-hybrid screening (Matchmaker I and II) as well as the
The yeast strain Y190 of S. cerevisiae was used as host for
the yeast two-hybrid screening, whereas HF7c and Y187 were used for
mating experiments. Yeast transformation was performed using the
lithium acetate method according to Gietz et al.
(47). Single transformants were spread on selection medium (SD
medium) lacking Leu or Trp. Cotransformants of strain Y190 were
propagated on SD medium containing Ade (10 mg/ml) and 50 mM
3-AT. Single transformants of Y187 were grown on SD medium lacking Trp.
The yeast strain HF7c was transformed with Gal4 activation domain
expressing His+Z+ clones from the library screen. Transformants were
propagated on SD/ Yeast Two-hybrid Screening of a Brain cDNA Library--
The
yeast strain Y190 was used to screen a rat brain Gal4/AD library to
identify genes encoding proteins that interact with ratPDE4D3. The
amino terminus (aa 1-133) of PDE4D3 as well as a truncated form of
ratPDE4D3 that included the autoinhibitory (UCR2), catalytic, and
COOH-terminal domain (aa 134-end of rPDE4D3) were used as a bait. The
cDNA corresponding to the truncated PDE (base 400-2018 of the
PDE4D3 ORF, accession no. U09457) was amplified by PCR using the
full-length ratPDE4D3 cDNA as a template. The following primers
with incorporated restriction sites were used: GUPA4, EcoRI
5'-CGGAATTCGAGGAGGCCTACCAGAAAC-3'; and GUPA3, 5'-TGAGTCGACTACGTGTCAGGACAACAATCGTC-3' SalI. The PCR product
was subcloned into the EcoRI/SalI site of pGBT9
downstream from the Gal4 DNA binding domain (plasmid pGBT9/1.6).
Amplifications were performed in the presence of recombinant
Pfu polymerase at a low cycle number to ensure high fidelity
amplification. The amplified fragment was sequenced in its entire
length to confirm the correct reading frame and sequence. Sequencing
was performed by Stanford University facilities using the ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA
polymerase, FS (PerkinElmer Life Sciences). Further truncations of
ratPDE4D3 were obtained as reported previously (18) to identify the PDE domain responsible for the interaction with positive AD/library clones.
These included PDEtruA (aa 164-end), PDEtruB (aa 179-end), and
PDEtruC (214-end). The construction of the plasmid containing the
amino terminus of PDE4D3 (0.4) has been described previously (18).
Under the experimental conditions used, the full-length PDE4D3 (93 kDa)
did not express well as a fusion protein in yeast nuclei.
A portion of the AD/cDNA plasmid library was amplified once to
obtain sufficient plasmid DNA for screening. The plasmid DNAs of bait
and library (concentration ratio 2:1) were simultaneously transformed
into Y190 cells on a large scale basis according to the manufacturer's
protocol. Protein expression in Y190 was induced on SD/Ade, 50 mM 3-AT (6.7 g/liter yeast nitrogen base without amino
acids, pH 5.8; 18 g/liter agar, 10 mg/liter adenine, 2% dextrose, and
50 mM 3-amino-1,2,4-triazole). Transcriptional
autoactivation of the pGBT9/1.6 was monitored by single and
cotransformation with control plasmids containing the Gal4
transcriptional activation domain alone (pGAD10) or with other in frame
cDNAs such as the SV40 large T-antigene (pTD1) and pGAD10/1.6. No
background for
After 7-19 days of incubation at 30 °C, the colonies growing on
His+ plates were counted and patched onto fresh SD/Ade, 50 mM 3-AT, together with controls. The first group of patched
His+ clones was checked again for positive growth 5 days after
incubation. At that stage, a
To isolate Leu+Trp+ yeast segregants with only the AD/library plasmid,
His+Z+ transformants were cultured in YPD medium overnight at 30 °C.
An aliquot of the sample was spread on SD/
To confirm the interaction of the isolated clones with the bait, yeast
strain Y190 and HF7c were retransformed with bait and AD/library
plasmid and tested for yeast mating and for Yeast Mating--
Yeast strain Y187 was transformed with pGBT9
and pGBT9/1.6 and selected for Trp+ transformants. Yeast strain HF7c
was single transformed with pGAD10, pGAD10/library positive clones.
Transformants were propagated on SD/ Cloning by Library Screening--
To obtain the complete
sequence of the PDE4D3-interacting protein myomegalin, the PBP46
cDNA isolated from the yeast library screening was used as a probe
to screen rat brain and skeletal muscle Northern Blot Analysis--
Two different DNA probes (probe 1, 1000 bp; probe 2, 665 bp) derived from the ORF of myomegalin were
labeled with [ RT-PCR Analysis--
Poly(A+) mRNA was isolated
from rat heart and skeletal muscle using the QuickPrep Micro mRNA
purification kit (Amersham Pharmacia Biotech). First strand cDNA
was prepared from mRNA with random hexanucleotide primers (20 pmol/µg of RNA) using murine reverse transcriptase and the conditions
recommended by the manufacturer's protocol (Amersham Pharmacia
Biotech). The cDNA obtained was amplified with the following
oligonucleotide primers designed against different regions of
myomegalin: MYO1-MYO2, forward primer (5'-GTGGAGAGCCTGAAAACGAGAG-3', corresponding to bases 163-183 (aa 55-61)) and reverse primer (5'-AGTCGTCTCAGAAGCAAGATACGA-3', corresponding to bases 830-844 (aa
274-282); predicted fragment, size 682 bp); MYO3-MYO4, forward primer
(5'-GAAGCTAGCTCTCCCCGCTACA-3', corresponding to bases 5266-5287 (aa
1756-1764)) and reverse primer (5'-CTACGAGTCCCTCTACGAAAAT-3', corresponding to bases 5987-6009 (aa 1995-2003; predicted fragment size, 743 bp)); KIA1-MYO2, forward primer
(5'-CTGTGGAATCTTTGGATGCAAGCGT-3', bp 1379-1403 of KIAA0477) and
reverse primer (5'-AGTCGTCTCAGAAGCAAGATACGA-3', fragment size, 587 bp).
Thirty cycles of PCR amplification were usually used (60 s at 94 °C,
60 s at 50 °C, and 2 min at 72 °C) and a final elongation
(10 min at 72 °C). The PCR products were separated on 1% agarose
gel containing 0.01% ethidium bromide and photographed under UV
irradiation at 320 nm.
Antibody Generation--
The peptide PBP4
(FASGHGRHVIGHVDDYDALQQQI) was conjugated to keyhole limpet hemocyanin
for immunizations in rabbits. The immunizations and bleeds were
performed at Strategic BioSolutions, Inc. (Ramona, CA). For affinity
purification, the PBP4 peptide was immobilized to 3 M
EmphazeTM Biosupport Medium AB 1 using the UltralinkTM immobilized carboxy kit from Pierce. The PBP4-specific IgG was eluted from the
column by sequential washes at low pH (ImmunoPure IgG elution buffer,
Pierce) and at high pH (0.1 M triethylamine, pH 11.5). The
antibodies were buffer exchanged to PBS and concentrated to 1 mg/ml
using an Ultrafree-15 Biomax-30 centrifugal filter device (Millipore
Corp., Bedford, MA).
Western Blot Analysis--
Tissues or cells were homogenized in
a Dounce homogenizer using a hypotonic buffer consisting of 20 mM Tris, pH 8.0, 1 mM EDTA, 0.2 mM
EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 50 mM benzamidine, 0.5 µg/m leupeptin, 0.7 µg/ml
pepstatin, 4 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 µM microcystine, and 1 µg/ml phenylmethylsulfonyl
fluoride (homogenization buffer). After homogenization the extract was
centrifuged for 40 min at 14,000 × g to separate
soluble and particulate fractions. Both fractions were diluted in 1×
sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2%
(w/v) SDS, 0.7 mM
Freshly excised skeletal and heart tissue was frozen in liquid
nitrogen. After pulverization, the tissue was extracted with buffer
containing 5 mM EGTA, 7 mM ATP, 130 mM propionic acid, 5 mM imidazole, 10 mM MOPS, pH 7.2, and a mixture of protease inhibitors,
following the method of Brandt et al. (20). After homogenization with all glass homogenizer, the samples were centrifuged at 100,000 × g. The pellets were extracted with 1×
sample buffer and centrifuged at 14,000 × g to remove
particulate material. After dilution in sample buffer, supernatants and
pellets were fractionated by SDS-PAGE as above.
Transfection and Immunofluorescence Localization--
The
truncated myomegalin (63 kDa) was tagged with flag epitope (DYKDDDDK)
at its NH2 terminus by transferring the insert PBP46 to the
BamHI/BglII of a modified vector (gift of Dr.
Louis Naumovsky, Stanford University, Stanford, CA) derived from pCEP4
(Invitrogen Corp., Carlsbad, CA). The construct was transfected in
COS-7 cells using the calcium phosphate method (19). Cells were grown
in Eagle's medium (Life Technologies) for 48 h, fixed with
ethanol-acetone solution (1:1) for 10 min, preincubated with PBS
containing 0.1% bovine serum albumin for 20 min, and then incubated
with specific primary antibodies in PBS for 1 h. Rabbit polyclonal
PBP4 antibody was used at 1:100 dilution, anti-flag at 1:80, CTR433
(median Golgi marker) (21) at 1:10, CTR453 (centrosomal marker) (22) at
1:500, and M3S1 at 1:50. After the cells were washed with PBS three
times, fluorescent fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies (1:100 dilution; Vector Laboratories, Inc., Burlingame, CA) were then added to the cells. The expressed proteins were localized using fluorescence microscopy after mounting the samples
with Vectashield mounting medium (Vector Laboratories, Inc.). Tissue
immunofluorescence localization was performed according to previously
published methods (23).
PDE Assay--
PDE activity was measured with 1 µM
cAMP as substrate according to the method of Thompson and Appleman (24)
with minor modification (16). Protein concentrations of the samples
were measured according to the method of Bradford (25).
Isolation of Clones Coding for Proteins That Interact with PDE4D in
the Yeast Two-hybrid Screen--
A yeast two-hybrid screening of a rat
brain library with baits corresponding to amino acids 1-133 and
134-end of PDE4D3 (see Fig. 1) yielded
several clones that interacted strongly with the PDE in both the
Cloning of the Full-length cDNA Coding for
Myomegalin--
Northern blot analysis with mRNAs from different
rat tissues indicated that PBP46 cDNA corresponds to a gene that is
expressed at a high level in skeletal muscle and heart (see below). A
rat skeletal muscle Myomegalin Structure--
A computer-assisted prediction of the
secondary structure of myomegalin indicated that this protein is mostly
composed of coiled-coil and Analysis of the Myomegalin Variants by Northern Blot and RT-PCR
Analysis--
To investigate whether different myomegalin transcripts
are present in rat and to analyze their tissue distribution, Northern blot analysis was performed using probes corresponding to the 3' end
(probe 1) and 5' end (probe 2) of the myomegalin ORF (Fig. 3A). The results indicated the
presence of abundant transcripts of ~7.5-8 kb (myomegalin-1) in
heart and skeletal muscle, which contains the entire ORF of myomegalin
(Fig. 3B). The 7.5-8-kb transcripts were also detected in
small amounts in brain, lung, and liver, and hybridized to both probe 1 and probe 2. Probe 1 also detected shorter transcripts, ~5.9 kb in
heart and ~2.5 kb in testis, that were not detected with probe 2 (Fig. 3C), indicating that these transcripts lack the 5' end
of the coding sequence. The use of probe 2 also revealed the presence
of a fourth transcript, ~4.3 kb, present in skeletal muscle (Fig.
3C). Thus, there are at least four different transcripts of
rat myomegalin: a 7.5-kb transcript expressed in heart and skeletal
muscle, a 5.9-kb transcript in heart, a 4.3-kb transcript in skeletal
muscle, and one specific for the testis (2.5 kb). The 2.5-kb variant,
which is the shortest form, roughly corresponds to the PBP46 clone
isolated in the yeast two-hybrid screening and is expressed exclusively
in rat testis. The presence of this shorter form was confirmed by the
isolation of two cDNA clones from a testis cDNA library (data
not shown), even though the initiation methionine of this ORF could not
be unequivocally identified. Finally, additional variants at both the
5' and 3' ends may be present on the basis of the cDNAs retrieved (data not shown).
A BLAST search of human nucleotide expressed sequence tag sequences
indicated a high degree of conservation of rat myomegalin and two
overlapping clones identified by sequencing randomly sampled clones
with insert sizes of 5-7 kb (KIAA0477 and KIAA0454) from a human brain
library (GenBankTM AB007946 and AB007923). The homology encompassed
most of the coding sequence except for the 5' end, again suggesting the
presence of splicing in the mRNA. To verify this possibility,
RT-PCR analysis was performed with mRNA from rat heart and skeletal
muscle. Different primers corresponding to 3' (MYO3 and MYO4) and 5'
end (MYO1 and MYO2) sequence of myomegalin, and one primer (KIA1)
corresponding to the 5' end of human sequence (Fig. 3A) were
used for amplification. This analysis indicated that transcripts
expressed in the heart and skeletal muscle most likely contain the
entire myomegalin ORF defined by cDNA cloning (Fig. 3, D
and E). It also uncovered the presence of an additional transcript containing the 5' end sequence orthologous to the human clone in heart but not in skeletal muscle. The sequencing of the KIA1-MYO2 PCR fragments from rat heart confirmed the presence of a
variant similar to that present in human (data not shown). This amino
terminus does not contain a leucine zipper domain.
Expression of the Myomegalin Protein--
To investigate the
properties of the protein, antibodies were raised against four peptides
corresponding to the carboxyl terminus region of myomegalin. Of the
different antisera generated, one was further characterized (PBP4).
This antibody recognized the tagged 64-kDa myomegalin in the
particulate fraction of transfected cells (Fig.
4A). This truncated myomegalin
form could not be solubilized with nonionic detergent but was partially
extracted with RIPA buffer (data not shown).
Western blot analysis with the PBP4 antibody recognized proteins of
~230-250 kDa in heart and skeletal muscle (Fig. 4B).
Additional immunoreactive bands of ~150 kDa and 115 and 180 kDa were
observed in heart and skeletal muscle, respectively. Although a
degradation of the 230-250-kDa species cannot be excluded, it is
possible that these additional bands correspond to splicing variants of myomegalin. A 62-kDa protein was consistently observed in rat testis
(Fig. 4C) or germ cell extracts (data not shown). These proteins were completely insoluble in standard homogenization conditions and could be partially solubilized from muscle only under
conditions that extract the myofibril components (Fig. 4).
Localization of Myomegalin in Cultured Cells--
Because of the
tight binding of myomegalin to particulate structures,
immunocytochemistry was used to further analyze the localization of
this protein in transfected COS-7 cells. The truncated form of
myomegalin (PBP46; aa 1765-2324) fused to a flag epitope was
transfected in COS-7 cells and the distribution detected with a
myomegalin-specific antibody (PBP4), and with an anti-flag antibody (Fig. 5A). An overlapping
pattern of immunofluorescence was obtained with both antibodies
confirming the specificity of the PBP4 antiserum for immunofluorescence
localization. Moreover, a signal with PBP4 was found in cells negative
for the flag antibody, indicating the presence of endogenous myomegalin
in COS-7 cells. This expression in COS-7 cells was confirmed by
detection of myomegalin transcripts by PCR amplification (data not
shown). The endogenous myomegalin localized in an inner
Golgi/centrosome region that corresponds to the location of the
transfected myomegalin. To further define the site of myomegalin
localization in these cells, centrosomal and Golgi markers were used to
demonstrate an overlapping localization with the two markers (Fig. 5,
B and C). Similar localization of myomegalin was
also observed in cultured FRTL-5 thyroid cells (data not shown). In
these latter cells, PDE4D is localized predominantly in a similar
Golgi/centrosomal region (16).
Colocalization of Myomegalin and PDE4D in Muscle Cells and
Testis--
Because myomegalin transcripts are highly expressed in
heart and skeletal muscle, myomegalin and PDE4D localization was
assessed in sections of cardiac (not shown) and skeletal muscle (Fig.
6). Staining with the PBP4 antibody
yielded a periodic pattern (~2 µm) of myomegalin localization along
muscle fibrils in skeletal (Fig. 6) and cardiac (data not shown)
tissues. Double staining using a myomegalin-specific antibody (PBP4)
and a PDE4D-specific antibody (M3S1) indicated colocalization of both
proteins in the same region of the myofibers (Fig. 6). This region
corresponds to the z band on the basis of a comparison with the
staining of the fibers with myosin, actin, and desmin antibodies (data
not shown).
In testis, myomegalin immunoreactivity was mostly present in germ cells
of the seminiferous tubules. The antibody stained a region in close
proximity to the nucleus, corresponding to the Golgi complex of
pachytene spermatocytes and round spermatids (Fig.
7). This predominant expression of the
short myomegalin variant in germ cells was confirmed by PCR analysis,
Northern blot, and Western blot of extracts from either seminiferous
tubules or isolated germ cells (data not shown).
Myomegalin and PDE4D3 Subcellular Localization in the Intact
Cell--
Solubilization of the full-length myomegalin could not be
achieved under nondenaturing conditions. However, the truncated myomegalin (PBP46), even though particulate after overexpression, could
be partially solubilized with RIPA buffer containing 0.1% SDS. To
determine whether this myomegalin variant interacts with PDE4D,
expression vectors for PDE4D3 and the truncated myomegalin (PBP46) were
cotransfected in COS-7 cells. Cells were solubilized with RIPA and the
extract subjected to immunoprecipitation with PDE4D-selective
antibodies. Under these conditions, the PDE4D-selective antibody
coimmunoprecipitated the truncated myomegalin in complex with the PDE
(Fig. 8B). The
immunoprecipitation was specific because, in the absence of PDE4D3, no
myomegalin was recovered in the immunoprecipitate even after
overexposure of the film (data not shown). Moreover, no myomegalin or
PDE was immunoprecipitated when unrelated IgG was used in this assay
(data not shown). Myomegalin was recovered in the immunoprecipitation
pellet when PDE4D1 and PDE4D3 were used for transfection, but not with
the PDE4D2 variant, which lacks the putative domain interacting with
myomegalin (Fig. 8C). This latter finding indicates that not
all PDE variants interact with this myomegalin form.
To further confirm the interaction between the two proteins in intact
cells, expression vectors for PDE4D3 and PBP46 were cotransfected in
COS-7 cells. After 24 h the cells were homogenized, the
particulate fraction separated by centrifugation, and the PDE activity
recovered in this fraction of the homogenate was measured.
Cotransfection of PDE4D3 and PBP46 led to a 2-4-fold increase in the
PDE activity recovered in the particulate fraction (Fig.
9A) as compared with the cells
transfected with PDE4D3 alone. Western blot analysis with
PDE4D-specific antibodies confirmed the PDE activity data. Although the
93-kDa PDE4D3 was at the limit of detection in the particulate fraction
of COS-7 cells transfected only with PDE4D3, cotransfection with
myomegalin produced an increase in the PDE4D3 protein recovered in the
particulate fraction (Fig. 9B). In agreement with previous
observations, the short myomegalin variant was recovered almost
exclusively in the particulate fraction of COS-7 cells (Fig.
9C).
With these studies, we report the identification and the initial
characterization of myomegalin, a novel protein with the properties of
a structural/scaffold protein. At least three potential splicing
variants of myomegalin detected in rat may serve distinct functions in
muscle and nonmuscle cells. Myomegalin is targeted to the
Golgi/centrosomal region in cultured COS-7 and FRTL-5 cells and in germ
cells of the testis, whereas in cardiac and skeletal muscle it is
associated with the sarcomere or the sarcoplasmic reticulum. We propose
that one of the functions of this protein is in cAMP signaling
compartmentalization because it targets a PDE component of the cAMP
signaling to these subcellular structures.
Myomegalin is structurally related to the Drosophila
centrosomin (cnn). Centrosomin was originally identified
while screening for genes regulated by antennapedia or other homeotic
genes (26) and is mostly composed of coiled-coil structures with three
leucine zipper domains. The first leucine zipper near the amino
terminus is very similar to the only leucine zipper found in one
variant of myomegalin. The extensive arrangement in coiled-coil
structures is also shared between centrosomin and myomegalin. The
Drosophila centrosomin is a component of the centrosomes and
the mitotic spindle (30) and is thought to play a crucial role during
morphogenesis of the central nervous system and midgut (26). A
testis-specific isoform of centrosomin has been described in fly (31).
Mutations that disrupt the spermatogenic-specific centrosomin impair
cytokinesis, kariokinesis, and organization of the sperm axoneme. In a
strikingly similar fashion, a splicing variant of the mammalian
myomegalin is expressed in germ cells of rat testis and is localized in
a Golgi/centrosomal region of pachytene spermatocytes and round spermatids; however, it could not conclusively be determined whether it
is located in the basal body of the developing flagellum. Thus, it is
likely that the mammalian myomegalin has functions during spermatogenesis that overlap with those of the centrosomin in Drosophila.
It should be pointed out that other mammalian proteins homologous to
the Drosophila centrosomin have been described previously. These include mouse centrosomin A and B and human 167 protein (32-34).
However, their similarity to the Drosophila centrosomin protein is low, and myomegalin is more related to centrosomin than to
these mammalian centrosomins. The function of mammalian centrosomin is
at present unknown.
Recently, several large coiled-coil proteins localized in the Golgi
apparatus have been classified as a distinct golgin family of proteins
(35-37). Among the many members identified, golgin-230/245/256, golgin
97, GM130, and giantin proteins have extensive coiled-coil structure
and a domain at the carboxyl terminus that specifies Golgi targeting
(grip domain). Myomegalin is weakly homologous to golgin 230/245/256
(20% identity, 40% conservative substitutions) in several coiled-coil
domains. However, a domain similar to the grip domain could not be
identified at the carboxyl terminus of myomegalin, even though the last
550 amino acids of this latter protein are sufficient to target it to
the Golgi/centrosome structures.
Although formal proof of an interaction of PDE4D with the full-length
myomegalin was precluded by the insolubility of the protein, we have
provided evidence of an interaction of the 62-64-kDa myomegalin
variant with PDE4D. Four independent lines of evidence indicate that
the two proteins may indeed exist in a complex in the intact cell. In
addition to the interaction detected with the yeast two-hybrid
screening, the 64-kDa variant of myomegalin and PDE4D interact
specifically in a coimmunoprecipitation assay. Furthermore,
cotransfection of myomegalin targeted the recombinant PDE4D3 to the
particulate fraction of COS-7 cells. Finally, myomegalin and PDE4D were
colocalized in cultured cells as well as in cardiac and skeletal muscle tissue.
Deletion mutagenesis of PDE4D indicates that a site of interaction with
myomegalin is in a domain that corresponds to the amino terminus of the
upstream conserved region 2 (UCR2). This domain is conserved in all the
long forms of PDE4 opening the possibility that other PDE4s may
interact with myomegalin. This interacting domain of PDE4D consists of
a region of predicted In view of its extensive coiled-coil composition, it is likely that
myomegalin either oligomerizes with itself or interacts with other
proteins. The presence of a leucine zipper domain, which is also known
to mediate protein/protein interaction, is consistent with this
conclusion. In addition to a PDE, other proteins may be interacting
with myomegalin. Using homology searches, a domain similar to the
domain involved in dynactin-centractin interaction, a helix-loop-helix
found in Clip170, and an uncharged domain that resembles an Src
homology 3 binding domain, were identified. Thus, the presence of these
putative interacting domains suggests that myomegalin is present in a
complex with several additional proteins, which may be involved in
vesicle transport or with their interaction with cytoskeletal
structures. This raises the possibility that myomegalin may play a role
in vesicle transport between the different layers of the Golgi stack.
Myomegalin is expressed at high levels in skeletal and cardiac tissue,
suggesting an important function in muscle cells. Upon staining with a
myomegalin antibody, a periodic pattern was observed indicating
localization either in the z region between sarcomeres or in the
sarcoplasmic reticulum. It could not be determined whether PDE4D and
myomegalin are in the z band proper or are associated with the
sarcoplasmic reticulum. Several other cAMP-signal transduction proteins
have been localized in the same region of the sarcomere including
adenylyl cyclase, L channels, and the regulatory subunit of PKA (39,
40). Thus, it is plausible that myomegalin brings PDE4D to a site where
several steps in the cAMP signaling cascade take place in muscle cells.
The colocalization of PDE4D and myomegalin in Golgi/centrosomal
structures points to a role of cyclic nucleotide signaling in the
function of these organelles. Targeting of a PDE to the Golgi may have
an important function in controlling cAMP diffusion to these regions.
Interestingly, PKA regulatory subunits, and anchoring AKAPs, have also
been localized in the Golgi apparatus and in the centrosome (8, 9, 41,
42). Thus, our findings suggest that PDE4D localization in these
regions serves to control the state of activation of PKAs and therefore
the cAMP-regulated phosphorylations in these domains. The exact role of
cAMP signaling in centrosome function and in vesicle transport in the
Golgi is largely unknown, even though there is ample indirect evidence that cAMP regulates both cytoskeleton assembly as well as vesicle transport (43, 44). Pigment granule redistribution in
Xenopus melanocytes is regulated by cAMP (45), and cyclic
nucleotides have long been implicated in the control of flagellar
movements (46). Thus, it is possible that strategic localization of a PDE in these regions via myomegalin interaction may be involved in the
control of organelle movements or cytoskeletal
assembly/disassembly.
-helical and
coiled-coil structures, with domains shared with microtubule-associated proteins, and a leucine zipper identical to that found in the Drosophila centrosomin. Transcripts of 7.5-8 kilobases
were present in most tissues, whereas a short mRNA of 2.4 kilobases
was detected only in rat testis. A third splicing variant was expressed
predominantly in rat heart. Antibodies against the deduced sequence
recognized particulate myomegalin proteins of 62 kDa in testis and
230-250 kDa in heart and skeletal muscle. Immunocytochemistry and
transfection studies demonstrate colocalization of PDE4D and myomegalin
in the Golgi/centrosomal area of cultured cells, and in sarcomeric structures of skeletal muscle. Myomegalin expressed in COS-7 cells coimmunoprecipitated with PDE4D3 and sequestered it to particulate structures. These findings indicate that myomegalin is a novel protein
that functions as an anchor to localize components of the
cAMP-dependent pathway to the Golgi/centrosomal region
of the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11 cDNA libraries (from rat brain and rat skeletal muscle)
were obtained from CLONTECH (Palo Alto, CA), yeast
media from Difco (Detroit, MI), and amino acids and 3-amino-1,2,4,-triazole (3-AT) from Sigma. Restriction enzymes were
purchased from Roche Molecular Biochemicals, DNA modifying enzymes and
PCR reagents from Life Technologies, Inc. or Stratagene (La Jolla, CA).
Leu.
-galactosidase activity was detected after overnight
incubation at 30 °C.
-galactosidase filter lift assay was
carried out to detect positive clones. Each His+Z+ clone was restreaked on SD/Ade, 50 mM 3-AT medium to generate single clones.
Single colonies were reassayed for
-galactosidase activity. The
clones obtained by screening with the amino terminus of PDE4D3 will be reported elsewhere.
Leu and incubated for 3 days. Colonies were replica-plated on SD/
Leu only and on SD/
Leu,
Trp plates. Colonies growing on SD/
Leu plates (+Trp) but
not on SD/
Leu,
Trp (
Trp) carry the AD/library plasmid; however, the plasmid containing the bait had been lost. These Trp auxotrophs were reassayed for
-galactosidase activity. Negative clones were saved. Plasmid DNA was isolated from Trp auxotrophs according to the
manufacturer's protocol and was used for transformation into E. coli strain HB101 using electroporation (Genepulser, Bio-Rad).
-galactosidase expression. To measure the
-galactosidase activity in transformants, the colony lift filter assay was done according to the manufacturer's protocol (CLONTECH). Blue colonies were developed
after incubation overnight at 30 °C.
Leu. Individual positive colonies
for Trp and Leu were streaked separately on the corresponding SD medium and grown for 3-4 days at 30 °C. The different transformants of Trp+ and Leu+ were replica-plated in a grid pattern on YPD plates and
incubated overnight at 30 °C. Colonies of diploid cells were replica-plated on SD medium lacking Leu, Trp, and His. After ~6 days,
His+ colonies were counted and LacZ expression was detected using the
-galactosidase assay.
gt11 libraries
(CLONTECH). Three of the more than 50 clones retrieved were sequenced, and the overlap with PBP46 was established by
multiple sequence alignment. Because the potential initiation codon was
absent in all three clones, oligonucleotides corresponding to the 5'
end of the new open reading frame were designed and used for further
screening of the same library. This procedure was repeated four times
until a clone containing in-frame stop codons upstream from the
putative initiation codon was retrieved. Overlapping cDNAs were
used to reconstruct the entire open reading frame of myomegalin. Melds
were confirmed by at least two overlapping clones. Three of the clones
retrieved contained 5' ends that could not be aligned with the
reconstructed open reading frame. Whether these clones encompass
splicing junctions or are cloning artifacts was not further investigated.
-32P]dCTP by the random primer procedure
(Life Technologies, Inc.). The labeled probes were purified using Quick
Spin G-25 Sephadex columns (Roche Molecular Biochemicals) and used to
hybridize a rat multiple tissue Northern blot
(CLONTECH). Hybridization and washing procedures
were carried out according to the manufacturer's protocol.
-mercaptoethanol, and 0.0025% (w/v)
bromphenol blue), boiled for 5 min, and subjected to electrophoresis on
8% SDS-polyacrylamide gel. The proteins were then blotted onto an
Immobilon membrane (Bio-Rad), followed by blocking of the membrane in
TBS-T (20 mM Tris-HCl, pH 7.6, 14 mM NaCl,
0.1% Tween 20) containing 0.1% (w/v) nonfat milk. After several
washes, the membrane was incubated with the primary antibody (M3S1,
PBP4 antiserum, or anti-flag tag antibody) in TBS-T for 60 min, then
washed with TBS-T followed by another 1-h incubation with
peroxidase-linked anti-rabbit or anti-mouse IgG. After several washes
with TBS-T, the membrane was incubated with the ECL detection reagent
(Amersham Pharmacia Biotech) and exposed to XAR-5 x-ray film for 5-120
s (Eastman Kodak Co.) to detect the peroxidase-conjugated secondary antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase and yeast growth assays. Of these positive clones,
one (PBP46) was further characterized because the sequence of this
cDNA demonstrated only weak similarity with known sequences. The
interaction with the PDE was evident whether the cDNA was fused to
the activation or the DNA binding domain, and when either a growth or
-galactosidase assay was used (Fig. 1). Analysis using constructs
containing the different PDE4D domains indicated that PBP46 interacted
with the amino terminus of PDE4D in a region that corresponds to the
upstream conserved region 2 (UCR2) of PDE4 (aa 134-164 of PDE4D3)
(Fig. 1). This conclusion is based on the observation that constructs
encoding aa 1-133 (0.4), aa 164-end (TruA), 179-end (TruB), and
214-end (TruC) of PDE4D3 (Fig. 1) did not interact with PBP46. This
PDE domain is upstream from an autoinhibitory domain that controls the
catalytic activity of the enzyme (18).
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Fig. 1.
Interaction between the carboxyl terminus
region of myomegalin and PDE4D using the yeast two-hybrid assay.
The PBP46 construct corresponds to amino acid 1765 to 2324 of
myomegalin. The PDE4D construct used (PDE1.6) contains the inhibitory
domain, the catalytic domain, and the carboxyl-terminal domain. The PDE
constructs with the different deletions and the myomegalin construct
(PBP46 cDNA) were subcloned in either pGBT9 in frame with the DNA
binding domain (BD) or pGAD10 in frame with AD. These
different constructs were used for transformation of the yeast Y190
strain using the lithium acetate method (see "Experimental
Procedures"). Interaction was assessed by growth in triple dropout
medium and by measuring the -galactosidase activity. A
representative experiment of the three performed is reported.
gt11 cDNA library was therefore repeatedly
screened using the PBP46 cDNA as a probe. To obtain the entire ORF,
the library was screened four times and 18 overlapping clones were characterized. A meld of these clones indicated the presence of an
uninterrupted ORF of 6975 bp, which encodes a protein of 2324 amino
acids with a calculated molecular mass of 262 kDa (Fig. 2). An additional 1000 bp of 3'-UTR and
more than 500 bp of 5'-UTR are consistent with the 7.5-8-kb transcript
detected by Northern blot analysis (see below). In view of the high
level of expression in muscle cells and the large size of the encoded
protein, this new gene was termed myomegalin (mmg, accession
no. AF139185).
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Fig. 2.
Structure of myomegalin. A diagram of
the structure of myomegalin is reported in panel
A. The regions of homology with the Drosophila
centrosomin (droCENT) dynactin (HumDYNAC), bovine
DAP-150 (Bov DAP-150), and Clip-170
(HumCLIP-170) are highlighted together with the alignment of
the homologous regions. Identical residues are in black,
while conservative substitutions are in gray. B,
deduced amino acid sequence of myomegalin. The gray
arrow in the sequence indicates the beginning of the PBP46
clone. The gray box corresponds to the sequence
of the peptide used to generate the PBP4 antibody. The coiled-coil
regions were identified using the Garnier program. The uncharged domain
contains a potential Src homology 3 binding domain.
-helical domains (Fig. 2A)
with 20% identity to myosin heavy chains, even though no sequences
similar to the ATP binding domain of the myosin heavy chain could be
identified. The full-length protein contains a putative leucine zipper
at the amino terminus between residues 8 and 31 and an uncharged region
rich in proline close to the COOH-terminal region (Fig. 2, A
and B). BLAST search comparison showed that the first domain is almost identical to the first leucine zipper of
Drosophila centrosomin, a protein localized in the
centrosome of Drosophila melanogaster (26). In addition, a
domain between amino acids 769 and 784 is homologous to a domain of
dynactin that is responsible for the binding to the actin-like protein
centractin/ARP1. Dynactin and centractin are part of the complex
implicated in vesicle transport and the interaction with the
microtubules in mammalian cells (27). Finally, myomegalin contains a
helix-loop-helix domain between amino acids 1079 and 1094. This domain
is homologous with regions found in human CLIP-170 (cytoplasmic linker
protein) and rat DAP-150 (dynein associated polypeptide) (Fig.
2A). CLIP-170 is a protein that binds microtubules and is
implicated in the process of endocytosis and organelle transport in
mammalians (28). The function of DAP-150 is still largely unknown (29).
The myomegalin domains included in the testis variant (corresponding to
the PBP46 clone, see below) contains mostly
-helices and two
coil-coil regions, as indicated in Fig. 2. The presence of coil-coil
regions and absence of putative transmembrane domains are consistent
with recovery of expressed PBP46, a truncated myomegalin, in
particulate fractions of transfected cells, and its resistance to
solubilization with nonionic detergents alone.
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Fig. 3.
Identification of the different myomegalin
variants and their tissue distribution. A, schematic
representation of the splicing boundary in the myomegalin sequence, the
probes used for Northern blot analysis, and the PCR primers used.
B and C, Northern blot analysis of different rat
tissues using probes at the 3' end (probe 1) and the 5' end (probe 2)
of myomegalin mRNA. He, heart; Br, brain;
Sp, spleen; Lu, lung; Li, liver;
Sk, skeletal muscle; Ki, kidney; Te,
testis. Exposure time was 17-20 h. D and E,
RT-PCR analysis of the expression of the two long variants of
myomegalin. RT-PCR was performed with the pair of primers reported
above the lanes. In some reactions, the reverse transcriptase was
omitted as a negative control ( RT). The identity of the
products was confirmed by sequencing.
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Fig. 4.
Western blot analysis of myomegalin
expression using the PBP4 antibody. A, COS-7 cells were
transfected with a pCEPflag-PBP46 construct or with empty vector
(mock). After 24 h, cells were harvested in homogenization buffer
and the extract centrifuged at 14,000 × g for 30 min.
Pellets (P) and supernatant (S) were fractionated
on SDS-PAGE, and the migration of the recombinant myomegalin was
monitored using anti-flag antibodies. B, rat heart and
skeletal muscle tissue was homogenized according to the procedure
detailed under "Experimental Procedures." Pellets and supernatants
were fractionated on SDS-PAGE. After transfer, membranes were probed
with the PBP4 antibody. C, total testis (TT) or
seminiferous tubules (ST) were homogenized and aliquots of
the homogenates fractionated on SDS-PAGE. After transfer, the membrane
was probed with the PBP4 antibody.
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Fig. 5.
Subcellular localization of endogenous and
recombinant myomegalin in COS-7 cells. A, COS-7
cells were transfected with the pCEPflag-PBP46 construct. Twenty-four h
after transfection, cells were stained with the myomegalin-specific
antibodies (PBP4) or with the flag epitope antibodies. Fluorescein
isothiocyanate-conjugated anti-rabbit secondary antibodies
(green) and rhodamine-conjugated anti-mouse IgG secondary
antibodies (red) were used to detect the PBP4 and the
monoclonal flag antibody, respectively. Pictures were captured with a
confocal microscope and merged. B, immunofluorescence
staining of untransfected COS-7 cells with the centrosomal marker
CTR453 and with PBP4 antibody. C, immunofluorescence
staining of untransfected COS-7 cells with the PBP4 antibody and with
the Golgi marker CTR433.
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Fig. 6.
Colocalization of myomegalin and PDE4D in
mouse skeletal muscle. Sections of mouse skeletal muscle were
stained with myomegalin (PBP4, red) and PDE4D-specific
(M3S1, green) antibodies. The distance between the two
contiguous bands was estimated to be 2 µm, consistent with the length
of a sarcomere. The staining with myomegalin and PDE4D antibodies
overlapped with that of desmin but not with either myosin or actin. The
periodic pattern of PDE4D staining was confirmed by a second polyclonal
anti-PDE4 antibody. In all instances, staining could be blocked by
preadsorption of the primary antibody with the corresponding peptide or
fusion protein. Similar results were obtained with rat skeletal
muscle.
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Fig. 7.
Cellular localization of testis-specific
myomegalin in germ cells. Sections of adult rat testis were
stained with the myomegalin antibody (PBP4) in the absence or presence
of peptide PBP4. DAPI was used to stain the nuclei of developing germ
cells and somatic cells.
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Fig. 8.
Coimmunoprecipitation of myomegalin and
PDE4D3. A, diagram of the domains present in the three
PDE4D variants used for the coimmunoprecipitation. B, COS
cells were transfected with either a PDE4D (pCMV5-PDE4D3) or a
myomegalin (pCEP4-PBP46) expression vector or a combination of the two.
After homogenization and centrifugation, the supernatant and the
RIPA-extracted supernatant were combined and subjected to
immunoprecipitation with PDE4D-selective monoclonal antibodies. The
pellets of the immunoprecipitation or an aliquot of the supernatant
before precipitation were analyzed by SDS-PAGE and Western blot using
either myomegalin- (PBP4) or PDE4D-selective (M3S1) antibodies. When
immunoprecipitation was carried out with nonimmune IgG as a control,
neither PDE4D3 nor myomegalin were recovered in the pellet (data not
shown). C, COS cells were cotransfected with vectors
containing PDE4D1, PDE4D2, or PDE4D3 cDNAs and a myomegalin
expression plasmid (pCEP4-PBP46). After 24 h, cell extracts were
prepared and processed for immunoprecipitation and Western blot
analyses as reported for panel B.
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Fig. 9.
Recovery of PDE4D3 in the insoluble fraction
after cotransfection with myomegalin. COS-7 cells were transfected
with plasmids pCDNA3-PDE4D3, pCEP4-PBP46 alone, or in combination.
Mock-transfected cells received an empty vector only. After 24 h,
cells were harvested, homogenized, and the soluble and particulate
fractions were isolated by centrifugation at 14000 × g
for 30 min. Pellets were washed twice and resuspended in the same
buffer. An aliquot of the pellet was used for the PDE assay
(A). Aliquots of the pellet and supernatants were
fractionated by SDS-PAGE, blotted, and probed with PDE4D-specific
antibodies (M3S1) (B) or with anti-flag antibodies
(C). Comparable amounts of soluble and particulate proteins
were loaded in each lane. Protein content was determined by the
Bradford method. A representative experiment of the three performed is
reported.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure with amphipathic
distribution of the charges. The carboxyl terminus of myomegalin also
contains
-helical domains that may mediate the interaction with a
PDE. Using the yeast two-hybrid assay, we have reported that this PDE4D
domain, which interacts with myomegalin, is also involved in
intramolecular interactions with the amino-terminal regulatory domain
and that it functions as an autoinhibitory domain that modulates the
catalytic activity of the enzyme (18). At present, it is not clear
whether the intramolecular interaction and the binding to
myomegalin are mutually exclusive or if they can occur at the same
time, nor is it known whether the binding of myomegalin alters the
catalytic activity of the PDE. We have reported that phosphorylation of
PDE4D3 alters the interaction of this domain with the amino terminus
(18); it is then possible that the state of phosphorylation also
affects the ability of the PDE4D to interact with myomegalin. Further studies are required to clarify this issue. It is possible that the
interaction of PDE4D with myomegalin, and therefore with the Golgi/centrosome, may be a dynamic process and that translocation of
this protein may occur during signaling. Indeed, data have recently
been reported indicating that PDE4D3 may translocate upon activation of
the mitogen-activated protein kinase pathway (38).
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ACKNOWLEDGEMENTS |
---|
We acknowledge Dr. Michel Bornens for constructive comments and for the gift of the centrosomal and Golgi antibodies, and Dr. Kjetil Tasken for helpful discussions and advice with these studies.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1-HD20788 and by a gift from Berlex (to G. P.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF139185.
§ These authors contributed equally to this study.
To whom correspondence should be addressed. Tel.:
650-725-2452; Fax: 650-725-7102; E-mail:
marco.conti@stanford.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006546200
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ABBREVIATIONS |
---|
The abbreviations used are: PKA, protein kinase A; AKAP, A kinase anchoring protein; PDE, phosphodiesterase; kb, kilobase pair(s); bp, base pair(s); ORF, open reading frame; RT, reverse transcriptase; PCR, polymerase chain reaction; AD, activation domain; 3-AT, 3-amino-1,2,4,-triazole; PAGE, polyacrylamide gel electrophoresis; SD, selection medium; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline with Tween 20; aa, amino acid(s); MOPS, 4-morpholinepropanesulfonic acid; RIPA, radioimmune precipitation assay.
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