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INTRODUCTION |
Modulation of the levels of the second messenger cAMP in cells is
important in the regulation of numerous physiological processes, including those in the immune/inflammatory systems, vascular smooth muscle, and the brain. Cyclic nucleotide phosphodiesterases
(PDEs)1 are a diverse family
of enzymes that hydrolyze cAMP and cGMP and thus play an important role
in modulating cAMP levels (1). The cAMP-specific phosphodiesterases
(PDE4s) can be differentiated from other PDEs by sequence homology of
the catalytic region of the enzymes (2) and by their ability to be
specifically inhibited by the drug rolipram. Rolipram and other
specific PDE4 inhibitors have been shown to have anti-depressant,
anti-inflammatory, and smooth muscle relaxant activity in humans (2).
The PDE4 enzymes are also characterized by the presence of unique
regions of amino acid sequence outside the catalytic region of the
proteins, which are called upstream conserved regions 1 and 2 (UCR1 and
UCR2) and are located in the amino-terminal half of the proteins (3). The PDE4s are comprised of a large family of isoforms, encoded by four
different genes (PDE4A, PDE4B, PDE4C, and PDE4D)
in humans, with additional diversity being generated by alternative
mRNA splicing (2).
We and other groups (3-5) have characterized five different isoforms
encoded by the human PDE4D gene, all of which appear to be
conserved among mammals (6, 7). The five isoforms differ by the
substitution of unique blocks of amino acids at the amino-terminal
regions of their respective proteins (5). The two smaller PDE4D
isoforms, PDE4D1 and PDE4D2, are located exclusively in the cytosolic
fraction of the cell (5, 7). The larger isoforms PDE4D3, PDE4D4, and
PDE4D5 are each found both in the cytosol as well as in association
with cellular particulate fractions (5, 7). The functional consequences
of this diversity are poorly understood. However, the PDE4D3 isoform is
a substrate for protein kinase A (PKA), which serves to activate this
isoform (6, 8-10).
In this study, we identify a novel property of a different PDE4D
isoform, PDE4D5. This human isoform was recently isolated by us and is
found in a variety of tissues and cell types, including the brain (5).
PDE4D5 can be distinguished from other PDE4D isoforms by the presence
of a unique amino-terminal region of 88 amino acids (5). We now
demonstrate that PDE4D5 interacts specifically and with high affinity
with the RACK1 protein (receptor for activated C-kinase). RACK1 is a
36-kDa WD-repeat protein (11) that binds to certain protein kinase C
(PKC) isoforms subsequent to their activation by diacylglycerol or
phorbol esters such as PMA. RACK1 appears to serve as a "scaffold"
or "anchor" protein for these PKC isoforms (12-15). Recently,
RACK1 has been shown to interact with the
subunit of integrins (16)
and the Src protein tyrosyl kinase (17). Scaffold and anchor proteins
physically connect various signal transduction components, such as
receptors, kinases, and elements of the cytoskeleton, into stable
complexes (18). Our data suggest that RACK1 may recruit the PDE4D5
isoform into a such a complex in a variety of cell types.
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EXPERIMENTAL PROCEDURES |
Materials--
A HeLa cell (human HeLa S3 cells; American
Type Culture Collection) two-hybrid library cloned into the
EcoRI and XhoI sites of the pGADGH vector (19)
was obtained from David Beach (Cold Spring Harbor Laboratory). This
vector expresses proteins as fusions with the activation domain of the
Saccharomyces cerevisiae GAL4 protein. Antibodies to RACK1
were obtained from Transduction Laboratories. The RACK1 antibody
detects a 36-kDa species in Jurkat cells (16, 17). A monoclonal
antibody to human PDE4D proteins, which does not cross-react with other
PDE4 species and which we have described previously (5), was obtained
from Sharon Wolda, ICOS Corp. This antibody interacts with PDE4D3 and
PDE4D5 with very similar affinity (5). The PDE4D antibody also detects
PDE4D species in rats and mice (5). cDNA clones for
Gs
and
'-COP were obtained from A. G. Gilman and
K. J. Harrison-Lavoie, respectively. The L40 S. cerevisiae
strain and the pBTM116 plasmid were obtained from A. B. Vojtek.
Two-hybrid Screens--
These were performed using methods we
have described previously (20). In brief, the full open reading frame
(ORF) of the pPDE79 cDNA (GenBankTM accession number
AF012073) encoding human PDE4D5 (5) was cloned into the NotI
site of pLEXAN, to generate pLEXAN79, which encodes a fusion between
PDE4D5 and the DNA binding domain of the Escherichia coli
LexA protein. These constructs were prepared by the addition of
NotI sites to the cDNAs by the use of PCR, as described
previously (3). pLEXAN is a derivative of pBTM116 (21), with a
NotI site inserted into the polylinker. Screens were
performed with the HeLa two-hybrid cDNA library in the S. cerevisiae strain L40 (22). To screen the HeLa cDNA library, positive clones were initially selected for growth in the absence of
histidine (without 3-aminotriazole) and then transferred to patches and
assayed for lacZ activity using a filter
-galactosidase assay, as described (20). Library plasmid DNA was then isolated from
the positives and then re-assayed for interaction with PDE4D5, using
methods described previously (20).
For additional two-hybrid experiments, the full ORFs of all five human
PDE4D isoforms (5) and other cDNAs were cloned into the
NotI site of pLEXAN, in a manner analogous to that described above for PDE4D5. Similarly, the full ORF of human RACK1 ((23) GenBankTM accession number M24194), obtained from a
positive isolated in the two-hybrid screen, was cloned into the
NotI site of pGADN, to produce pGADNRACK1. pGADN is a
derivative of pGADGH but with a NotI site inserted into the polylinker.
Generation of Bacterial Expression Constructs--
The full ORFs
of PDE4D5, PDE4D3, and RACK1 were cloned into the NotI site
of pMALN, to generate pMALPDE4D5 (also called pMALP79) and pMALPDE4D3
(also called pMALN43), respectively. pMALN is a derivative of pMALc2
(New England Biolabs (24)), with a NotI site inserted into
the polylinker. The full ORF of RACK1 was cloned into the
NotI site of pGEX-5X-3 (Amersham Pharmacia Biotech (25)) to
generate pGEXRACK. All constructs named "pMAL ... " generate fusions between the maltose-binding protein (MBP) and the amino terminus of the protein encoded by the insert. Constructs named "pGEX ... " constructs generate fusions between glutathione
S-transferase (GST) and the amino terminus of the protein
encoded by the insert.
Generation of COS7 Cell Expression Constructs--
The full ORF
of PDE4D5 was cloned into the NotI site of pcDNA3
(Invitrogen), to create pcDNAPDE4D5VSV (also called
pcDNAN79VSV). In this construct, the insert is placed under the
control of the cytomegalovirus intermediate early gene promoter. The
full ORF of PDE4D3 was cloned into pCIneo to produce pCIneo-PDE4D3 (5). In both cases, a sequence corresponding to the vesicular stomatitis virus (VSV) glycoprotein epitope (26) was added immediately downstream
from the last codon of the PDE to encode a carboxyl-terminal fusion.
The native PDE4D stop codon was removed in this process, but a
synthetic stop codon was placed immediately downstream from the epitope
sequence. The full ORFs of RACK1, PDE4D1, PDE4D2, PDE4D4, PDE4B1,
PDE4B2, PDE4B3, and PDE4C2 were cloned into the NotI site of
pcDNA3 (without a carboxyl-terminal epitope).
Generation of cDNAs Encoding Mutant Forms of PDE4D5--
To
generate deletions in the amino-terminal region of PDE4D5, PCR was used
to amplify various regions of PDE4D5, which were then cloned into
pLEXAN or pcDNA3. NotI sites were added to the PCR
primers to aid in cloning. To generate point mutations in PDE4D5, the
full-length PDE4D5 cDNA was subjected to site-directed mutagenesis
with the QuikChange site-directed mutagenesis kit (Stratagene).
Verification of Two-hybrid, Expression, and Mutagenesis
Constructs--
All PCR-generated or mutant constructs were verified
by sequencing prior to use.
Growth of Cell Lines--
All cell lines used in this study were
obtained from the American Type Culture Collection. The lines were
grown in Dulbecco's modified Eagle's medium, supplemented with fetal
calf serum and antibiotics.
Co-immunoprecipitations--
COS7, SK-N-SH, Jurkat, 3T3-F442A,
or HEK293 cells were harvested in 0.5 ml of lysis buffer (55 mM Tris-HCl, pH 7.4, 132 mM NaCl, 22 mM sodium fluoride, 11 mM sodium pyrophosphate,
1.1 mM EDTA, 5.5 mM EGTA) containing complete
protease inhibitor mixture (Roche Molecular Biochemicals) and lysed
with 8 strokes of a 261/2-gauge needle attached to a disposable
syringe. This method was used because we wanted to process a large
number of samples quickly. It produced complete lysis of cells on the
basis of vital dye staining and absence of latent lactate dehydrogenase
activity. After lysis, cell debris was removed by centrifugation at
12,000 × g for 10 min.
For immunoprecipitation of RACK1, 500 µg of cleared cytosol was
mixed with 30 µl of pre-equilibrated anti-mouse IgM-agarose (Sigma)
and incubated for 30 min at 4 °C. The beads were removed by
centrifugation at 2,000 × g for 5 min, and the cleared
lysates were incubated with 16 µl of anti-RACK1 antibody
(Transduction Laboratories) in the presence of anti-mouse IgM agarose
beads for 3 h at 4 °C. For immunoprecipitation of PDE4D5 from
COS7 cells, 500 µg of cleared cytosol was mixed with 30 µl of
pre-equilibrated protein A-agarose beads (Sigma) and incubated for 30 min at 4 °C. The beads were removed, and lysates were incubated for
3 h at 4 °C with 16 µl of monoclonal anti-PDE4D antibody (5)
in the presence of protein A-agarose beads. In both cases, the beads were then collected by centrifugation (2,000 × g for 5 min) and washed three times with lysis buffer. Co-immunoprecipitation
of PDE4D with RACK1 was analyzed by immunoblotting with the anti-PDE4D monoclonal antibody and the anti-RACK1 antibody.
Expression of Glutathione S-Transferase (GST) and Maltose-binding
Protein (MBP) Fusions in E. coli--
Cultures of E. coli
JM109 containing pGEXRACK1, pMALPDE4D3, or pMALPDE4D5 were induced with
1 mM isopropyl-
-D-thiogalactopyranoside (Roche Molecular Biochemicals) for 4 h at 30 °C. Bacteria were harvested by centrifugation at 2,500 × g for 10 min at
4 °C, and the bacterial pellet was frozen at
80 °C overnight.
The bacterial pellets were resuspended in 10 ml of ice-cold
resuspension buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10 mM
-mercaptoethanol, and complete protease inhibitor mixture) and
sonicated with 4 × 30-s bursts at maximal setting. Triton X-100
was added to a final concentration of 0.02%, and cell debris was then
removed by centrifugation at 15,000 × g for 10 min at
4 °C. The cleared supernatant was incubated with 1/10th volume of
pre-equilibrated glutathione-Sepharose beads (for GST fusions) or
amylose resin (for MBP fusions) on an orbital shaker for 30 min at
4 °C. The beads were collected by centrifugation at 2,000 × g for 1 min and washed three times with ice-cold
resuspension buffer. The fusion proteins were eluted by the addition of
5 mM glutathione, 50 mM Tris-HCl, pH 8.0 (for
GST fusions), or 10 mM maltose, 50 mM Tris-HCl,
pH 8.0 (for MBP fusions), and dialyzed three times against 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5%
glycerol. The purified fusion proteins were stored at
80 °C until required.
RACK1 Pull-down Assays--
COS7 cells were transfected with
either 10 µg of plasmid pcDNAPDE4D5VSV or 10 µg of plasmid
pCIneo-PDE4D3. Transfections were done as described previously (5, 27).
After 72 h, cells were harvested and lysed with 8 strokes of a
261/2-gauge needle in 0.5 ml of lysis buffer. Cell debris was
removed by centrifugation (12,000 × g for 10 min at
4 °C), and 500 µg of cleared lysate was incubated with GST or
GST-RACK1 and 60 µl of glutathione-Sepharose beads for 1 h at
4 °C. Beads were pelleted by centrifugation at 2,000 × g for 5 min at 4 °C and washed three times in lysis
buffer. Protein complexes were eluted by the addition of 5 mM glutathione, 50 mM Tris-HCl, pH 8.0, and
co-precipitation of PDE4D5 was analyzed by immunoblotting with an
anti-VSV monoclonal antibody (26). In two cases (see below)
immunoblotting was performed with the PDE4D monoclonal antibody.
ELISA Protein Interaction Assay--
Reacti-Bind
glutathione-coated ELISA plates (Pierce) were treated with 1 mg of
purified GST or GST-RACK1 for 16 h at 4 °C and then washed
three times with 100 µl/well wash buffer (10 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 0.05% Tween 20). Dilutions of the MBP
fusions of PDE4D3 and PDE4D5 were incubated with immobilized GST or
GST-RACK1 for 3 h at room temperature, and the wells were then
washed three times with 100 µl/well ice-cold wash buffer. Protein
complexes were fixed with the addition of 100 µl/well 4% (v/v)
paraformaldehyde in phosphate-buffered saline for 30 min at 4 °C.
Paraformaldehyde fixation was used at this step because of concern that
the complexes might dissociate during the subsequent detection
procedure. However, it was determined subsequently that the binding of
PDE4D5 and RACK1 was so avid that the addition of paraformaldehyde made
little difference. After fixation, protein complex formation was
detected by the addition of anti-PDE4D monoclonal antibody (1:10,000
(v/v) in dilution buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl)) for 1 h at room temperature, followed by
alkaline phosphatase-conjugated anti-mouse IgG (Sigma; 1:2000 (v/v) in dilution buffer) for a further hour at room temperature.
Immunoreactivity was visualized with the BCIP Microwell 2 Component
Phosphatase Substrate System (Kirkegaard & Perry Laboratories)
following the manufacturer's instructions and quantified using an MRX
microplate reader (Dynex Technologies) set at a test wavelength of 630 nm. RACK1 binding was stated as being the amount of PDE4D
immunoreactivity found associated with GST-RACK1 minus that associated
with GST alone and was expressed as arbitrary units.
Preparation and Fractionation of Tissue Homogenates--
These
were performed as we have described previously (5, 27-31). Confluent
cultures of COS7 cells were scraped into 0.8 ml of lysis buffer and
lysed with 20 strokes of a Dounce homogenizer equipped with a
tight-fitting pestle. These homogenates were then fractionated as
follows. For the low speed pellet (P1), they were centrifuged at
1000 × gav for 10 min. The supernatant
from this step was then centrifuged at 100,000 × gav for 1 h to yield a high speed pellet
(P2, particulate) fraction and a supernatant (SN, cytosol) fraction.
The pellets were then resuspended in 0.5 ml of lysis buffer.
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
These were as described previously (32). In brief,
samples were resuspended in Laemmli buffer and boiled for 5 min.
Membranes were blocked in 5% (w/v) low-fat milk powder in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) overnight at
room temperature. They were then incubated with anti-PDE4D monoclonal
antibody or anti-RACK1 antibody diluted in 1:5000 (v/v) in 1% (v/v)
low-fat milk powder in TTBS (TBS plus 0.1% (v/v) Tween 20) for 3 h at room temperature. Detection of the bound antibody was with
anti-mouse IgG peroxidase (for PDE4D5) or anti-mouse IgM peroxidase
(for RACK1) secondary antibodies (both from Sigma) and the enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Biotech).
PDE Assays--
PDE activity was assayed as described previously
(33). All assays were conducted at 30 °C, and in all experiments a
freshly prepared slurry of Dowex:H2O:ethanol (1:1:1; v/v)
was used. In all experiments, initial rates were taken from linear time
courses of activity. Km values were determined over
a substrate range of 0.25-25 µM cAMP (7 different
concentrations). Dose-dependent inhibition by rolipram was
determined in the presence of 1 µM cAMP and over a range
(8-10 different values) of 10 nM to 100 mM
rolipram. The IC50 was then determined from these values,
using a least squares fitting algorithm. Rolipram was dissolved in
100% Me2SO as a 1 mM stock and diluted in 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2 to
provide a range of concentrations in the assay. The residual levels of
Me2SO were shown not to affect PDE activity over the ranges
used in this study. To define Km values, data from
PDE assays were analyzed by computer fitting to the hyperbolic form of
the Michaelis-Menten equation using an iterative least squares
procedure (Ultrafit; with Marquardt algorithm, robust fit, experimental
errors supplied; Biosoft). Relative Vmax values could be calculated using the Michaelis equation and the experimentally derived Km values, as described previously (29).
Measurement of Protein Concentrations--
Protein
concentrations were measured by the method of Bradford, using bovine
serum albumin as a standard (34).
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RESULTS |
Isolation of RACK1 as a Protein Interacting with PDE4D5 in a
Two-hybrid Screen--
We have recently described a novel PDE4D
isoform, called PDE4D5 (5). PDE4D5 differs from other PDE4D isoforms by
the presence of a unique amino-terminal end, 88 amino acids in length
(5), which has no detectable sequence homology to any other
phosphodiesterase (Fig. 1). To determine
more about the properties of PDE4D5, we wished to investigate whether
specific proteins might bind to it. For this purpose, we used
full-length PDE4D5 as a "bait" in a two-hybrid screen. Two
independent screens were performed, with identical results. The results
of one screen are shown (Table I). In
both screens, a large number of cDNA clones were obtained, all of
which encoded the full ORF of the RACK1 protein (12-15, 23).

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Fig. 1.
Schematic of the amino-terminal region of
PDE4D5. Top, a line diagram representing the five human
PDE4D isoforms (5). The numbers 1-5 represent isoforms
PDE4D1 through PDE4D5, respectively. The heavy bar indicates
sequences homologous to those in other PDE4 isoforms, with the
strongest regions of conservation (the catalytic region and UCR1 and
UCR2) indicated by the cross-hatched areas. The thin,
branched lines adjacent to the numbers indicate
sequence regions unique to each isoform. The thin lines
merge where the sequences of the various isoforms join that of the
shared sequence. The PDE4D2 isoform begins in the middle of UCR2, at a
methionine that is internal to the other four isoforms.
Bottom, the amino acid sequence of the unique 88 amino-terminal region of PDE4D5. The numbers immediately
above the sequence indicate the amino acid coordinates. Amino acids
subjected to site-directed mutagenesis (Figs. 6c and
7B, below) are underlined. The small
arrows immediately above the sequence indicate amino acids that,
when mutated, block the interaction with RACK1 (Figs. 6c and
7b). The numbers below the sequence indicate the
amino-terminal ends of the nested deletion constructs (N1, N2, etc.)
used in Figs. 6b and 7a. WT indicates
the full-length construct. Regions of amino acids removed from deletion
constructs (D1 and D2) are also shown.
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Specificity of the PDE4D5-RACK1 Interaction--
To obtain
preliminary evidence that the PDE4D5-RACK1 interaction was specific, we
used two-hybrid
-galactosidase assays to test the interaction of
RACK1 with a variety of "baits" expressed as LexA fusions. These
included lamin (22), casein kinase II, Ras, Raf, several transcription
factors, and LexA itself (i.e. not as a fusion). In a
similar manner, we tested PDE4D5 for its ability to bind to these
proteins expressed as GAL4 fusions and also to the GAL4 activation
domain itself (i.e. not as a fusion). No interaction was
detected under conditions where we could demonstrate an interaction
between PDE4D5 and RACK1 (data not shown). We also tested PDE4D5 for
its ability to bind to two other WD-repeat proteins, the G-protein
Gs
subunit and the coatomer subunit protein
'-COP. Our rationale for testing
'-COP was that, like RACK1, it has also
been shown to bind to PKC isoforms, although with selectivity for
different PKC isoforms than RACK1 (35). We were unable to detect an
interaction between PDE4D5 and either of these two WD-repeat proteins,
using
-galactosidase assay conditions that did detect the
interaction between PDE4D5 and RACK1 (The results for
'-COP are
shown in Fig. 2.) These data suggest that
PDE4D5 interacts specifically with RACK1 and not with WD-repeat
proteins generally.

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Fig. 2.
PDE4D5 interacts with RACK1 but not with the
related WD-repeat protein '-COP. The
PDE4D5 cDNA was cloned into pLEXAN to produce fusions with the LexA
DNA binding domain. Various WD-repeat proteins were cloned into pGADN
to produce fusions with the GAL4 activation domain. S. cerevisiae cells containing the appropriate plasmids were patched
onto plates that selected for both plasmids and subjected to a filter
-galactosidase assay, as described (20). Positive results in the
assay produce a change in the color of the patches from pink
to blue. The bottom two patches serve as internal
positive and negative standards, respectively (the oncoproteins
RASVal-12 and RAF (22), and the vectors without inserts).
As a positive control, the GAL4 activation domain fusion of '-COP
was shown to bind to LexA- -COP in a two-hybrid test (data not
shown), consistent with results reported previously (50).
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Endogenous Expression of RACK1 and PDE4D5 in COS7 Cells--
To
confirm and expand our two-hybrid data, we tested whether PDE4D5 and
RACK1 could be co-immunoprecipitated from mammalian cells. We performed
experiments first on COS7 cells (this and the next two sections), which
were later expanded to the study of other cell types (see below).
We have reported previously that PDE4D exists both in the cytosol and
in the cellular particulate fraction of various tissues and cell lines
(5). To determine whether this was also true in COS7 cells, COS7 cells
were disrupted and fractionated to yield a high speed (S2) supernatant
fraction, reflecting cytosolic components, and also P1 and P2
particulate fractions (see "Experimental Procedures"). The
fractions were subjected to SDS-PAGE and immunoblotted with antibodies
specific for either PDE4D or RACK1. Both specific polyclonal and
monoclonal PDE4D antibodies were used, with similar results (5). The
PDE4D antibodies were all generated to a carboxyl-terminal region of
the PDE4D protein and have been shown to detect all five PDE4D species
(5), as the PDE4D isoforms differ solely by their distinct
amino-terminal regions (Fig. 1).
Analysis of untransfected COS7 cells showed that they contain two PDE4D
immunoreactive species of 95 ± 3 and 103 ± 2 kDa (Fig. 3a), consistent with the
endogenous expression of the PDE4D3 and PDE4D5 isoforms, respectively
(5). Recombinant PDE4D3 and PDE4D5 isoforms expressed transiently in
COS7 cells (5) co-migrated on SDS-PAGE with these two endogenous PDE4D
species (data not shown). As demonstrated previously (5), when COS7
cells were transfected with cDNAs encoding these two isoforms, so
that the recombinant enzymes represented greater than 98% of total PDE activity in these cells, the major fraction of these enzymes was located in the cytosol (S2) fraction, but some immunoreactivity was
also seen in the pellet fractions.

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Fig. 3.
Endogenous expression and
co-immunoprecipitation of RACK1 and PDE4D5 in COS7 cells.
a, extracts from untransfected COS7 cells were fractionated
(see "Experimental Procedures") and then immunoblotted with the
PDE4D antibody (5). This identified in the high speed supernatant (S2)
fraction two immunoreactive species of 95 ± 3 and 105 ± 2 kDa. On the basis of co-migration with recombinant species, these
represented PDE4D3 and PDE4D5, respectively (5). PDE4D5 and to a lesser
extent PDE4D3 were also found in the low speed (P1) and high speed (P2)
pellet fractions. b, extracts from untransfected COS7 cells
were fractionated and then immunoblotted with the RACK1 antibody. This
identified in the high speed supernatant fraction a single
immunoreactive species of 36 ± 1 kDa consistent with RACK1 (12).
A similar immunoreactive species was also found in both the low speed
(P1) and high speed (P2) pellet fractions. c, cytosolic (S2)
fractions were prepared from untransfected COS7 cells and either
immunoblotted directly (lanes Total) or subjected
to immunoprecipitation with either the PDE4D antibody (lanes
4D-IP) or the RACK1 antibody (lanes
RACK1-IP). The fractions/immunoprecipitates were then run on
SDS-PAGE. The upper half of the gel was immunoblotted with
the PDE4D-specific antibody. The arrows indicate the
location of PDE4D3 and PDE4D5 as 95- and 105-kDa species, respectively.
The lower half of the gel was immunoblotted with the
RACK1-specific antibody, and the position of the 36-kDa RACK1 species
is indicated. In each case, analyses were performed on cells treated
(lanes +) or not treated (lanes ), with the
PKC-activating phorbol ester PMA (10 µM). d,
immunoblots of the combined pellet (lanes p) and
S2 fractions (lanes s) of untransfected COS7
cells treated with 10 µM PMA. Cells were harvested at the
indicated times (in minutes), fractionated as in a-c, and
subjected to SDS-PAGE, followed by immunoblotting for PKC (top
panel), RACK1 (middle panel), and PDE4D (bottom
panel). All data are typical of experiments done at least three
times. Each lane on the gels represents 50 µg of protein, with PDE
activities in the range of 25-35 pmol/min/mg protein. Equal amounts of
protein from the P1, P2, and S2 fractions were analyzed. The PDE4D
antibodies (either polyclonal or monoclonal) did not immunoprecipitate
purified GST-RACK1. The RACK1 antibody did not immunoprecipitate
purified MBP-PDE4D5 (data not shown).
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The specific antibody to RACK1 detected in untransfected COS7 cells was
a single 36 ± 1-kDa species (Fig. 3b), indicative of
the presence of endogenous RACK1 (12). As also found for PDE4D5, the
major fraction of RACK1 was located in the cytosolic (S2) fraction,
although RACK1 immunoreactivity was also evident in the pellet
fractions, to a similar extent to the level seen for endogenous PDE4D5
(Fig. 3a).
PDE4D5 and RACK1 Can Be Co-immunoprecipitated from COS7
Cells--
In order to determine whether endogenous PDE4D5 and RACK1
interacted in COS7 cells, we took the cytosolic (S2) fraction from COS7
cells and subjected it to an immunoprecipitation protocol using either
the RACK1 antibody (Fig. 3c; lanes RACK1-IP) or
the PDE4D antibody (Fig. 3c; lanes
4D-IP). The resulting immunoprecipitates were then
subjected to SDS-PAGE and immunoblotting. As PDE4D5 migrates as a
105-kDa protein (5) and RACK1 as a 36-kDa protein (12), we treated the
top half of the blot with the PDE4D antibody and the bottom half with
the RACK1 antibody. Cytosolic extracts (i.e. not subjected
to immunoprecipitation) were immunoblotted as controls (Fig.
3c; lanes Total). From this analysis, it was clear that the RACK1 antibody not only immunoprecipitated RACK1 but
that it also co-immunoprecipitated PDE4D5 (Fig. 3c).
Conversely, the PDE4D antibody not only immunoprecipitated PDE4D3 and
PDE4D5 but it also co-immunoprecipitated RACK1. These data demonstrate that endogenously expressed PDE4D5 and RACK1 are in a complex in the
cytosol of COS7 cells. We were also able to show that particulate RACK1
and PDE4D5, when solubilized by an Nonidet P-40 detergent system
described by others (17), could be similarly co-immunoprecipitated (data not shown).
PMA Does Not Affect Binding of RACK1 to PDE4D5--
It has been
demonstrated previously that treatment of cells with phorbol esters,
which activate PKC, is a necessary prerequisite for the interaction of
RACK1 with both PKC (12) and the integrin
subunit (16) but not for
the interaction of RACK1 with Src (17). Our co-immunoprecipitation data
suggest that PDE4D5, like Src, can interact with RACK1 without PKC
activation, as the treatment of COS7 with the phorbol ester PMA did not
affect the ability of PDE4D5 and RACK1 to be co-immunoprecipitated,
regardless of which antibody was used (Fig. 3c). In order to
ascertain that PMA was able to activate PKC in COS7 cells, we treated
COS7 cells with PMA, harvested them at various times, prepared
cytosolic (S2) fractions, and subjected them to SDS-PAGE, followed by
immunoblotting with antibodies for PKC
, RACK1, or PDE4D (Fig.
3d). These experiments showed clearly that endogenous PKC
was rapidly and completely translocated from the cytosol to the
particulate fraction within 5-10 min (Fig. 3d, top panel).
These data also indicate that PKC
was not constitutively activated
in COS7 cells, as PKC
was clearly cytosolic prior to challenge with
PMA. These observations suggest that PDE4D5, like Src, can interact
with RACK1 without PKC activation. Additionally, they suggest that PMA
does not trigger any translocation of either RACK1 or PDE4D5 to the
particulate fraction of COS7 cells (Fig. 3d, bottom two
panels).
PDE4D5 and RACK1 Can Be Co-immunoprecipitated from Various Cell
Types--
To determine whether the PDE4D5-RACK1 interaction occurred
in cells generally, we examined a number of other cell lines,
specifically HEK293 (human embryonic kidney), 3T3-F442A (mouse
fibroblast/pre-adipocyte), SK-N-SH (human neuroblastoma), and Jurkat
(human T-cell). Cytosolic extracts from these cell lines were subjected
to SDS-PAGE with subsequent immunoblotting. The top half of the
immunoblot was treated with a PDE4D antibody and the bottom half with
the RACK1 antibody. With the PDE4D antibody, we detected two
immunoreactive species of 105 ± 3 kDa and 95 ± 2 kDa in
extracts from both HEK293 and 3T3-F442A cells (Fig.
4, lanes T). These isoforms
co-migrated (data not shown) with recombinant PDE4D5 (105 kDa) and
PDE4D3 (95 kDa (5)), respectively, and migrated very distinctly from the other PDE4D isoforms (5), namely PDE4D1 (68 kDa), PDE4D2 (68 kDa),
and PDE4D4 (119 kDa). We did observe a faint band indicative of an
~68-kDa immunoreactive species in immunoblots of 3T3-F442A cells
(Fig. 4), which may reflect either or both of the PDE4D1/2 isoforms
(5). In contrast, a single ~105-kDa immunoreactive species,
indicative of PDE4D5 (5), was noted in SK-N-SH cells, and no
immunoreactive PDE4D species was noted in cytosolic extracts from
Jurkat cells (Fig. 4). Immunoblotting of the lower half of the gel with
an antibody specific for RACK1 identified a single 36 ± 2-kDa
species present in all of these cell types.

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Fig. 4.
PDE4D5 and RACK1 can be co-immunoprecipitated
from various cell lines. Cytosolic extracts were prepared from the
following cell lines: Jurkat, HEK293, 3T3-F442A, and SK-N-SH. They were
either immunoblotted directly (lanes T) or
subjected to immunoprecipitation with the RACK1 antibody
(lanes rIP or i) or a nonspecific
mouse antiserum (lanes n). The
fractions/immunoprecipitates were then run on SDS-PAGE. The upper
half of the gel was immunoblotted with the PDE4D-specific
antibody. The arrows indicate the location of PDE4D3 and
PDE4D5 as 95- and 105-kDa species, respectively. The lower
half of the gel was immunoblotted with the RACK1-specific
antibody, and the position of the 36-kDa RACK1 species is indicated.
Each lane on the gels represents 50-µg protein, with PDE activities
in the range of 25-35 pmol/min/mg protein. RACK1 was also
co-immunoprecipitated with PDE4D5 when the initial immunoprecipitation
was performed with the PDE4D-specific antibody, as demonstrated by
immunoblotting of PDE4D5 immunoprecipitates with the RACK1 antibody
(data not shown).
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To test for an interaction between PDE4D5 and RACK1 in cytosolic
extracts from these cell lines, we subjected them to an
immunoprecipitation protocol with either the RACK1 antibody (Fig. 4;
lanes i or rIP) or a mouse nonspecific antiserum
(Fig. 4, lanes n). The resulting immunoprecipitates were
then subjected to immunoblotting with the PDE4D and RACK1 antibodies,
as described above. These experiments showed that RACK1 could be
immunoprecipitated from all cells by the RACK1 antibody but not by the
nonspecific antiserum. In the HEK293, 3T3-F442A, and SK-N-SH cell
lines, all of which express the PDE4D5 isoform, a single 105-kDa
species in the RACK1 immunoprecipitates was detected upon
immunoblotting with a PDE4D antibody (Fig. 4). No PDE4D immunoreactive
species was detected in RACK1 immunoprecipitates from Jurkat T-cells,
which did not express PDE4D5 (Fig. 4). These data indicate that
natively expressed cytosolic RACK1 is complexed with natively expressed
cytosolic PDE4D5 in both human (HEK293 and SK-N-SH) and mouse
(3T3-F442A) cell lines. Our data are consistent with PDE4D5 being
quantitatively immunoprecipitated with RACK1 from HEK293 and 3T3-F442A
cells. Additionally, the data support the concept that PDE4D3 does not
interact with RACK1, as PDE4D3 was not immunoprecipitated from HEK293
and 3T3-F442A cells using the RACK1 antibody (Fig. 4).
PDE4D5 and RACK1 Interact Directly in Vitro in a
Dose-dependent Manner--
We wished to determine whether
RACK1 and PDE4D5 interacted directly, rather than through an
intermediate protein. For this purpose, we studied the interaction
between recombinant RACK1 and PDE4D5 as synthesized in E. coli. PDE4D5 was expressed as a maltose-binding protein fusion
(MBP-PDE4D5) in E. coli. It was then purified (Fig.
5a) on a maltose affinity
column (see "Experimental Procedures") and had an activity of
29 ± 2 pmol/min/mg protein (with 1 µM cAMP as
substrate) and a Km value of 5.1 ± 0.7 µM cAMP (mean ± S.D., n = 3 experiments). RACK1 was generated and purified as a GST fusion in
E. coli (GST-RACK1; see "Experimental Procedures").

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Fig. 5.
Interaction of recombinant forms of PDE4D5
and RACK1, as purified from E. coli. a, fusions
between GST and RACK1, and also between MBP and PDE4D3 or PDE4D5, were
expressed and purified from E. coli (see "Experimental
Procedures"). GST alone (i.e. not as a fusion) was
expressed and purified in an identical manner. Cell lysates
(lanes b) and purified proteins (lanes
e) obtained after elution from the appropriate affinity column
were run on SDS-PAGE and stained with Coomassie Blue. The species were
purified to apparent homogeneity as analyzed by SDS-PAGE. The positions
of the arrows marks the relative molecular weight of the
purified proteins as follows: GST, 27.3 ± 1.l kDa;
GST-RACK1, 59.8 ± 1.4 kDa; MBP-4D3,
128 ± 3.8 kDa; MBP-4D5, 135.3 ± 2.6 kDa. These
data are typical of experiments done at least three times.
b, the interaction of E. coli purified recombinant PDE4D5 and RACK1 was tested in an ELISA as
described under "Experimental Procedures." The MBP-PDE4D5 fusion
bound to GST-RACK1 in a dose-dependent manner, with an
EC50 of 7.4 ± 1.1 pM (mean ± S.D.;
n = 3 separate experiments). As a control, parallel
experiments were performed for MBP-PDE4D3. c, dose-response
curves were calculated for the inhibition of PDE4D5 by rolipram, at a
concentration of substrate (cAMP) of 1.0 µM. Assays were
performed on MBP-PDE4D5 ("4D5-MBP") alone, and also on MBP-PDE4D5
complexed with GST-RACK1. Assays were performed using an excess of
GST-RACK1 so that all of the PDE4D5 would be complexed with RACK1 (see
"Experimental Procedures"). In pull-down experiments, all of the
PDE4D5 could be shown to complex with GST-RACK1 under these conditions
(data not shown). As a control, assays were performed with GST alone,
added at comparable levels. The IC50 values for rolipram
inhibition were 0.13 ± 0.1, 0.16 ± 0.05, and 0.52 ± 0.07 µM for MBP-PDE4D5 alone, MBP-PDE4D5 mixed with GST,
and MBP-PDE4D5 mixed with GST-RACK1, respectively (mean ± S.D.,
n = 3). These values are significantly different
(MBP-PDE4D5 alone compared with MBP-PDE4D5 complexed with GST-RACK1;
p < 0.005, t test). Protein assays were
performed, and molar concentrations were determined on the basis of the
calculated molecular weights.
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To measure the interaction of PDE4D5 with RACK1, the purified protein
was used in a capture plate assay. This demonstrated (Fig.
5b) that PDE4D5 bound to RACK1 in an apparent
dose-dependent fashion. From these experiments we
determined the affinity of the interaction between RACK1 and PDE4D5 as
having an EC50 of 7.4 ± 1.1 pM (mean ± SD; n = 3 experiments). These data demonstrate that
RACK1 and PDE4D5 can interact directly without any intermediary protein
being involved and that the interaction is unlikely to require any
post-translational modification of the two proteins. The high affinity
for interaction is also compatible with a direct interaction in
vivo.
We also demonstrated that PDE4D3, as a purified MBP fusion protein
(Fig. 5a), was unable to bind to RACK1 when the capture assay was performed under conditions identical to those that
demonstrated interaction between RACK1 and PDE4D5 (Fig. 5b).
This is consistent with the lack of interaction between PDE4D3 and
RACK1 in intact cells (Figs. 3 and 4).
Enzymatic Activity of RACK1-bound PDE4D5--
In order to evaluate
whether interaction with RACK1 altered the catalytic activity of
PDE4D5, we tested the effect on PDE4D5 activity of complexing E. coli-purified recombinant PDE4D5 with E. coli-purified
recombinant RACK1. Under conditions (using a "pull-down" procedure;
see Experimental Procedures) where all of the PDE4D5 could be shown to
be complexed with RACK1, we found that RACK1 produced little or no
change in either the Km or
Vmax for the hydrolysis of cAMP by PDE4D5.
Specifically, PDE4D5 exhibited a Km of 5.1 ± 0.7 µM cAMP when free from RACK1 and a
Km of 6.9 ± 0.9 when complexed with RACK1
(mean ± S.D.; n = 3 experiments). The ratio of
maximal catalytic activity for cAMP hydrolysis between RACK1-bound and
free forms of PDE4D5 was 0.95 ± 0.06 (mean ± S.D.;
n = 3 experiments). We also determined the effect of
RACK1 on the thermal denaturation profile of PDE4D5, which was measured
upon incubation of the enzyme at 50 °C. Whether complexed or not to
RACK1, the activity of PDE4D5 decayed as a single exponential (data not
shown), indicative of a single homogenous enzyme in both instances. The
half-lives for decay of PDE activity were very similar for both the
RACK1-complexed and uncomplexed forms of PDE4D5 (38.3 ± 2.1 and
34.3 ± 2.6 min; mean ± SD; n = 3 experiments). This suggests that interaction of PDE4D5 with RACK1 does
not elicit a major conformational change affecting enzyme activity.
A number of investigators have noted that PDE4 isoforms can undergo
conformational changes, which alter their sensitivity to inhibition by
rolipram and other PDE4-selective inhibitors (31, 36, 37). Therefore,
we analyzed the sensitivity of E. coli-purified MBP-PDE4D5
to rolipram when both free and complexed with E. coli-purified GST-RACK1. Dose-effect analyses (Fig. 5c) showed that rolipram inhibited the cAMP PDE activity of MBP-PDE4D5 in a
dose-dependent manner, with an IC50 value of
0.16 ± 0.5 µM. In contrast, when this enzyme was
complexed with GST-RACK1 a small shift in sensitivity to rolipram
inhibition was seen (Fig. 5c), which was reflected in an
increase in the IC50 to 0.52 ± 0.07 µM.
This effect was unlikely to be caused by the GST portion of GST-RACK1,
as the addition of GST alone to the assays, at a level identical to
that of GST-RACK1, had negligible effect on rolipram inhibition (Fig.
5c; IC50 of 0.19 ± 0.02 µM).
RACK1 Interacts with PDE4D5 but Not with Other PDE4
Isoforms--
Five PDE4D isoforms have been identified to date ((5)
Fig. 1). We have already demonstrated that RACK1 does not interact with PDE4D3 (Figs. 3c, 4, and 5b). To determine
whether any of the PDE4D1, PDE4D2, PDE4D3, or PDE4D4 isoforms interact
with RACK1, cDNAs encoding these isoforms were expressed in
S. cerevisiae as LexA fusions, and two-hybrid assays were
used to test for their ability to interact with a GAL4-RACK1 fusion. No
interactions were detected (data not shown), indicating that RACK1
interacts specifically with the PDE4D5 isoform.
To confirm this observation in mammalian cells, we also tested the
ability of RACK1 to associate with recombinant PDE4 isoforms expressed
in COS7 cells. For these experiments, we utilized a GST "pull-down"
method, as modified by us previously (38). In brief, we transiently
transfected COS7 cells with cDNAs encoding various PDE4 isoforms.
Cytosolic fractions were prepared from the transfected cells and
incubated with GST-RACK1, and the resulting complex was absorbed onto
glutathione-agarose beads (see "Experimental Procedures"). The
beads were then harvested by centrifugation, washed, and immunoblotted
with the PDE4D antibody (Fig. 6).

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Fig. 6.
RACK1 can pull-down recombinant forms of
PDE4D5 expressed in COS7 cells. In these experiments, various
PDE4D isoforms (or mutant forms thereof) were expressed in COS7 cells.
Cytosolic extracts were prepared from the cells, mixed with GST-RACK1,
and then subjected to affinity absorption on glutathione-agarose beads,
followed by SDS-PAGE and immunoblotting. a, pull-down
experiments performed with wild-type recombinant PDE4D isoforms. PDE4D3
or PDE4D5 were expressed in COS7 cells and then subjected to
"pull-downs" with GST-RACK1 (lanes rg) or GST
alone (lanes g). After affinity absorption, the material on
the beads was immunoblotted with the PDE4D antibody. Cytosolic extracts
from the cells (i.e. not subjected to pull-downs) were run
as standards (lanes ly). In cells transfected with vector
alone (mock), GST-RACK1 pulled down a single 105-kDa species
consistent with PDE4D5. In cells transfected with a construct
expressing PDE4D3, a 105-kDa band was seen, whose intensity was not
different from that seen in the mock-transfected cells. In cells
transfected with a construct expressing PDE4D5, there was a dramatic
increase in the intensity of the PDE4D immunoreactive species pulled
down by GST-RACK1. Expression of the PDE4D isoforms in the COS7 cells
was measured by quantitative immunoblotting with the PDE4D monoclonal
antibody, as described previously (5). The expression of the various
PDE4D constructs was similar, and identical amounts were used in
comparative experiments. b, pull-down experiments performed
with constructs encoding deletions in PDE4D5. The regions of PDE4D5
included in the various constructs are shown in Fig. 1. All the
constructs encoded a VSV epitope at the carboxyl-terminal end of the
protein (5, 10), which could be detected by an anti-VSV antibody (26),
using methods described by us previously (5, 10). The expression of the
construct was monitored by immunoblotting of cytosolic extracts with
the anti-VSV antibody (upper panel, lysate). This allowed
detection of the transfected species without background from endogenous
PDE4D5. Pull-down experiments were performed with GST-RACK1, followed
by immunoblotting of the material on the beads with the VSV-antibody
(lower panel, Rack1 bound). Lane m
represents mock transfections (vector only). c,
co-immunoprecipitations of RACK1 and PDE4D5 mutants. Constructs
encoding mutants of PDE4D5 with Asn-22 mutated to Ala (lanes
22) or with Trp-24 mutated to Ala (lanes
24) were expressed in COS7 cells. Wild-type (unmutated)
PDE4D5 was expressed in parallel transfections (lanes
wt). Cytosolic fractions were immunoblotted directly
(lanes lysate) or were immunoprecipitated with
the RACK1 antibody (lanes RACK ippt) or with a
nonspecific mouse antiserum (lanes ns-Ab ippt). The samples
were then immunoblotted with the PDE4D antibody. The migration of
PDE4D5 on the gel is indicated. All data are typical of experiments
done at least three times.
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As shown before by us (5, 27, 29-31, 39), in homogenates of the
transfected COS7 cells the activity of the transfected PDE4D species
accounted for over 98% of the total cellular cAMP PDE activity (Fig.
6a, lanes ly). When cytosolic extracts from PDE4D5-transfected cells were subjected to "pull-downs" with
GST-RACK1, a 105-kDa PDE4D immunoreactive species consistent with
PDE4D5 was identified on the immunoblots (Fig. 6a, lanes
rg). In contrast, when pull-downs were performed with GST alone,
no PDE4D species was seen on the immunoblots (Fig. 6a, lanes
g). In parallel experiments, we performed pull-downs from COS7
cells transfected with a cDNA encoding PDE4D3. We were unable to
pull down any immunoreactive PDE4D3 with GST-RACK1 (Fig.
6a). However, endogenous PDE4D5 was pulled down in these
experiments to a level similar to that seen using an equivalent amount
of cytosol from mock-transfected cells.
In similar experiments, we determined that PDE4D1, PDE4D2, and PDE4D4,
when expressed in COS cells (5), could not be pulled down by GST-RACK1
(data not shown). In still additional experiments, we determined that
the human PDE4A4/5 isoform (3, 31), the human PDE4B isoforms PDE4B1,
PDE4B2, and PDE4B3 (39), and also the human PDE4C2 isoform (41), when
expressed in COS7 cells, could not be pulled down with GST-RACK1. These
data imply that, of the known PDE4 isoforms, RACK1 binds selectively to PDE4D5.
The Interaction of PDE4D5 with RACK1 Is Mediated by Specific Amino
Acids in the PDE4D5 Amino-terminal Region--
As RACK1 interacts
specifically with PDE4D5, and not with other PDE4 isoforms, it is
likely that RACK1 interacts with regions of sequence that are unique to
PDE4D5. PDE4D5 differs from all known PDE4 isoforms (2, 5) in the
presence of a unique region of 88 amino acids at its amino terminus
(Fig. 1). We wished to determine which specific amino acids within this
region are essential for the binding of PDE4D5 to RACK1.
As a first step, we created a two-hybrid construct containing just the
unique 88-amino acid amino-terminal region of PDE4D5, and we
demonstrated that it could interact with RACK1 (Fig.
7a, patche NT). We
then created constructs encoding deletion mutations in the
amino-terminal region of PDE4D5, and we tested them for their ability
to interact with RACK1. The interaction was tested by both a two-hybrid
assay (Fig. 7a) and by pull-down experiments (Fig.
6b). For the two-hybrid experiments, constructs encoding fusions between LexA and various amino-terminal deletions of PDE4D5 were tested for interaction with GAL4-RACK1. For the pull-down experiments, we tested the ability of mutant PDE4D5 forms expressed in
COS7 cells to interact with GST-RACK1. For the COS7 cell experiments, it was essential that we could distinguish between the transfected mutant protein and endogenous PDE4D5 proteins in COS7 cells. For this
purpose, we utilized PDE4D5 constructs that encoded an epitope of the
vesicular stomatitis virus envelope (26), attached to the
carboxyl-terminal end of the protein (5, 10). Expression of these
VSV-tagged PDE4D5 species in COS7 cells could be detected by
immunoblotting with a monoclonal antibody specific for the VSV epitope
(26). Preliminary experiments demonstrated that COS7 cell-expressed,
VSV-tagged unmutated PDE4D5 could be pulled down with GST-RACK1 (Fig.
6b, lane wt). In contrast, COS7 cells transfected with the
vector alone demonstrated no detectable VSV immunoreactivity (Fig.
6b, lane m). We then tested the ability of the various
PDE4D5 amino-terminal deletion mutants to interact with GST-RACK1. The
pull-down experiments (Fig. 6b) demonstrate that a single
region of the PDE4D5 amino terminus, comprising amino acids 12-29, is
necessary for interaction with RACK1 but that other regions appear to
be dispensable. Identical conclusions were obtained using two-hybrid
methods (Fig. 7a).

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Fig. 7.
Yeast two-hybrid analysis of PDE4D5 deletions
and point mutations. a, plasmids encoding fusions
between the DNA binding domain of LexA and various amino-terminal
deletions of PDE4D5 were tested for their ability to interact with
RACK1, expressed as a fusion with the GAL4 activation domain
(right column, pGADN-RACK1). The identical LexA
fusions were tested for their ability to interact with the GAL4
activation domain alone (left column, pGADN). The
regions of PDE4D5 included in the various constructs are shown in Fig.
1. Also shown is the interaction generated by a LexA fusion containing
the unique 88-amino acid amino-terminal region of PDE4D5
(NT). The interactions were tested with the filter
-galactosidase assay used in Fig. 2. b, individual amino
acids in the amino-terminal region of PDE4D5 were mutated to alanine,
and the resulting constructs were expressed as LexA fusions and tested
for their ability to interact with pGADN-RACK1. Also included as
controls are LexA fusions of unmutated PDE4D5 (wt) and the
signal produced with pGADN-RACK1 and LexA alone (nb).
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To obtain additional data localizing the region of interaction, we
created two mutants with deletions in the middle of the PDE4D5
amino-terminal region. One deletion (D2, Fig. 1) removed amino acids 22-27 and the other (D1) amino acids 19-51.
Neither of these two mutations interacted with GST-RACK1 in a pull-down assay (Fig. 6b). These data narrowed down a region essential
for interaction with RACK1 to a small region within the PDE4D5 amino terminus. Secondary structure analyses performed with the Wisconsin package of DNA analysis programs (data not shown) demonstrated that
this region of PDE4D5 (approximately amino acids 19-50) may form an
-helix but otherwise did not have any distinguishing sequence
motifs. It has no obvious primary sequence homology to PKC, Src, or integrins.
We then analyzed the effects of mutations in individual amino acids in
the region defined by the deletion mutation analysis. Site-directed
mutagenesis was used to mutate individual codons in this region to
alanine. The effect of each of these mutations on the interaction with
RACK1 was tested with the two-hybrid assay. Ten codons were
individually mutated, and mutations of four of these (Asn-22, Pro-23,
Trp-24, or Asn-26) were each shown to block completely the interaction
(Fig. 7b). Two of these mutations (Asn-22 and Trp-24) were
then expressed in COS7 cells and tested for their ability to
co-immunoprecipitate with RACK1. Both the mutations blocked the
interaction in this assay as well (Fig. 6c). These mutations
were clustered in a very small region of the protein, which presumably
serves as the major, or possibly only, region of interaction between
PDE4D5 and RACK1.
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DISCUSSION |
Regulation of the levels of the second messenger cAMP has the
potential to influence multiple processes in cells and tissues. PDE4
enzymes have been implicated in numerous cellular functions in the
brain, airway smooth muscle, endocrine tissues, and in the
immune/inflammatory systems (for review see Ref. 2). However, the
regulation of many PDE4 isoforms is poorly understood. We have recently
isolated a novel PDE4 isoform, PDE4D5, which is expressed in numerous
cell lines and also in the brain (5). In the present study, we have
demonstrated that PDE4D5 interacts specifically and at high affinity
with the RACK1 WD-repeat scaffold protein. The interaction between
these two proteins was demonstrated by multiple independent methods,
including two-hybrid screening, pull-down assays with recombinant
RACK1, and binding studies with purified recombinant proteins. We have
also demonstrated by co-immunoprecipitation studies that native,
endogenously expressed RACK1 and PDE4D5 interact in cells.
The term RACK1 stands for receptor for activated protein C kinase, and
the first identified functional role for RACK1 was as a protein capable
of binding to various PKC isoforms after they had been activated
through treatment of cells with either diacylglycerol or phorbol esters
(12-15). It is a member of the large family of WD-repeat proteins
(11), which have numerous functions in cells. It is most homologous to
the Gs
subunit, which activates certain isoforms of
adenylyl cyclase and various ion channels (11, 42) and which also
serves as an anchor for the
-adrenergic receptor kinase (43). X-ray
diffraction studies of the Gs
subunit have demonstrated
that it is has a "propeller" structure (44, 45), and the
"blades" of the propeller are believed to be sites of interaction
between Gs
and other proteins. It is likely that RACK1
has a similar structure (46). The coatomer protein
'-COP is also a
WD-repeat protein that also interacts with PKC isoforms (35). However,
we have shown that PDE4D5 does not bind to either Gs
or
'-COP, demonstrating that PDE4D5 interacts specifically with RACK1
and not with WD-repeat proteins in general.
More recently, RACK1 has been shown to interact with the integrin
subunit (16) and the protein tyrosyl kinase Src (17). RACK1 may serve
as a scaffold or "adaptor" protein for either Src or the integrin
subunit. Overexpression of RACK1 inhibits the tyrosine kinase
activity of Src and inhibits the growth of NIH-3T3 cells (17). The
physiologic role of RACK1 in integrin function remains to be determined
(see below).
Although RACK1 has been shown to interact with at least four different
proteins, the mechanisms for these interactions appear to be quite
different. PKC isoforms need to be activated by treatment with phorbol
esters and Ca2+ before they can interact with RACK1 (47).
This treatment is believed to produce a conformational change in the
PKC enzyme, which exposes its C2 region, allowing it to interact with
RACK1 (13-15). A number of domains within the C2 region of PKC are
involved in its binding to RACK1 (13, 14). These domains show no
obvious sequence homology to the region in the amino terminus of PDE4D5 that we have shown to be essential for its interaction with RACK1 (Figs. 6 and 7). The interaction of RACK1 with the
subunit of integrins also requires the stimulation of cells with PMA (16). This
suggests that PKC activation is necessary for the interaction of the
integrin
subunit with RACK1 or that PMA can directly promote the
interaction. This interaction may involve a conformational change in
RACK1, as the interaction between RACK1 and the integrin
subunit
can only be demonstrated in vitro if a truncated form (WD-repeats 5-7, inclusive) of RACK1 is used.
In some ways, the interaction that we observe between PDE4D5 and RACK1
appears to resemble that between RACK1 and the SH2 region of Src (17).
Src interacts with full-length GST-RACK1 in pull-down assays, which is
also true for PDE4D5 (Figs. 3, 4, and 7). Co-immunoprecipitation of Src
and RACK1 from cell lysates did not require PMA, which is also true for
the interaction between RACK1 and PDE4D5. Therefore, RACK1 may interact
with different proteins through different mechanisms, some of which
involve phorbol ester-induced conformational changes and some of which
apparently do not.
Several other PDE4 isoforms have also been demonstrated to bind to
other signaling proteins, and it is of interest that these interactions
also appear to be mediated by sequences in the unique amino-terminal
ends of the PDE4 proteins. The PDE4A4/5 isoform has been demonstrated
to bind to proteins containing SH3 domains (38). The PDE4A1 isoform is
targeted to membranes, and the membrane targeting of this isoform is
mediated by specific amino acids in its amino-terminal region (27-29,
48).
The physiologic implications of the RACK1-PDE4D5 interaction may be
related to the ability of RACK1 to serve as a scaffold or adaptor
protein that mediates the recruitment of PDE4D5 into a protein complex.
A single scaffold or adaptor protein may interact with multiple
different proteins, all of which can potentially be recruited into the
complex. For example, protein kinase A-anchoring proteins can interact
with protein kinase A, PKC, and protein phosphatase 1 (18, 49).
Scaffold, anchor, and adaptor proteins physically connect various
signal transduction components, such as receptors, kinases, and
elements of the cytoskeleton, into stable complexes. These complexes
bring enzymes closer to their regulatory components or substrates or
closer to other components of a signaling network (18). Therefore, the
multidomain protein RACK1 may serve as a scaffold able to recruit a
variety of signal transduction proteins. Some partners of RACK1, such
as PKC isoforms and integrins, may be recruited to the complex only
upon activation by phorbol esters, whereas others, such as PDE4D5 and
Src, may be constitutively associated with RACK1 in those cell types
where both proteins are expressed. The functional significance of the association of PDE4D5 with RACK1 remains to be elucidated. The recruitment of PDE4D5 to a signaling complex may provide a potential mechanism for the modulation of cAMP levels in the vicinity of the
complex. This could in turn regulate the activity of protein kinase A,
which could influence the activity or function(s) of the complex, or of
other adjacent cellular components.