Characterization of RII
and
D-AKAP1 in differentiated adipocytes
Archana
Chaudhry,
Chen
Zhang, and
James G.
Granneman
CNS Molecular Sciences, Pfizer Global Research and Development,
Ann Arbor 48105; and Department of Psychiatry and Behavioral
Neurosciences, Wayne State University School of Medicine, Detroit,
Michigan 48201
 |
ABSTRACT |
A-kinase anchoring proteins (AKAPs) have been
proposed to regulate cAMP-dependent signaling in the cell by targeting
RII subunits of protein kinase A (PKA) to specific subcellular
compartments. RII
is the predominant PKA subtype in
adipose tissue. In gel overlay assays of C3H/10T1/2 adipocytes and
adipose tissue, RII
bound to several proteins including
a prominent 132-kDa band, which was strongly induced upon
differentiation of C3H/10T1/2 cells into adipocytes. Immunoblotting and
nuclease protection analysis of C3H/10T1/2 cellular extracts identified
this band as D-AKAP1/S-AKAP84, a putative AKAP. Immunocytochemical
analysis of C3H/10T1/2 adipocytes revealed that most of
D-AKAP1/S-AKAP84, but not RII
, was colocalized
with a mitochondrial-selective dye, MitoTracker red. These
findings were further confirmed in studies where D-AKAP1/ S-AKAP84,
but not RII
, were localized in purified mitochondria
made from C3H/10T1/2 adipocytes. Moreover, D-AKAP1, which is
upregulated after differentiation, did not recruit RII
to membrane fractions enriched in mitochondria. These results
demonstrate that D-AKAP1/S-AKAP84 does not interact with PKA in
differentiated C3H/10T1/2 adipocytes under the conditions tested.
anchoring proteins; adipose tissue; protein kinase A; adenosine
3',5'-cyclic monophosphate
 |
INTRODUCTION |
VARIOUS RECEPTORS,
including
-adrenergic receptors (
-AR), regulate
important functions in adipocytes through the generation of cAMP and
the activation of protein kinase A (PKA) (9), and growing
evidence suggests that the PKA signaling pathway is functionally compartmentalized in adipocytes. For example, catecholamine activation of lipolysis in both brown and white adipocytes requires far lower levels of cAMP than does activation by forskolin, suggesting that adrenergic receptors more effectively target cAMP generation to activation of lipolysis (12). The molecular basis for the
functional compartmentalization of cAMP-mediated responses in
adipocytes, however, is presently unknown.
One potential means of physically compartmentalizing cAMP signaling is
through the subcellular targeting of PKA. In this regard, A-kinase
anchoring proteins (AKAPs) have been proposed to target the holoenzyme
to subcellular targets via specific interactions with the regulatory
subunit RII (7). RII
is most abundantly expressed in brown and white adipose tissue, and targeted disruption of
RII
produces lean mice that are resistant to obesity
(8). Little is known about the subcellular distribution of
RII
in fat cells or whether interactions with AKAPs
tether PKA to specific subcellular locations. The present study was
undertaken to determine whether adipose tissue expresses potential
AKAPs and whether these proteins are involved in tethering PKA to
specific subcellular locations. The C3H/10T1/2 cell line was chosen for this study because these cells can be easily differentiated into adipocytes that exhibit appropriate PKA-mediated responses, including lipolysis to
3-AR agonists (19). The
results show that adipocytes express several proteins that bind
RII
in vitro. The predominant protein that binds to
RII
in gel overlay assays was identified as
D-AKAP1/S-AKAP84, a putative AKAP that is targeted to mitochondria (6, 11, 14, 15, 18). However, although D-AKAP1 interacts with RII in vitro, the in vivo distribution of these proteins is
distinct. Thus D-AKAP1/S-AKAP84 is unlikely to play a role in PKA
signaling in adipocytes.
 |
METHODS |
Materials.
Paraformaldehyde solution was from Electron Microscopy Sciences (Ft.
Washington, PA). Monoclonal antibodies to RII
and the
catalytic subunit (C) of PKA were from Transduction Laboratories (Lexington, KY). Monoclonal antibody to prohibitin was from RDI (Flanders, NJ). Dr. Susan Taylor (University of California, San Diego)
provided polyclonal antibodies to D-AKAP1 and D-AKAP1 core peptide
(amino acids 284-408). MitoTracker red and Oregon green-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).
All other secondary antibodies were from Jackson Labs (West Grove, PA).
Nitrocellulose and 4-15% precast gels were obtained from
Novex/Invitrogen (Carlsbad, CA). Tissue culture reagents were from
GIBCO (Gaithersburg, MD). All other reagents were from Sigma (St.
Louis, MO).
Western blot analysis.
Proteins (10-25 µg) were subjected to electrophoresis in a
4-20% precast gel and transferred to nitrocellulose. The
nitrocellulose membranes were blocked for 1 h in Tris-buffered
saline (TBS) containing 5% milk (Carnation) and 0.1% Tween 20. The
blots were incubated with antibodies to D-AKAP1/S-AKAP84 (1:4,000),
prohibitin (1:200), RII
(1:5,000), or C (1:1,000). After
extensive washing in TBS containing 0.1% Tween 20, the blots were
incubated in horseradish peroxidase-conjugated anti-rabbit or
anti-mouse secondary antibody for 1 h. Immunoreactive proteins
were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).
Immunocytochemistry.
C3H/10T1/2 adipocytes were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) at 4°C for 20 min. Before fixation, some cells were incubated at 37°C for 30 min with 0.1 µM
MitoTracker red, a rosamine dye that is selectively incorporated into
mitochondria. Fixed cells were quenched in 100 nM glycine,
permeabilized in 0.2% Triton X-100, and blocked for 1-2 h in PBS
containing 1% each of ovalbumin, bovine serum albumin (BSA), and goat
serum. Cells were then incubated either for 1 h with
anti-RII
antibody or overnight at 4°C with
anti-D-AKAP1/ S-AKAP84 antibody. When RII
and
D-AKAP1/S-AKAP84 immunoreactivity was examined for colocalization,
cells were incubated overnight with the two antibodies. The next day,
cells were washed three times in PBS and incubated for 1 h in
Oregon green- or Cy3-conjugated anti-rabbit or anti-mouse secondary
antibody. Control samples were incubated either with secondary antibody
alone or with purified RII
or the peptide representing
the core sequence of D-AKAP1/ S-AKAP84, which was used to generate
the D-AKAP1/ S-AKAP84 antibody (14). Fluorescence signals were detected with an Olympus Fluoview laser scanning confocal microscope.
Nuclease protection assay.
A fragment of D-AKAP1/ S-AKAP84 cDNA representing the
core sequence of D-AKAP1/S-AKAP84 was amplified by PCR and cloned
into PCR 2.1 vector, and T7 polymerase was used to generate a probe for
nuclease protection assay (NPA). NPA was performed as previously described (5).
Cell culture.
C3H/10T1/2 cells were grown in basal Eagle's medium (BME) supplemented
with 10% fetal calf serum in 5% CO2 in air. The medium was changed every 2-3 days. On reaching confluence, cells were placed in differentiating medium that consisted of BME supplemented with 10% fetal calf serum, 1 µM insulin, 1 µM
9-cis-retinoic acid, and 1 µM BRL-49653. Cells were used
8-12 days later, at which time they were over 80% differentiated.
Preparation of total lysates and cell fractionation.
Brown and white adipose tissue was obtained from male 200- to 250-g
Sprague-Dawley rats (Hilltop, Scottsdale, PA). Total cell lysates were
prepared by homogenizing C3H/10T1/2 cells or adipose tissue in TES
buffer (50 mM Tris, 2 mM EDTA, and 254 mM sucrose, pH 7.5) containing
protease inhibitors (Roche Diagnostics, Mannheim, Germany).
Isolation of mitochondria.
Mitochondria were isolated from C3H/10T1/2 adipocytes as described
previously (13) with some modifications. Briefly,
C3H/10T1/2 adipocytes were homogenized in buffer A (250 mM
mannitol, 0.5 mM EGTA, and 5 mM HEPES, pH 7.4) and centrifuged at 3,000 g. Mitochondria were then pelleted by centrifuging the
supernatant for 10 min at 10,000 g. The resulting pellet was
suspended in a small volume of buffer A, layered on top of
20 ml of 30% (vol/vol) Percoll in 225 mM mannitol, 1 mM EGTA, and 25 mM HEPES (pH 7.4), and centrifuged for 30 min at 95,000 g in
a Beckman 60Ti rotor. Mitochondria were collected from the lower part
of the dense, brownish yellow mitochondrial band by centrifuging this
fraction at 6,300 g.
Expression, purification, and 32P
phosphorylation of RII
.
The expression plasmid pET11c containing the rat RII
(kindly provided by John Scott, Vollum Institute, Oregon Health Sciences University) was transformed into Escherichia coli
BL21(DE3) cells (Novagen). RII
protein was isolated
as described previously (10) with the modification that
cells were lysed in buffer containing 0.1% Triton X-100.
RII
was 32P phosphorylated by PKA
(Calbiochem, La Jolla, CA) as described previously (3,
16).
[32P]RII overlay assays.
Overlay assays were performed as described previously (3, 10,
16). Briefly, proteins in cell lysates were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then blocked overnight in TBS containing 1% BSA, 5% milk, and
0.05% Tween 20. Blots were then probed with 100,000 cpm/ml of
[32P]RII
in blotto for 3-4 h, washed
extensively with TBS containing 0.1% Tween 20, and visualized by autoradiography.
 |
RESULTS |
AKAPs are structurally diverse proteins that have been defined by
their ability to bind RII in vitro (7). To identify
potential AKAPs in adipose tissue, recombinant RII
was
phosphorylated with [32P]ATP and used to screen adipocyte
extracts in gel overlay assays. RII
bound several
proteins in brown adipose tissue homogenates, including a prominent
132-kDa band (Fig. 1). The binding of
RII
to several of these proteins, including the 132-kDa
band, was blocked by Ht 31 peptide, a peptide thought to mimic the
amphipathic helix of AKAPs and thus serve as a competitive inhibitor of
RII-AKAP interaction (7). Binding was not blocked by Ht
31-P peptide, in which the mutation of two proline residues disrupts
amphipathic helix formation (Fig. 1). RII
also bound to
the 132-kDa band in white adipose tissue and in C3H/10T1/2 cells (Fig.
2). In C3H/10T1/2 cells, the 132-kDa
protein was strongly induced upon differentiation into adipocytes. In
white adipose tissue, RII
also bound another lower
molecular weight protein that appeared to be the major RII binding
protein in this tissue. Although Ht 31 peptide completely blocked the
binding of RII to the 132-kDa band, it failed to completely block the
binding of RII to this lower molecular weight protein. This protein
also was present in C3H/10T1/2 cells, although to a much lesser extent
than the 132-kDa band.

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Fig. 1.
RII overlay analysis of total cellular proteins from
brown adipose tissue. Total cell lysates were prepared, subjected to
SDS-PAGE, transferred to nitrocellulose, and probed for A-kinase
anchoring proteins (AKAPs) as described in METHODS. Blotted
proteins were probed with [32P]RII in the presence (+) or
absence ( ) of Ht 31 peptide or in the presence of Ht 31-P.
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Fig. 2.
RII overlay analysis of total cellular proteins from white adipose
tissue (A) and C3H/10T1/2 cells (undifferentiated and
differentiated adipocytes) (B). Total cell lysates were
prepared, subjected to SDS-PAGE, transferred to nitrocellulose, and
probed for AKAPs as described in METHODS. Blotted proteins
were probed with [32P]RII in the presence or absence of
Ht 31 peptide.
|
|
The size of the 132-kDa band was similar to that described for
D-AKAP1/S-AKAP84 (6, 11, 15, 18). The presence of D-AKAP1/S-AKAP84 in C3H/10T1/2 adipocytes was examined by both immunoblotting and NPA (Fig. 3). An
antibody directed against the core sequence of D-AKAP1/S-AKAP84
(14) recognized the 132-kDa band identified in the gel
overlay assay. Furthermore, D-AKAP1/S-AKAP84 immunoreactivity was
strongly induced after differentiation of C3H/10T1/2 cells into
adipocytes (Fig. 3A) and completely abolished by
preabsorption with the immunizing peptide (not shown). NPA of RNA from
C3H/10T1/2 adipocytes indicated that D-AKAP1/S-AKAP84 mRNA
expression is strongly induced upon adipocyte differentiation (Fig.
3B). Thus adipocyte differentiation strongly induced
D-AKAP1/S-AKAP84 protein and mRNA expression.

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Fig. 3.
Identification of D-AKAP1/S-AKAP84 protein and mRNA in
C3H/10T1/2 adipocytes. Confluent C3H/10T1/2 cells were grown for
8-12 days in either regular medium (undifferentiated cells)
or adipocyte-differentiating medium (differentiated adipocytes).
A: total cellular proteins were subjected to SDS-PAGE and
transferred to nitrocellulose. Blots were probed with
D-AKAP1/S-AKAP84 antibody. B: detection of
D-AKAP1/S-AKAP84 mRNA in undifferentiated and differentiated
adipocytes by nuclease protection assay. NT,
nucleotides.
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Quantitative immunoblotting of recombinant RII
and
D-AKAP1 core peptide demonstrated that they are expressed in nearly equal amounts in differentiated adipocytes (Table
1), indicating that putative AKAP is
present in amounts that might target a significant fraction of
adipocyte PKA. D-AKAP1/S-AKAP84 has been localized to mitochondria
in cultured cell lines and in spermatids (6, 11, 15, 18).
We therefore examined the subcellular distribution of D-AKAP1 and
RII
in C3H/10T1/2 adipocytes by confocal microscopy. Mitochondria were visualized with the fluorescent dye MitoTracker red,
whereas D-AKAP1 and RII
were visualized by indirect immunofluorescence. D-AKAP1 was strongly colocalized to mitochondria, as indicated by the appearance of yellow fluorescence upon merging of
the double-labeled images (Fig.
4A). In contrast,
RII
fluorescence showed no evidence of mitochondrial
localization (Fig. 4B). Line scan analysis of confocal
images demonstrated a very strong colocalization of MitoTracker red and
D-AKAP1 fluorescence, whereas RII fluorescence did not localize
to mitochondria (Fig. 4C). Immunocytochemical
double-labeling of RII
and D-AKAP1 indicated
little, if any, colocalization of these proteins (Fig. 5A). Consistent with these
results, line scan analysis indicated that RII
and
D-AKAP1/ S-AKAP84 were not significantly colocalized in these cells
(Fig. 5B).

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Fig. 4.
Immunolocalization of MitoTracker red, D-AKAP1/S-AKAP84, and
RII in C3H/10T1/2 adipocytes. Confocal images of D-AKAP1
(A) or RII (B) are shown with
Oregon green-conjugated antibodies costained with MitoTracker red, a
mitochondrial fluorescent marker. Individual staining patterns as well
as overlapped localization is shown. C: line scan analysis of images in
A and B. D-AKAP1 (a) but not
RII fluorescence (b) correlates with
MitoTracker red fluorescence.
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Fig. 5.
Immunolocalization of D-AKAP1/S-AKAP84 and
RII in C3H/10T1/2 adipocytes. A: confocal
images of D-AKAP1/S-AKAP84 with
anti- D-AKAP1/S-AKAP84 antibody (red) costained with
anti-RII antibody (green). Individual staining patterns
as well as overlapped localization are shown. B: line scan
analysis of images in A. RII fluorescence
does not correlate well with that of D-AKAP1.
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The above results indicate that D-AKAP1/S-AKAP84, but
not RII
, is targeted to mitochondria. To verify this
finding with an independent biochemical technique, we subjected
C3H/10T1/2 adipocyte homogenates to differential centrifugation and
Percoll gradient purification of mitochondria. As shown in Fig.
6, the distribution pattern of
D-AKAP1/S-AKAP84 was virtually identical to that of the
mitochondrial marker prohibitin (17). Specifically, D-AKAP1/S-AKAP84 was restricted to heavy particulate fractions and
highly enriched in purified mitochondria. In contrast,
RII
was largely excluded from the heavy particulate
fraction and nearly completely absent from purified mitochondria. As a
positive control for an authentic RII
-interacting
protein, we monitored the distribution of the C subunit of PKA and
found that its distribution was identical to that of
RII
.

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Fig. 6.
Western blot analysis of purified mitochondrial fractions
obtained from C3H/10T1/2 adipocytes. Mitochondrial fractions were
prepared as described in METHODS. The various fractions
were subjected to immunoblotting and probed with antibodies directed
against RII , C, D-AKAP1/S-AKAP84, and the
mitochondrial marker prohibitin. Hom, homogenate; sup, supernatant.
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We also examined whether the induction of D-AKAP1/S-AKAP84 that
occurs upon adipocyte differentiation alters the subcellular distribution of RII
. Homogenates of undifferentiated and differentiated C3H/10T1/2 cells were fractionated at 40,000 g into supernatant and membrane fractions (Fig.
7). In undifferentiated adipocytes,
RII
and C were found in the membrane fraction. D-AKAP1
was absent in undifferentiated cells, and prohibitin was expressed at
low levels. Differentiation caused a marked induction of all four
proteins; however, their subcellular distribution was distinct. After
differentiation, both RII
and C were nearly exclusively
localized to the high-speed supernatant, whereas both D-AKAP1 and
prohibitin were exclusively localized in the high-speed pellet. Very
low levels of RII (<1% of total) could be found in membrane
fractions. Because D-AKAP1 has been reported to bind both RI and
RII (14), we examined the distribution of RI as well as
RII
in the 40,000 g supernatant and membrane fractions of differentiated C3H/10T1/2 adipocytes. Both R1 and RII
immunoreactivity was found primarily in the
supernatant (Fig. 8).

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Fig. 7.
Western blot analysis of 40,000 g pellet and
supernatant fractions from C3H/10T1/2 adipocytes. Undifferentiated and
differentiated C3H/10T1/2 adipocytes were homogenized in
Tris-EDTA-sucrose (TES) buffer and centrifuged at 40,000 g.
The pellet and supernatant proteins were subjected to SDS-PAGE and
transferred to nitrocellulose. Blots were probed with antibodies
directed against RII , C, D-AKAP1/S-AKAP84, and the
mitochondrial marker prohibitin.
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Fig. 8.
Western blot analysis of 40,000 g pellet and
supernatant fractions from C3H/10T1/2 adipocytes. Undifferentiated and
differentiated C3H/10T1/2 adipocytes were homogenized in TES buffer and
centrifuged at 40,000 g. The pellet and supernatant proteins
were subjected to SDS-PAGE and transferred to nitrocellulose. Blots
were probed with antibodies directed against RI (A) or
RII (B).
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To determine whether membrane-associated RII represented a weak
interaction with any AKAP, we prepared C3H/10T1/2 adipocyte membranes
in the presence and absence of Ht 31 peptide, which abolishes AKAP-RII
interactions in vitro. Fractionation of C3H/10T1/2 adipocytes in the
presence of Ht 31 peptide did not significantly alter the relative
distribution of D-AKAP1 and RII
in membrane fractions
(Fig. 9).

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Fig. 9.
Effect of Ht 31 peptide on RII and D-AKAP1
immunoreactivity in 40,000 g pellets obtained from
C3H/10T1/2 adipocytes. C3H/10T1/2 adipocytes were homogenized in TES
buffer, incubated in the presence or absence of Ht 31 peptide (1.5 µM), and centrifuged at 40,000 g. Pellet proteins were
subjected to SDS-PAGE and transferred to nitrocellulose. Blots were
probed with antibodies directed against RII and D-AKAP1.
Results are expressed as the ratio of RII -to-D-AKAP1
immunoreactivity (means ± SE, n = 5).
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 |
DISCUSSION |
Growing evidence suggests that PKA signaling in adipocytes is
functionally compartmentalized, although the basis of that
compartmentalization is unknown. One possibility is that PKA activity
is directed to specific subcellular locations through interactions with
targeting proteins (7). Numerous proteins that bind RII
have been identified, although their role in targeting PKA in vivo is
largely lacking. D-AKAP1/ S-AKAP84 was first identified on the basis
of its ability to interact with RII in vitro (14,
18). Utilizing gel overlay assays, we determined that
D-AKAP1/S-AKAP84 is the major RII binding protein present in
brown adipose tissue and in C3H/10T1/2 adipocytes. Furthermore,
D-AKAP1/S-AKAP84 protein and mRNA expression were strongly induced
upon adipocyte differentiation. Quantitative immunoblotting
demonstrated that D-AKAP1/S-AKAP84 and RII
are present
in nearly equal amounts in C3H/10T1/2 adipocytes. Given the high
affinity of RII
for D-AKAP1/S-AKAP84 in vitro, these
observations raised the possibility that subcellular targeting of PKA
might be achieved through this interaction in vivo.
Analysis of the subcellular distribution of D-AKAP1/S-AKAP84 by
confocal microscopy and subcellular fractionation clearly demonstrated that D-AKAP1/S-AKAP84 is targeted to mitochondria in
C3H/10T1/2 adipocytes. These observations confirm previous results
demonstrating that D-AKAP1/S-AKAP84 is targeted to the outer
mitochondrial membrane in spermatids and transfected HEK-293 cells
(6, 11, 15, 18). The present results, however, do not
support a role for D-AKAP1/S-AKAP84 in the subcellular targeting of
PKA. Thus fractionation studies demonstrated that the distribution of
RII
correlated perfectly with that of C, and both
proteins were excluded from fractions that were greatly enriched in
D-AKAP1/S-AKAP84. Immunocytochemical analysis of intact adipocytes
confirmed the mitochondrial targeting of D-AKAP1/S-AKAP84 in intact
cells, as well as the exclusion of RII from this organelle. Although it
has been reported that overexpression of D-AKAP1 in HEK-293 cells
increases the association of RII with mitochondria (6),
our results indicate that D-AKAP1 does not sequester RII
to specific subcellular compartments in adipocytes. Indeed, adipocyte differentiation, which strongly upregulated D-AKAP1/S-AKAP84
expression in membranes, was correlated with a cytosolic distribution
of PKA subunits. Very small levels of RII could be found in
mitochondrial fractions, raising the possibility that D-AKAP1 might
play a very modest role in mitochondrial targeting. However, this does
not appear to be the case, because fractionation in the presence of Ht
31 peptide, which abolishes RII-AKAP interactions in vitro, had no
effect on the low amounts of RII present in membrane fractions. Therefore, the presence of RII in membrane fractions most likely represents minor cytosolic contamination.
D-AKAP1 can bind both RI and RII subunits (14, 18),
and it might be argued that D-AKAP1 plays a role in PKA signaling by
tethering RI or RII
subunits to specific locations
inside the adipocyte. This seems unlikely, however, for several
reasons. Most significantly, the catalytic subunit C of PKA, which
binds all R subunits, was colocalized with RII
and not
with D-AKAP1. This finding is not surprising given that adipocytes
express only low levels of RI or RII
subunits
(8). Nonetheless, the subcellular distribution of RI and
RII
paralleled that of the predominant
RII
.
Putative AKAPs have been defined by the ability of these proteins to
interact with RII in artificial systems such as gel overlay and yeast
two-hybrid assays (4, 11, 18, 20). This binding activity
is blocked by the AKAP-inhibitory peptides such as Ht 31, indicating a
common mode of interaction with RII. However, the observation that some
tissues contain dozens of proteins that bind RII in vitro in a Ht
31-competitive fashion (4, 20) challenges the specificity
of the overlay approach and its relevance to PKA targeting in vivo. For
example, despite the high abundance of D-AKAP1/S-AKAP84 in sperm,
modulation of sperm motility by Ht 31 peptide appears to be independent
of both PKA targeting (1) and activity (20).
The recent discovery of proteins other than RII that bind AKAPs in
sperm further questions the role of AKAPs as PKA targeting proteins, at
least in some tissues (2).
The expression of D-AKAP1 appears to be associated with
mitochondrial function. D-AKAP1 is highly expressed in brown fat, a
tissue known for its high mitochondrial content, and D-AKAP1 gene
expression is strongly correlated with mitochondriogenesis that takes
place upon differentiation of C3H/10T1/2 cells. The observation that
D-AKAP1 does not target RII in vivo despite its high affinity in vitro
suggests that the determinants of this interaction are unavailable in
vivo. Future studies are necessary to determine whether these proteins
have additional binding partners that confer their distinct
distribution in vivo.
 |
FOOTNOTES |
Address for correspondence: A. Chaudhry, Pfizer Global Research
and Development, 2800 Plymouth Road, Ann Arbor, MI 48105 (E-mail: archana.chaudhry{at}pfizer.com).
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.
Received 2 April 2001; accepted in final form 7 September 2001.
 |
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