(Received for publication, December 5, 1995; and in revised form, January 19, 1996)
From the
Stimulation of rat adipocytes with insulin and isoproterenol
results in serine phosphorylation and activation of the adipocyte
cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be
important in the antilipolytic action of insulin (Degerman, E., Smith,
C. J., Tornqvist, H., Vasta, V., Manganiello, V. C., and Belfrage,
P.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 533-537).
Here we demonstrate, by two-dimensional phosphopeptide mapping, that
the major phosphopeptide generated by trypsin, or trypsin followed by
Asp-N protease digestion of [P]cGI PDE
phosphorylated in adipocytes in response to isoproterenol and/or
insulin, in each case co-migrates with the phosphopeptide released by
the same treatment of M
FRRPS(P)LPCISREQ
.
This peptide was synthesized based on the deduced sequence of the
cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent
protein kinase (protein kinase A). Radiosequencing of authentic and
synthetic tryptic
P-peptides showed that a single site in
cGI PDE (Ser
) was phosphorylated in adipocytes incubated
with isoproterenol and/or insulin. The more than additive
phosphorylation and activation of cGI PDE in response to the two
hormones found in this report and previously (Smith, C. J., Vasta, V.,
Degerman, E., Belfrage, P., and Manganiello, V. C.(1991) J. Biol.
Chem. 266, 13385-13390) is proposed to reflect cross-talk
between their respective signal transduction pathways at the level of
the cGI PDE serine protein kinase or upstream regulatory component(s).
Insulin controls cell metabolism and proliferation through
several multicomponent diverging pathways, which are only partially
understood (1, 2, 3) . One important and well
known metabolic effect is to antagonize hormone-activated lipolysis in
adipose tissue, brought about mainly via insulin-mediated activation of
a membrane-associated cGMP-inhibited phosphodiesterase (cGI PDE, ()referred to as the PDE 3 gene family; (4) ), which
leads to a reduction in cAMP, a decrease in cAMP-dependent protein
kinase (protein kinase A) activity, and a lowering of the
phosphorylation and thereby the activity of the hormone-sensitive
lipase, the rate-limiting enzyme in the regulation of
lipolysis(5, 6, 7, 8, 9, 10, 11) .
Insulin and cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes(12, 13) . An insulin-stimulated cGI PDE serine protein kinase activity in rat adipocytes has previously been partially characterized(14, 15) . In the presence of both isoproterenol and insulin, the phosphorylation/activation of cGI PDE is more than additive(13, 16) , suggesting cross-talk between the two signal transduction pathways.
To further dissect the
components of the antilipolytic signaling pathway controlling the cGI
PDE, we used two-dimensional tryptic phosphopeptide mapping and other
methods to analyze the site(s) phosphorylated in this enzyme in P-labeled rat adipocytes incubated with insulin,
isoproterenol, or both. Since the amount of labeled phosphopeptides
that could be obtained from experiments with intact cells was far less
than that required for amino acid sequencing, the phosphorylation
site(s) was identified by comparison of properties of phosphopeptides
from cGI PDE phosphorylated in adipocytes, or from recombinant cGI PDE
expressed in NIH 3006 human fibroblasts (
)(rcGI PDE)
phosphorylated by protein kinase A, with peptides synthesized on the
basis of the deduced sequence of rat adipocyte cGI PDE (17) and
phosphorylated by protein kinase A. Peptides chosen for these studies
contained consensus sequences for phosphorylation by protein kinase A (18) , since preliminary results indicated that the site(s)
phosphorylated during incubation of adipocytes with insulin or
isoproterenol was located in the same tryptic phosphopeptide.
Figure 1:
Linear
structure of cGI PDE. Boxes indicate putative protein kinase A
consensus sequences; tryptic cleavage sites are marked by arrows. Tryptic fragments derived from protein kinase
A-phosphorylated synthetic peptides P and
P
are denoted tPP
/tPP
and tPP
,
respectively. Tryptic fragments derived from protein kinase
A-phosphorylated P
are not
indicated.
Particulate adipocyte cGI PDE,
corresponding to 1 ml packed cell volume, was prepared and
solubilized(12, 13) . NIH-3006 cells (NIH-3T3 cells
that stably overexpress the human insulin receptor; (19) ) were
stably transfected with full-length cGI PDE cDNA and cultured as
described. Confluent cells were washed rapidly with
ice-cold phosphate-buffered saline, suspended in 50 mM Tris,
pH 7.4, containing 5 mM MgCl
, 1 mM EDTA,
5% glycerol, 1% C
E
(heterogeneous non-ionic
detergent; (18) ), 10 µg/ml antipain and leupeptin, and 1
µg/ml pepstatin A (1 ml/dish) and harvested with a rubber
policeman. The cell suspension was sonicated (three times for 20 s
each, on ice in a Branson Sonifier 250, output control 1.5, constant
duty cycle) and thereafter centrifuged (33,000
g, 60
min, 4 °C). Solubilized adipocyte cGI PDE (360 pmol/min; PDE
activity was determined as described previously) (12, 13, 20) or solubilized rcGI PDE (40
nmol/min), derived from one dish, was incubated with 50 mM HEPES, pH 7.4, containing 10 mM MgCl
, 1
mM dithioerythritol, 40 µM [
-
P]ATP, and 100 units/ml protein
kinase A, in final volumes of 12 or 2 ml respectively, for 30 min at 30
°C.
Figure 2:
Two-dimensional tryptic phosphopeptide
maps of cGI PDE from intact rat adipocytes, stimulated with
isoproterenol and/or insulin. A, solubilized cGI PDE from P-labeled rat adipocytes (2 ml of 10% by volume of
cells/condition) stimulated with isoproterenol (ISO), insulin (INS) or in combination (ISO+INS), as indicated,
was immunoprecipitated and subjected to SDS-PAGE, followed by digital
imaging of
P. B-D, solubilized cGI PDE from
P-labeled rat adipocytes (20 ml of 10% by volume of cells
with 0, 5-1 mCi/ml) stimulated with isoproterenol and/or insulin
was immunoprecipitated, subjected to SDS-PAGE, transferred to a
nitrocellulose membrane, and digested with trypsin.
P-Phosphopeptides were separated by two-dimensional
TLE/TLC and visualized by digital imaging of
P. The major
tryptic phosphopeptides are indicated as tPP
, tPP
, or tPP
. Minor tryptic
phosphopeptides are indicated with arrows. X marks
the origin. The electrophoresis direction indicated is from anode to
cathode.
We have previously identified
Ser as the major site in the solubilized rat adipocyte
cGI PDE phosphorylated by protein kinase A in
vitro(18) . Ser
is located within one of the
three consensus sequences for phosphorylation by protein kinase A
(-RRXS-) in the rat adipocyte cGI PDE (17) (Fig. 1). This serine was not, however,
phosphorylated in cGI PDE in intact adipocytes during isoproterenol
stimulation (Fig. 2). Fig. 3(A and B)
is a representative example of two-dimensional tryptic phosphopeptide
maps of isolated, solubilized adipocyte cGI PDE and rcGI PDE, maximally
phosphorylated by protein kinase A to an estimated total
phosphorylation stoichiometry of 0.9 ± 0.2 (mean ± S.E., n = 5) (cf. (18) ). When compared to
the two-dimensional tryptic phosphopeptide map from
[
P]cGI PDE phosphorylated in intact adipocytes (Fig. 2B), two major in vitro phosphopeptides
tPP5 and tPP6 (Fig. 3, A and B) clearly
migrated differently from the tPP
. tPP5 and tPP6 were
identified as tPP
and tPP
(Fig. 1) by two approaches. (i) After elution from the TLC
plates, tPP5 and tPP6 cross-reacted with anti-P
as judged from Tricine-SDS/urea-PAGE of immunopellets and
immunosupernatants (Fig. 3C, lanes 5 (tPP
) and lane 6 (tPP
)). Minor phosphopeptides (denoted
tPP1-tPP4) did not react (Fig. 3C, lanes 1, 2,
and 4). (ii) tPP5 and tPP6 co-migrated with
tPP
and tPP
respectively, as judged from mixing experiments in which the
respective phosphopeptide was eluted from the TLC plate, mixed, and
re-run together using two-dimensional phosphopeptide mapping (data not
shown). tPP5 (tPP
) and tPP6
(tPP
) were previously shown to be generated
from P
by tryptic cleavage C-terminal to
Arg
or Arg
and Arg
,
respectively(18) . Thus, tPP
was clearly distinct
from tPP
or tPP
, i.e. the major site phosphorylated in cells by isoproterenol
was different from that phosphorylated in vitro by protein
kinase A (Ser
).
Figure 3:
Two-dimensional tryptic phosphopeptide
maps of solubilized cGI PDE phosphorylated in vitro by protein
kinase A. Rat adipocyte cGI PDE (A) and rcGI PDE (B)
were phosphorylated with protein kinase A in the presence of
[-
P]ATP. Tryptic phosphopeptides were
analyzed by two-dimensional TLE/TLC. C, major tryptic
phosphopeptides from protein kinase A-phosphorylated rcGI PDE
(tPP1-tPP6), indicated as 1-6, were eluted from
the TLC plates and incubated with anti-P
.
Immunosupernatants and immunopellets were then subjected to
Tricine-SDS/urea-PAGE. Lanes 1-6 represent
tPP1-tPP6. X marks origin.
One of the minor phosphopeptides
(tPP1) (Fig. 3) appeared to migrate similarly to
tPP. Eluted tPP1, derived from solubilized rcGI PDE
phosphorylated with protein kinase A in vitro, co-migrated
with tPP
in mixing experiments (data not illustrated).
Thus, although most of the protein kinase A-catalyzed phosphorylation
of solubilized authentic adipocyte cGI PDE took place on Ser
as reported previously(18) , with an estimated
phosphorylation stoichiometry of 0.4 ± 0.04 (mean ± S.E., n = 5), some also occurred in the same tryptic peptide
as that phosphorylated in intact cells during isoproterenol stimulation
(estimated in vitro phosphorylation stoichiometry 0.2 ±
0.04 (mean ± S.E., n = 7)). tPP1 was therefore
used to further identify the site phosphorylated in cells, as described
below.
The minor tryptic phosphopeptides tPP2, tPP3, and tPP4 migrated differently from the major tryptic phosphopeptide derived from cGI PDE phosphorylated in cells. The presence of tPP3 was variable, being absent in some experiments. The positions of these tryptic phosphopeptides in the enzyme were not identified.
Figure 4:
Comparison of tryptic phosphopeptides from
rat adipocyte cGI PDE phosphorylated in cells with
tPP. P
was
phosphorylated by protein kinase A, trypsinized, and subjected to
two-dimensional TLE/TLC (A). Major tryptic phosphopeptides
tPP
, tPP
, and tPP
derived from adipocyte cGI PDE phosphorylated in cells were
eluted from the TLC plates and re-run without (B-D) or
with (E-G) eluted tPP
(A). X marks
origin.
As shown in Fig. 4,
tPP and tPP
, which migrated very
similarly to tPP
in the two-dimensional phosphopeptide
mapping system (Fig. 2, B-D), co-migrated with
tPP
. Thus, stimulation of adipocytes with
isoproterenol, insulin, or the combination of both appeared to result
in phosphorylation of cGI PDE within the amino acid sequence
Arg
-Arg
. To verify this, the tryptic
phosphopeptides were cleaved once more with Asp-N protease (known to
cleave N-terminal to cysteic acid or aspartic acid). As shown in Fig. 5, this treatment generated one new phosphopeptide, which
in each case again co-migrated with the one obtained from Asp-N
protease-treated tPP
.
Figure 5:
Comparison of Asp-N protease-treated
tryptic phosphopeptides from cGI PDE phosphorylated in rat adipocytes
with Asp-N protease-digested tPP.
tPP
and tryptic phosphopeptides from cGI PDE
phosphorylated in cells stimulated with isoproterenol, insulin, or both
hormones were prepared and subjected to two-dimensional TLE/TLC. Major
tryptic phosphopeptides, corresponding to tPP
,
tPP
, tPP
, and tPP
,
were eluted from the TLC plates, re-run (A, D, G, and J), or digested with Asp-N protease and
subjected to two-dimensional TLE/TLC as indicated (B, E, H, and K). These Asp-N protease-generated
tryptic phosphopeptides from adipocytes (E, H, and K) were eluted and mixed with eluted Asp-N protease-digested
tPP
(B) and subjected to
two-dimensional TLE/TLC as indicated (F, I, and L). The generation of a new phosphopeptide is illustrated by
mixing of Asp-N protease-digested and undigested tPP
(C). X marks
origin.
Figure 6:
Separation of tryptic phosphopeptides by
Mono Q, C8 reversed phase chromatography, or Tricine-SDS/urea-PAGE.
rcGI PDE and P were phosphorylated in vitro by protein kinase A in the presence of
[
-
P]ATP. Tryptic phosphopeptides were
separated by two-dimensional TLE/TLC. tPP1 (
), derived from
protein kinase A-phosphorylated rcGI PDE and tPP
(
), were eluted from the TLC plates and subjected to
chromatography on a Mono Q HR 5/5 (Pharmacia) column (A) or on
a reversed phase C8 column (Kromasil C8 5µ, 4.6
150 mm) (B), either separately or in combination (
). The Mono Q
column was equilibrated in 10 mM Tris-HCl, pH 8.0, and eluted
with a linear gradient of 0-500 mM NaCl (1 ml/min). The
C8 reversed phase column was equilibrated in 0.1% trifluoroacetic acid
and eluted with a linear gradient of 0-60% acetonitrile (0.5
ml/min). The elution-position of the
P-labeled peptides
were detected by liquid scintillation counting. C, tPP1 from
protein kinase A-phosphorylated rcGI PDE and tPP
were eluted from the TLC plates and subjected to
Tricine-SDS/urea-PAGE.
Figure 7:
Radiosequencing of tryptic phosphopeptides
obtained from cGI PDE phosphorylated in adipocytes. Tryptic
phosphopeptides from solubilized cGI PDE from P-labeled
rat adipocytes stimulated with isoproterenol and/or insulin were
analyzed as described under ``Experimental Procedures'' and
in Fig. 2. The major phosphopeptides (tPP
,
tPP
, and tPP
) were eluted and
coupled to Sequelon-AA membranes and sequenced on an Applied Biosystems
gas phase sequencer (model 470A). Released phenylthiohydantoin amino
acid-derivatives from each cycle were spotted on TLC plates, and the
radioactivity was quantified by digital imaging and volume integration
of each spot after subtraction of background
P.
Using several experimental approaches and fractionation
procedures, we have demonstrated that stimulation of adipocytes with
insulin, isoproterenol, or the combination of both results in
phosphorylation of cGI PDE at a single site (Ser) and
activation of the enzyme. Ser
is localized within the
sequence MFRRPS
LPCISREQ, one of three consensus
sequences (-RRXS-) for putative protein kinase A-catalyzed
phosphorylation of rat adipocyte cGI PDE (Fig. 1). The
insulin-induced phosphorylation was presumably mediated by the
previously described (14, 15) insulin-stimulated cGI
PDE kinase activity found in cytosolic extracts of rat adipocytes
following their stimulation by insulin. For technical reasons, it has
not yet been possible to identify the serine(s) in cGI PDE
phosphorylated in vitro during incubation with such extracts
from insulin-treated adipocytes. Several insulin-stimulated serine
protein kinases, which appear to have one or several properties in
common with insulin-stimulated cGI PDE kinase (e.g. activation
by serine/threonine phosphorylation that is blocked by wortmannin
treatment of cells), have been found in adipose
tissue(28, 29, 30) .
Protein kinase A is
likely to be responsible for the isoproterenol-induced phosphorylation
of cGI PDE in intact cells. In cell-free preparations, Ser rather than Ser
was found, as described
previously(18) , to be the major target for protein kinase
A-catalyzed phosphorylation of cGI PDE. Ser
was found to
be phosphorylated by protein kinase A in vitro, less
extensively than Ser
but to the same extent as in intact
cells in response to isoproterenol or insulin (phosphorylation
stoichiometry approximately 0.2). This, and the fact that
membrane-associated adipocyte cGI PDE has been reported to become
activated(31) , and platelet and hepatocyte cGI PDEs
phosphorylated and activated in vitro by protein kinase A (32, 33, 34) , indicates that this kinase
directly (and not through another kinase) phosphorylates and activates
the enzyme in intact cells, although this still remains to be
definitely established. The reason for the preferential phosphorylation
of Ser
rather than Ser
by protein kinase A in vitro is not known. However, adipocyte cGI PDE is
membrane-bound and in fat cell extracts requires salt and detergent for
efficient solubilization. The cDNA structure of the enzyme predicts it
to be an intrinsic membrane protein with six putative transmembrane
sequences in its N-terminal region(17) . In spite of several
attempts with intact and solubilized membrane preparations of cGI PDE,
we have so far been unable to establish conditions during which
Ser
is not extensively phosphorylated by protein kinase A in vitro. (
)This may indicate that correct membrane
insertion of the enzyme is a critical determinant for exposing
Ser
and not Ser
for phosphorylation.
Previous work showing different time-courses for phosphorylation and activation of cGI PDE in rat adipocytes stimulated with isoproterenol or insulin (12, 13) and more than additive phosphorylation/activation during stimulation with both hormones in combination(13, 16) , was tentatively interpreted as indicating phosphorylation at two sites in cGI PDE(10) . Clearly, the present result is not consistent with this hypothesis and instead indicates that the signal pathways interact upstream of cGI PDE. Since the insulin-stimulated cGI PDE kinase appears to be activated in fat cells via phosphorylation during insulin stimulation ((10) ; cf.(35) ), this kinase could be dually regulated by an insulin-stimulated kinase kinase (or an insulin-inactivated phosphatase) and protein kinase A, respectively. However, phosphatidylinositol 3-kinase (36) and insulin receptor substrate-1 (and related proteins)(1, 37) , known upstream links in the antilipolytic signal chain, could also be merging points. cAMP-enhancing agents have recently been reported to modulate the signals from insulin and other growth factors(38, 39, 40, 41, 42, 43) , generally by attenuating them, but at least in one case by enhancing the effect of growth factors(43) , consistent with the findings in this report. The identification reported here of the site in cGI PDE phosphorylated in cells in response to both insulin and cAMP-enhancing agents should be useful for the further dissection of the signal transduction pathway, linking the hormone receptor events to the activation of cGI PDE in the control of adipose tissue lipolysis.