(Received for publication, April 24, 1997, and in revised form, June 2, 1997)
From the Department of Cell Biology, Parke-Davis
Pharmaceutical Research Division, Warner-Lambert Company, Ann
Arbor, Michigan 48105 and the § Department of Physiology,
University of Michigan School of Medicine, Ann Arbor, Michigan
48109
We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32P-labeled phosphorylase a by decreasing the Km of PP1 for phosphorylase 5-fold, while having no effect on the Vmax of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.
While much attention has been focused on the activation of protein kinase signaling cascades, many enzymes involved in glucose and lipid metabolism are regulated by dephosphorylation (2). As the main physiological hormone controlling glucose utilization, insulin exerts many of its effects through the activation of type 1 protein phosphatase (PP1).1 However, insulin treatment of cells results in the dephosphorylation of only a limited number of proteins, while simultaneously other proteins are phosphorylated (3). This paradox suggests that mechanisms must exist whereby insulin activates discrete pools of PP1, leading to the dephosphorylation of specific target proteins.
PP1 is found in many cellular compartments, including the nucleus, plasma membrane, and glycogen particle. It is thought that the cellular localization of this enzyme is mediated by its association with targeting proteins (4, 5). We have recently identified a novel PP1 glycogen-targeting subunit from 3T3-L1 adipocytes, termed PTG for protein targeting to glycogen (1). PTG is the third member of a family of PP1 glycogen-targeting subunits, which also includes RGL, isolated from muscle (6, 7), and the hepatic GL protein (8, 9). In contrast to the restricted localization of RGL and GL, PTG is highly expressed in all insulin-sensitive tissues.2 In addition to targeting PP1 to the glycogen particle, PTG can also form complexes with PP1 substrate enzymes that regulate glycogen metabolism, namely glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. Overexpression of PTG in the metabolically inactive CHO-IR cell line dramatically increased the levels of basal and insulin-stimulated glycogen synthesis (1). PTG may therefore serve as a scaffolding protein, assembling the proteins involved in glycogen metabolism and priming them for the reception of intracellular signals.
The mechanism by which insulin specifically activates glycogen-targeted PP1 activity remains poorly understood. The proposed phosphorylation and activation of the PP1-RGL complex by pp90RSK (10) has subsequently been challenged (11-15). Further, the two putative PP1 regulatory phosphorylation sites of RGL are not conserved in PTG (1). Therefore, other mechanisms must exist for the regulation and activation of PP1 activity targeted to glycogen by PTG. The results presented here demonstrate that PTG is not a direct target for insulin-activated protein kinases. However, PTG does increase PP1-specific activity against phosphorylase a by three separate mechanisms: by targeting the phosphatase to glycogen, by directly binding and co-localizing PP1 substrates, and by reducing the affinity of PP1 for inhibitor peptides such as DARPP-32.
Cell culture reagents, IPTG, glycogen
phosphorylase b, and phosphorylase kinase were from Life
Technologies, Inc. Okadaic acid was purchased from Calbiochem.
[32P]Orthophosphate was obtained from ICN, whereas
[-32P]ATP (3000 Ci/mmol) and
[
-35S]ATP (1250 Ci/mmol) were from NEN Life Science
Products. [
-32P]dCTP (3000 Ci/mmol) and ECL reagent
were obtained from Amersham. Glutathione-Sepharose 4B beads and Ni-NTA
agarose beads were purchased from Pharmacia Biotech Inc. and Qiagen,
respectively. Polyhistidine-tagged DARPP-32 construct was the kind gift
of Drs. J. Bibb and A. Nairn (Rockefeller University), while chicken
affinity-purified anti-PP1
antibody was generously provided by Dr.
J. Lawrence (University of Virginia). GluT-4 antibody was from Dr. G. Lienhard (Dartmouth). Horseradish peroxidase-conjugated rabbit
anti-chicken IgG was obtained from Accurate Chemical Corp. (Westbury,
NY).
3T3-L1 fibroblasts and CHO-IR cells were maintained as described previously (14, 16). 3T3-L1 fibroblasts were differentiated into adipocytes by a standard protocol (14); adipocytes were routinely used 6-10 days after completion of differentiation. Primary rat adipocytes were isolated from epididymal fat pads and fractionated as described previously (17).
Northern Blot AnalysisTotal RNA was isolated from 3T3-L1
fibroblasts and fully differentiated adipocytes by acid guanidinium
thiocyanate-phenol-chloroform extraction (Rnasol; Biotex Laboratories).
RNA samples (15 µg) were electrophoresed in 1.2% agarose, 2.2 M formaldehyde, 1 × MOPS, and transferred by
capillary diffusion to nylon membranes (Hybond, Amersham). Membranes
were pre-hybridized for 1 h in FBY hybridization buffer (10%
polyethylene glycol, 1.5 × SSPE, 7% SDS) and then hybridized
overnight at 65 °C with the 1.0-kilobase EcoRI fragment
of PTG, which had been gel-purified and labeled with
[-32P]dCTP by random priming (>1 × 109 cpm/µg). The blot was washed for 15 min at 65 °C
in 2 × SSC, 0.1% SDS, then washed twice at 65 °C in 0.1 × SSC, 0.1% SDS for 15 min each time. Blots were analyzed by
autoradiography. RNA loading was determined by ethidium bromide
staining and by probing for
-actin.
PTG was
subcloned into the pGEX-5X-3 expression vector (Pharmacia), and fusion
protein was expressed in Escherichia coli DH5. One liter
of 2X-YT media plus 100 µg/ml ampicillin was seeded with 10 ml of a
saturated overnight culture and allowed to grow at 37 °C for
3.5 h (A600 = 0.5-0.7). Protein expression
was induced with 1 mM IPTG for 3 h at 37 °C.
Bacteria pellets were resuspended in 20 ml of PBS containing 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin added just before use and were lysed
by two passes through a French press (1,000 p.s.i.). 2 ml of PBS plus
10% Triton X-100 were added to the lysate which was then gently mixed
at 4 °C for 15 min. The supernatant from a 27,000 × g, 15-min centrifugation spin was applied to 1 ml of
glutathione-Sepharose beads equilibrated in PBS. After 30 min of mixing
at 4 °C, the beads were washed five times with PBS and analyzed by
SDS-PAGE. This protocol was designed to maximize GST-PTG yield and
solubility. Typically, 0.5-1 mg of GST-PTG was isolated per liter of
culture. GST-PTG was batch-eluted three times with 5 ml of PBS plus 20 mM glutathione (pH 7.8), followed by concentration with a
Centriprep-30 (Amicon). A GST fusion protein comprising the 40-kDa N
terminus of RGL (GST-G40) was expressed, purified, and
eluted in an identical fashion. E. coli DH5
containing a
full-length hormone-sensitive lipase GST fusion protein construct
(GST-HSL; provided by Dr. C. Baumann, Parke-Davis) were grown to an
A600 of 1, and then protein expression was
induced with 0.5 mM IPTG for 3 h at 30 °C. GST-HSL
was purified as above, and immobilized GST-HSL was labeled using
purified protein kinase A catalytic subunit and 50 µM
[
-32P]ATP (2500 cpm/pmol) at room temperature for 45 min. The beads were washed extensively with PP1 homogenization buffer,
and 10 µl of a 50% slurry were used as substrate in the PP1
assay.
A saturated culture of bacteria containing a polyhistidine-tagged DARPP-32 construct was diluted and grown in LB media at 37 °C for 3-4 h until the A600 = 0.4. Protein expression was induced by an overnight incubation with 1 mM IPTG at 30 °C. Bacterial pellets were lysed as above, and the clarified lysate was applied to Ni-NTA agarose affinity resin. After batch elution in 50 mM Tris (pH 6.8), 150 mM NaCl, and 0.5 M imidazole, DARPP-32 was concentrated using a Centriprep-10.
In Vivo and in Vitro Phosphorylation AssaysCHO-IR cells were transiently transfected with a FLAG epitope-tagged PTG construct as described previously (1). 48 h post-transfection, cells were serum-deprived for 3 h in phosphate-free Dulbecco's modified Eagle's medium plus 0.5% calf serum. Cells were then incubated for 1 h in the same media containing 1 mCi/1.5 ml of [32P]orthophosphate. After stimulation, cells were washed three times with ice-cold PBS and lysed in HNTG buffer (14, 18) plus protease inhibitors, and anti-FLAG immunoprecipitations were performed as described (1). Replicate culture plates were treated in parallel without [32P]orthophosphate, and in vitro kinase assays were performed on the cell extracts as below.
3T3-L1 adipocytes were serum-starved for 3 h in Krebs-Ringer
buffer with 30 mM Hepes (pH 7.4) plus 0.5% BSA and 2.5 mM glucose. After treatment, cells were washed three times
with ice-cold PBS and were then collected in kinase lysis buffer (10 mM Hepes (pH 8.0), 50 mM -glycerophosphate,
70 mM NaCl, 1% Triton) plus 1 mM sodium
vanadate, and protease inhibitors were added before use. After
centrifugation at 15,000 × g for 10 min, 15-35 µg
of the lysates were assayed in duplicate for kinase activities. GST-PTG and MAP kinase assays were performed at 37 °C for 10 min in the presence of 50 mM Hepes (pH 7.4), 10 mM
MgCl2, and 40 µM [
-32P]ATP
(3000 cpm/pmol), using as substrate 10 µg of GST-PTG or 2.5 µg of
MAP2 protein, respectively. Protein kinase A assays were performed at
37 °C for 2 min as above, using 5 µg of GST-G40 as substrate, in
the absence and presence of 1 mM dibutyryl cAMP. Reactions
were terminated by the addition of SDS-sample buffer. Samples were
analyzed by SDS-PAGE and autoradiography, substrate proteins were
excised from the dried gels, and 32P incorporation was
measured by liquid scintillation counting.
3T3-L1 adipocytes were washed three
times with ice-cold PBS and were then harvested in PP1 homogenization
buffer (50 mM Hepes (pH 7.2), 2 mM EDTA, 0.2%
-mercaptoethanol, and 2 mg/ml glycogen) plus protease inhibitors.
Cells were lysed by sonication, and nuclei and cell debris were
pelleted by centrifugation at 2,500 × g for 5 min.
Where indicated, the resulting post-nuclear supernatant (PNS) was
subjected to sequential centrifugation to prepare plasma membranes
(10,000 × g, 15 min) and to separate glycogen-enriched pellets from the cytosol (100,000 × g, 1 h).
Particulate fractions were resuspended in homogenization buffer using a
23-gauge needle. For PP1 assays, 1-3 µg of cellular fraction was
preincubated in PP1 homogenization buffer (20-µl final volume)
containing 4.5 nM okadaic acid for 2 min at 37 °C.
Reactions were initiated by the addition of 10 µl (15 µg, 5 µM final) of 32P-labeled phosphorylase
a (3 nM okadaic acid, 5 mM caffeine
final). After 5-10 min, the reactions were terminated by the addition of 90 µl of ice-cold 20% trichloroacetic acid and 5 µl of 2% BSA (modified from Ref. 19). Samples were incubated for 10 min on ice, and
precipitated protein was pelleted by a 2-min 15,000 × g centrifugation spin. Phosphate released into the
supernatant was measured by liquid scintillation counting. For
Lineweaver-Burk analysis, PP1 activity was measured using 0.5 µg of
PNS fraction as above and 1, 2, 3, 4, and 6 µM final
concentrations of phosphorylase substrate. In the absence of GST-PTG,
samples were incubated at 37 °C for 2.5, 5, 7.5, and 10 min; in the
presence of GST-PTG, samples were incubated for 1, 2, 4, and 5 min to
ensure that maximal dephosphorylation did not exceed 25% of the
substrate added. Reactions were terminated and quantitated as above,
and results were analyzed using Cricket Graph (Cricket software).
[32P]Phosphorylase a (~2000 cpm/pmol) was
prepared as described previously (14).
For phosphorylase
a binding measurements, 5 µg of immobilized GST-PTG was
resuspended in PP1 homogenization buffer plus 200 mM NaCl
and 0.2% BSA. 32P-Labeled phosphorylase a was
added to final concentrations of 1, 5, 10, and 15 µM
(final volume 30 µl). Samples were incubated at 4 °C with gentle
mixing for 30 min and were then washed three times with buffer.
Phosphorylase a binding was determined by liquid scintillation counting. Parallel incubations using immobilized GST-PTP1B protein were subtracted as blanks for each phosphorylase concentration. For PP1 binding measurements, immobilized GST-PTG was
incubated with increasing amounts of bacterial lysate in PP1 homogenization buffer containing recombinant PP1. Samples were treated as above, except that binding was analyzed by anti-PP1 immunoblotting and ECL detection. Standard curves of PP1 protein were
included on the immunoblots. Autoradiograms were analyzed by
computer-assisted video densitometry using a Bio Image system (Millipore).
Eluted DARPP-32 was thiophosphorylated as described (20). Following thiophosphorylation, polyhistidine-tagged DARPP-32 was rebound to Ni-NTA agarose beads, washed extensively, and was then eluted and concentrated as above.
We
have recently identified by two-hybrid screening a novel
glycogen-targeting subunit of PP1 from 3T3-L1 adipocytes, termed PTG
(1). This protein appears to act as a molecular scaffold for glycogen
metabolism and can dramatically increase glycogen synthesis upon
overexpression in tissue culture cells. To determine whether PTG
expression is correlated with the increase in metabolic activity and
insulin responsiveness observed following 3T3-L1 differentiation, PTG
expression was examined in fibroblasts and fully differentiated
adipocytes by Northern analysis. A single hybridizing mRNA species
of 3 kilobases was identified, which was dramatically up-regulated
following adipogenesis (Fig. 1). These
results correlate PTG expression with 3T3-L1 adipocyte differentiation, suggesting a critical role for PTG in the regulation of glycogen synthesis in 3T3-L1 adipocytes.
PP1 and Phosphorylase a Bind GST-PTG in Vitro
We have
previously demonstrated (1) that PTG can form specific complexes with
PP1, phosphorylase a, phosphorylase kinase, and glycogen
synthase, the key enzymatic regulators of glycogen synthesis. To better
characterize the protein-protein interactions of these enzymes with
PTG, the binding affinities of GST-PTG fusion protein for PP1 and
phosphorylase a were determined. Immobilized GST-PTG was
incubated with varying amounts of recombinant PP1, the beads were
washed and subjected to SDS-PAGE, and binding was quantitated by
densitometry scanning of anti-PP1
immunoblots. Binding was saturated
at 850 nM PP1, with an EC50 of approximately 335 ± 18 nM (Fig.
2A). The affinity of PTG for
glycogen phosphorylase was measured next. Varying amounts of
32P-labeled phosphorylase a were incubated with
immobilized GST-PTG, the beads were washed extensively, and binding was
determined by liquid scintillation counting. As seen in Fig.
2B, phosphorylase a bound to PTG with an
EC50 of 5.45 ± 0.14 µM. In contrast to the results of Doherty et al. (21), these data confirm
earlier results demonstrating that PTG can directly associate with
phosphorylase a (1).
PTG Increases the Affinity of PP1 for Phosphorylase a
We
examined the role of PTG in both the subcellular targeting and the
regulation of PP1 specific activity. GST-PTG addition to a 3T3-L1
adipocyte PNS fraction, followed by differential centrifugation, resulted in a dose-dependent, 4-6-fold increase in PP1
activity in the glycogen-enriched fraction (Fig.
3A, GP). PTG
addition to 3T3-L1 lysates also reduced the amount of PP1 activity
targeted to the plasma membrane fraction (Fig. 3A,
PM), indicating that changes in the level of PTG expression
can impact on the cellular distribution of PP1 activity (1).
Since PTG forms stable complexes with PP1 substrate proteins, it is
possible that PTG can also modulate PP1 specific activity independently
of glycogen localization. To test this possibility, varying amounts of
soluble GST-PTG were added to 3T3-L1 adipocyte PNS fractions, and then
PP1 activity was measured. PTG caused a dose-dependent
2-fold increase in PP1 specific activity against phosphorylase
a (Fig. 3B), whereas addition of 500 nM GST protein had no effect on PP1 activity (data not
shown). Lineweaver-Burk analysis revealed that PTG decreased the
Km of PP1 for phosphorylase a 5-fold,
while having no effect on the Vmax (Fig. 3B). Similar results were obtained in a glycogen-free,
cytosolic fraction (data not shown), indicating that the stimulation of PP1 activity was independent of glycogen targeting. GST-PTG also caused
a dose-dependent 3-fold increase in bacterially expressed PP1 activity against phosphorylase a (data not shown).
GST-PTG was completely soluble and did not pellet upon
ultracentrifugation in the absence of glycogen, possibly explaining
differences with the results of Doherty et al. (21).
The effects of PTG on PP1 activity were dependent on the substrate used in the phosphatase assay. HSL is a lipid-metabolizing enzyme, which is dephosphorylated by PP1 in response to insulin stimulation of adipocytes. Although GST-PTG addition to a 3T3-L1 adipocyte PNS fraction increased PP1 specific activity 2-fold against phosphorylase a (Fig. 3D, Phos a), in parallel assays, there was no change in PP1 activity when 32P-labeled hormone-sensitive lipase was used as substrate (Fig. 3D, HSL). Thus PTG regulates PP1 specific activity by both targeting the phosphatase to glycogen and also by selectively binding and co-localizing certain substrates with PP1.
PTG Is Not a Target of Intracellular SignalingBecause PTG is
likely to play a critical role in the regulation of glycogen synthesis
by insulin, we examined whether PTG might be phosphorylated in response
to hormone treatment. Although the putative phosphorylation sites
previously suggested for RGL (10, 22) are not conserved in
PTG (1), the possible phosphorylation of other residues on PTG was
examined. CHO-IR cells transiently transfected with FLAG-PTG were
labeled with [32P]orthophosphate and exposed to either
100 nM insulin or 10 µg/ml forskolin for 5 min. PTG was
immunoprecipitated with anti-FLAG antibodies and subjected to SDS-PAGE
followed by autoradiography. PTG exhibited a low basal state of
phosphorylation, which was not changed by either treatment (Fig.
4A). In replicate cultures, insulin or forskolin exposure increased MAP kinase or protein kinase A
activity 3- and 5-fold, respectively (Fig. 4B and data not
shown).
To determine whether 3T3-L1 adipocytes contained a protein kinase
capable of phosphorylating PTG, in vitro phosphorylation assays were performed using PTG as substrate. Cells were exposed to a
variety of agents, and cellular lysates were prepared and incubated
with GST-PTG and [-32P]ATP. Samples were then analyzed
by SDS-PAGE and autoradiography. As seen in Fig. 4C, GST-PTG
was not phosphorylated in vitro by basal extracts
(lane 1 versus 2). Further stimulation of 3T3-L1 adipocytes
with either insulin or forskolin did not lead to the activation of a
PTG kinase (Fig. 4C, lanes 3 and 4).
EGF or TPA treatment of cells also did not result in any measurable
phosphorylation of GST-PTG in vitro (Fig. 4C,
lanes 5 and 6). Finally, PTG was also not
phosphorylated in vitro by purified MAP kinase or protein kinase A catalytic subunit (data not shown), further indicating that
PTG is not a physiological substrate for insulin- or cAMP-activated kinases.
Translocation of PP1 to and from the glycogen particle in
response to the phosphorylation of RGL has been suggested
to underlie hormonal regulation of PP1 activity (10, 22). Although PTG is not phosphorylated in response to external stimuli, possible changes
in the cellular distribution of PP1 following insulin treatment were
examined. Primary rat adipocytes were isolated and exposed to 10 nM insulin for 30 min, and cellular fractions were prepared
by differential centrifugation. Insulin induced a translocation of
GluT-4 protein from the low density microsomal fraction to the plasma
membrane fraction (Fig. 5A).
However, insulin stimulation did not modulate PP1 protein levels in
any fraction, including the low density microsomal fraction, which
contains the glycogen pellet (Fig. 5B); identical results
were obtained using an anti-PP1
antibody (data not shown). Further
insulin or forskolin treatment of 3T3-L1 adipocytes also did not cause a detectable translocation of PP1 between cellular fractions (data not
shown). Taken together, these data indicate that the regulation of PP1
activity by insulin, or agents that elevate intracellular cAMP levels,
occurs independently of PP1 translocation.
PTG Decreases the Inhibition of PP1 by DARPP-32
PP1 is
maintained in a low activity state in 3T3-L1 adipocytes by the binding
of phosphorylated DARPP-32.3
Previous studies (23) suggested that insulin may activate PP1 in
primary rat adipocytes by inducing the dephosphorylation and disassociation of this peptide. The role of PTG in modulating the
regulation of PP1 activity by DARPP-32 was investigated.
Thiophosphorylated DARPP-32 specifically inhibited PP1 activity in
3T3-L1 lysates (Fig. 6), with a
Ki of 3 nM, consistent with previous results (20). Addition of purified GST-PTG to the lysates caused a
rightward shift in the inhibition curve of DARPP-32
(Ki 30 nM, Fig. 6), indicating that PTG
lowers the binding affinity of DARPP-32 for PP1. These data suggest
that a decrease in the cellular phospho-DARPP-32 concentration in
response to insulin might result in the preferential activation of PP1
bound to PTG.
The regulation by insulin of enzymes involved in glycogen synthesis is primarily mediated by the activation of PP1 (2). The activities of glycogen synthase, phosphorylase a, and phosphorylase kinase are modulated by insulin via a mechanism involving their net dephosphorylation, resulting in an increase in glucose storage as glycogen. The paradoxical dephosphorylation of only a limited number of proteins by insulin, despite the ubiquitous presence of PP1 in nearly all cellular compartments, has yet to be explained. Mechanisms must exist for the establishment of discrete pools of PP1 which are preferentially activated by insulin.
PP1 is maintained in discrete subcellular compartments by association with specific targeting subunits. Three proteins have been identified which bind both glycogen and PP1, thus localizing PP1 at the glycogen particle. RGL was first purified from skeletal muscle (6, 7), and GL was subsequently purified from liver (8, 9). We have recently identified a third PP1 targeting subunit from 3T3-L1 adipocytes, termed PTG, for protein targeting to glycogen (1). PTG binds not only to PP1 and glycogen, but also to the primary enzymatic regulators of glycogen synthesis, namely glycogen synthase, phosphorylase kinase, and phosphorylase a (1). By co-localizing PP1 with its substrates at the glycogen particle, PTG acts as a scaffolding protein, assembling metabolic enzymes for the localized reception of intracellular signals.
Studies in different cell lines indicate that the level of PTG expression correlates with cellular metabolic activity. CHO-IR cells contain no endogenous PTG protein and exhibit a low basal rate of glycogen synthesis. Overexpression of PTG in these cells resulted in a 7-10-fold increase in glycogen synthesis (1). Differentiation of 3T3-L1 fibroblasts into lipid-containing adipocytes caused a dramatic increase in insulin-stimulated glycogen synthesis.3 PTG mRNA levels were strongly up-regulated during this differentiation protocol (Fig. 1), further indicating an important role for this protein in the regulation of glycogen synthesis by insulin.
PTG appeared to be capable of regulating PP1 specific activity in vitro by several mechanisms. Firstly, a GST-PTG fusion protein bound to PP1 with high affinity. Moreover, addition of GST-PTG to 3T3-L1 lysates resulted in a concentration-dependent translocation of PP1 to the glycogen-enriched pellet (Fig. 3A). The level of cellular expression of PTG would therefore presumably dictate the localization of PP1 at the glycogen particle (1). Secondly, GST-PTG also bound directly to phosphorylase a (Fig. 2B). Addition of GST-PTG to 3T3-L1 lysates, with no subsequent fractionation, caused a 2-fold increase in PP1 specific activity against phosphorylase a in vitro. Since PTG addition decreased the Km of PP1 for phosphorylase a 5-fold, without affecting the Vmax of the reaction, this increase in phosphatase activity resulted from the formation of a trimeric complex between PTG, PP1, and its substrate phosphorylase a. This effect of PTG on PP1 activity was restricted to specific proteins. Although HSL is a physiological substrate for PP1 in adipocytes and in vitro, PTG did not affect PP1 activity against this enzyme (Fig. 4D). Thus, PTG regulates PP1 activity against glycogen metabolic enzymes, both by targeting PP1 to glycogen and also by directly binding to and co-localizing specific PP1 substrate proteins.
The precise mechanism by which insulin activates glycogen-targeted PP1 activity remains unclear. Dent et al. (10) reported that the phosphorylation state of two protein kinase A consensus sites on the RGL glycogen targeting subunit regulated PP1 activity in vitro. However, this model has subsequently been disputed (11-15). PTG does not share the putative regulatory phosphorylation sites of RGL (1), and PTG was not phosphorylated in response to either insulin or forskolin treatment of CHO-IR cells (Fig. 4A). Additionally, neither agent could activate a kinase from 3T3-L1 adipocytes capable of phosphorylating exogenous PTG in vitro (Fig. 4C). Finally, insulin treatment had no effect on PP1 binding to PTG in CHO-IR cells (1), and insulin did not increase PP1 localization at the glycogen particle in either primary rat adipocytes (Fig. 5B) or 3T3-L1 adipocytes.3 Taken together, these results indicate that phosphorylation of PTG and/or changes in the affinity of PTG for PP1 do not mediate the hormonal regulation of PP1 activity targeted to glycogen.
In 3T3-L1 adipocytes, PP1 is maintained in a low basal activity state by DARPP-32 binding. DARPP-32 expression is dramatically induced upon differentiation of 3T3-L1 fibroblasts into adipocytes and correlates with a decrease in PP1 basal activity and increase in stimulation by insulin.3 Furthermore, DARPP-32 is expressed in pig brown fat (24), bovine adipose tissue (25), and in primary rat adipocytes, where it has been reported to be dephosphorylated in response to insulin (23). PP1 bound to PTG was resistant to inhibition by DARPP-32 (Fig. 6), in agreement with results with RGL (26). Since PTG reduces the affinity of DARPP-32 for PP1, glycogen-targeted PP1 activity may be more sensitive to possible insulin-induced dephosphorylation of inhibitor peptides in vivo. PTG may therefore not only increase PP1 specific activity against glycogen-targeted enzymes, but also may partially underlie the specific activation of glycogen-targeted PP1 by insulin. Additional work is needed to fully test this proposed model.