(Received for publication, June 26, 1995; and in revised form, September 8, 1995)
From the
We have shown previously (Nishimura, M., Fedorov, S., and Uyeda,
K.(1994) (J. Biol. Chem. 269, 26100-26106) that the
administration of high concentrations of glucose stimulates
dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase in
perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation
reaction. To characterize the protein phosphatase, we have purified the
Xu 5-P-activated protein phosphatase to homogeneity from livers of rats
injected with high glucose. Several protein phosphatases in the livers
were separated by DEAE-cellulose chromatography, but only one peak of
the enzyme was activated by Xu 5-P. The protein phosphatase was
inhibited by okadaic acid (IC = 1-3
nM) and did not require Mg
or
Ca
, suggesting that the enzyme was type 2A. The
enzyme was a heterotrimer (M
= 150,000) and
consisted of structural (A, 65 kDa), catalytic (C, 36 kDa), and
regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic
peptides derived from the B subunit showed similarity with those of the
B
isoform of rat protein phosphatase 2A, but five out of 73
residues were different. The protein phosphatase catalyzed
dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase a, and pyruvate kinase, and the K
values were 0.8 µM, 3.7 µM, and
2.2 µM, respectively. Among these substrates
dephosphorylation of only the bifunctional enzyme was activated by Xu
5-P, and the K
value for Xu 5-P was 20
µM. Xu 5-P was the only sugar phosphate which activated
the PP2A among all the sugar phosphates examined.
These results
demonstrated the existence and isolation of a unique heterotrimeric
protein phosphatase 2A in rat liver which catalyzed the
dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated
specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A
explains the increased Fru 2,6-P level in liver after high
glucose administration.
Many cellular processes and signaling transductions are
controlled by reversible phosphorylation of proteins. Fru 2,6-P(
)is the most potent activator of phosphofructokinase
and plays an important role in regulation of glycolysis, especially in
liver (reviewed in (1) ). Synthesis and degradation of Fru
2,6-P
are catalyzed by a bifunctional enzyme,
Fru-6-P,2-kinase:Fru-2,6-bisphosphatase. Liver
Fru-6-P,2-kinase:Fru-2,6-Pase is phosphorylated by cAMP-dependent
protein kinase(2, 3, 4) . When blood glucose
level falls, glucagon level increases which raises the cAMP level in
hepatic cells. The elevated cAMP activates cAMP-dependent protein
kinase, which phosphorylates Fru-6-P,2-kinase:Fru-2,6-Pase, leading to
the inhibition of Fru-6-P,2-kinase and the activation of Fru-2,6-Pase.
This results in a rapid decrease in Fru 2,6-P
, inhibition
of phosphofructokinase and glycolysis, and activation of
gluconeogenesis.
Mechanism for regulation of the dephosphorylation
of Fru-6-P,2-kinase:Fru-2,6-Pase remains unclear. Pelech et al.(5) sought to identify the nature of protein phosphatases
in liver involved in dephosphorylation of some of the known regulatory
enzymes of carbohydrate metabolism, such as the bifunctional enzymes,
phosphofructokinase, and fructose-1,6-bisphosphatase. In rat liver
extract, they detected four classes of protein phosphatase (PP1, PP2A,
PP2B, and PP2C) by DEAE chromatography and determined their activities
toward the substrates in vitro. All of these protein
phosphatases catalyzed the dephosphorylation of all the substrates, but
they concluded, based on the differences in hydrolysis rates, that PP2A
and PP2C appear to be the major phosphatases toward the
glycolytic/gluconeogenic enzymes in liver but could not assess the
significance of these results in vivo. Previous work from this
laboratory has shown that perfusion of the liver with high
concentrations of glucose results in an increased Fru 2,6-P level which is due to dephosphorylation of the bifunctional
enzyme(6) . This is in contrast to the earlier view (7) which attributed to the increased Fru 6-P concentration,
which is the substrate for the kinase. This dephosphorylation of the
bifunctional enzyme was a result of activation of a protein
phosphatase, and the activation appears to be caused by two factors (6) . One factor is Xu 5-P, and the other is yet unidentified
but may involve a covalent modification of the protein phosphatase.
These results suggested the existence of a specific protein phosphatase
in liver which dephosphorylates the bifunctional enzyme and which is
activated by Xu 5-P in response to the high glucose.
In the present study, we purified the Xu 5-P-activated protein phosphatase to homogeneity from rat liver and investigated the properties of the protein phosphatase.
Figure 1:
DEAE-Sepharose chromatography of Xu
5-P-activated protein phosphatase. The enzyme fraction from the
ammonium sulfate precipitation (step 3 of purification) was applied to
the column (3 40 cm) at a flow rate of 270 ml/h, and fractions
of 15 ml were collected as described under ``Experimental
Procedures.'' The protein phosphatase activity was assayed in the
absence and presence of Xu 5-P (50 µM). The solid line indicates the NaCl gradient. (
-
) protein
phosphatase activity in the absence of Xu 5-P;
(
-
) protein phosphatase activity in the
presence of Xu 5-P; (circo]-
) absorbance at 280
nm. mU, milliunits.
Figure 2:
SDS-polyacrylamide gel electrophoresis of
Xu 5-P-activated protein phosphatase. The electrophoresis was performed
as described under ``Experimental Procedures.'' Lane
1, marker proteins (from the top): phosphorylase b (M = 94,000); bovine serum albumin (M
= 67,000); ovalbumin (M
= 43,000); carbonic anhydrase (M
= 30,000); and trypsin inhibitor (M
= 20,100). Lane 2, Xu 5-P-activated protein
phosphatase; lane 3, immunoblotting of the same gel using
antibodies against the catalytic subunit of PP2A as described under
``Experimental Procedures.''
Okadaic acid is a potent inhibitor of
PP2A (IC = 1 nM) but requires much higher
concentrations to inhibit PP1 (IC
= 15-40
nM). It is, therefore, useful in distinguishing PP1 and
PP2A(16) . The Xu 5-P-activated protein phosphatase activity
was inhibited 45 and 53% by 1 nM okadaic acid in the absence
and presence of Xu 5-P, respectively (Fig. 3). These results
indicated that the Xu 5-P-activated protein phosphatase was PP2A,
consistent with the immunoblotting analysis.
Figure 3:
Okadaic acid inhibition of Xu
5-P-activated protein phosphatase. Xu 5-P-activated protein phosphatase
activity was determined in the absence () and the presence
(
) of Xu 5-P (50 µM) with various concentrations of
okadaic acid. Data are the mean of results from three
experiments.
When phosphorylase a and pyruvate
kinase were used as substrates, the K value was
five and three times, respectively, higher than that with rat liver
bifunctional enzyme as a substrate, while the V
remained the same. Interestingly, addition of Xu 5-P (50
µM) had no effect on either K
or V
when phosphorylase a or pyruvate
kinase was used as a substrate. Although the Xu 5-P-activated protein
phosphatase catalyzed the dephosphorylation of those substrates, the
activation by Xu 5-P occurred only with Fru-6-P,2-kinase:Fru-2,6-Pase
as a substrate.
To examine the specificity for Xu 5-P, effects of various sugar phosphates including the major intermediates of the pentose phosphate pathway were examined at near physiological concentrations(17) . These compounds included glyceraldehyde 3-phosphate (50 µM), erythrose 4-phosphate (5 µM), ribose 5-phosphate (50 µM), ribulose 5-phosphate (50 µM), 6-phosphogluconate (100 µM), glucose 6-phosphate (200 µM), Fru 6-P (50 µM), and sedoheptulose 7-phosphate (100 µM). None of these sugar phosphates activated more than 1.2 times while under the same conditions Xu 5-P activation was 2.2 times.
Figure 4:
Comparison of amino acid sequences of the
tryptic peptides derived from the 52-kDa subunit of Xu 5-P-activated
PP2A with the B subunit of rat PP2A. Mismatched amino acid
residues are boxed. B
, deduced amino acid sequence of
B
subunit of PP2A from rat library(19) ; peptides, amino acid sequence of the tryptic peptides obtained
from the 52-kDa subunit of Xu 5-P-activated
PP2A.
In the present study we have purified Xu 5-P-activated
protein phosphatase to apparent homogeneity from rat liver administered
with high glucose. Based on the cation dependencies, the dose response
to okadaic acid, and the reaction to antibody, this protein phosphatase
is likely to be an isozyme of PP2A. Although PP2A is the most abundant
isozyme among protein phosphatases, this PP2A is new because no other
PP2A holoenzyme (or other protein phosphatase) reported thus far is
activated by a sugar phosphate. The enzyme is a heterotrimer,
consisting of subunits with M = 65,000,
52,000, and 36,000. The known PP2A holoenzymes are heterotrimeric
complexes consisting of a catalytic subunit (C, M
= 36,000-38,000), a structural subunit (A, M
= 60,000-65,000), and a third
subunit termed B or phosphatase regulatory (PR) subunit (M
= 54,000-55,000 or
72,000-74,000) (15, 18, 20) . Molecular
cloning thus far has identified two isoforms (
and
) of both
A (21, 22) and C (23, 24) subunits,
and the amino acid sequences of these subunits are highly conserved
during evolution (25) . Because of the highly conserved nature
of these subunits, we assigned the 65-kDa subunit of the PP2A as A
subunit of Xu 5-P-activated protein phosphatase and the 36-kDa subunit
as the C subunit based on its M
and
immunoreactivity toward the catalytic subunit. Multiple families and
isoforms of the regulatory subunits (B), however, increase the
diversity and complexity of the PP2A enzyme. Various holoenzymes
containing different B subunits are thought to play important roles in
diverse physiological functions by determining substrate specificity
and by regulating catalytic activities of
PP2A(26, 27, 28, 29) . While the
same A and C subunits are present in different cells, the B subunits
comprise at least three distinct families of proteins. These B subunits
include M
= 55,000 (B/PR55), M
= 54,000 (B`), and M
= 72,000-74,000 (B"/PR72). Biochemical and
immunological analyses have shown that these regulatory subunits are
distinct polypeptides(15, 18, 20) . Three
cDNAs for B/PR55 (
,
, and
) have been cloned from
mammalian sources(19, 30, 31) , and these
isoforms of B subunits have been shown to be also highly conserved
during evolution(18) . The amino acid sequences of a limited
number of tryptic peptides derived from the B subunit of Xu
5-P-activated PP2A revealed that, although it is closest to the rat
neuronal B
subunit(19) , several amino acids are
different. Moreover, since this B subunit was smaller (52 kDa) than any
of the known B subunits, it is possible that this is a new and unique
subunit.
A question may be raised whether Xu 5-P-activated PP2A
could account for the dephosphorylation of the bifunctional enzyme in vivo. The concentration of the bifunctional enzyme in rat
liver is 0.4 µM dimer(32) , containing 0.8
µM phosphorylation sites, which is the same as the K of the PP2A for this substrate. The PP2A
activity (V
) in rat liver is 2.4 and 6
milliunits/g of liver in the absence and the presence of Xu 5-P,
respectively. Thus, there is sufficient Xu 5-P-activated PP2A activity
to dephosphorylate the bifunctional enzyme completely in less than 20 s
in the presence of Xu 5-P, taking into account that the activity is 0.5 V
. Furthermore, we believe that 2-3 times
activation by Xu 5-P is significant because the substrate itself, i.e. the bifunctional enzyme, is a catalyst which is being
activated by the protein phosphatase and consequently should not
require large changes in the protein phosphatase activity. This
represents another example of a cascade system of signal amplification.
The mechanism by which Xu 5-P activates this protein phosphatase is
currently unknown. One possibility is that since Xu 5-P activates the
dephosphorylation of only Fru-6-P,2-kinase:Fru-2,6-Pase as a substrate,
the pentose-P may bind to the bifunctional enzyme, and the complex
could be a better substrate than the free enzyme for the protein
phosphatase. Another possibility is that Xu 5-P may bind to the B
subunit and dissociate B from the heterotrimer, resulting in
activation. Pelech et al.(5) studied the substrate
specificity of the catalytic subunit of PP2A and reported about the
same activity toward Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase a, and pyruvate kinase. Thus, it is possible that the B
subunit inhibits the potential activity of the C subunit in the trimer,
specifically with Fru-6-P,2-kinase:Fru-2,6-Pase as substrate and that
Xu 5-P reacts with B to release this inhibition. These possibilities
and others are currently under investigation. It is generally thought
that PP2A is always in an active state in vivo and inhibitors
regulate the PP2A activities in vivo. As far as we are aware,
the activation of PP2A by Xu 5-P in rat liver described herein
represents one of only two examples of metabolite (or small M compounds) activation of PP2A. The other is
activation of a heterotrimeric form of PP2A of rat T9 glioma cells by
ceramide(33, 34) . However, a more recent report (35) indicates that even the catalytic subunit is activated by
ceramide and lacks substrate specificity. This is in contrast to the Xu
5-P-activated PP2A, because activation by Xu 5-P shows the substrate
specificity, thus reflecting the differences in the activation
mechanism. Since PP2A is not only the most abundant form of protein
phosphatase, especially in cytoplasm, but also shows broad substrate
specificities, it is possible that other PP2As may be regulated by
specific metabolites.
It is becoming increasingly clear that Fru
2,6-P is one of the most important activators of
phosphofructokinase and glycolysis in liver. The concentration of this
sugar bisphosphate in liver is responsive to hormones and nutritional
states (reviewed in (1) ). Consequently, the bifunctional
enzyme, Fru-6-P,2-kinase:Fru-2,6-Pase, which catalyzes synthesis and
degradation of Fru 2,6-P
, is one of the most important
enzymes in regulation of glycolysis and gluconeogenesis. Although this
enzyme is regulated by several metabolites based on in vitro studies(1, 7) , the
phosphorylation/dephosphorylation of the enzyme is probably more
important in vivo. The relative activities of the kinase and
the phosphatase, which determine the Fru 2,6-P
level, are
directly related to the phosphorylation state of the bifunctional
enzyme. The latter then is controlled by the relative activities of
protein kinase and protein phosphatase (Fig. 5). The
phosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase is catalyzed by a
cAMP-dependent protein kinase which results in inhibition of
Fru-6-P,2-kinase and activation of Fru-2,6-Pase, leading to decreased
Fru 2,6-P
and inhibition of glycolysis. Thus, the hormonal
control of the bifunctional enzyme is by cAMP-mediated phosphorylation
of the bifunctional enzyme.
Figure 5: A scheme for regulation of Fru-6-P,2-kinase:Fru-2,6-Pase by phosphorylation and dephosphorylation in vivo. (P)-F6P,2K:2P, phosphorylated form of Fru-6-P,2-kinase:Fru-2,6-Pase; F6P,2K:2P, dephosphorylated form of Fru-6-P,2-kinase:Fru-2,6-Pase; PKA, cAMP-dependent protein kinase.
Dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase is catalyzed by Xu 5-P-activated PP2A described here, which is regulated in part by the Xu 5-P level in liver. This conclusion is based on the following observations: (a) Xu 5-P is an intermediate of both oxidative and nonoxidative parts of the hexose monophosphate shunt pathway and is shown to rise with high glucose administration in liver (17) ; (b) among the protein phosphatases in the liver extract, only this PP2A was activated specifically by Xu 5-P. The observation that Xu 5-P-activated protein phosphatase also catalyzes dephosphorylation of phosphorylase a and pyruvate kinase in the absence of Xu 5-P may be explained by the possible differences in the locations of these substrates and the PP2A in the cells; and (c) Xu 5-P-activated PP2A is activated by Xu 5-P only with the bifunctional enzyme as a substrate. It is, therefore, tempting to suggest that Xu 5-P serves as a second messenger, sensing the glucose level in circulation and attenuating the effects of cAMP. Thus, it is possible that cAMP and Xu 5-P are the key signals in regulating the relative activities of the protein kinase and the protein phosphatase in a reciprocal manner to maintain glucose homeostasis. Obviously, further investigation is necessary to determine the significance of the roles that Xu 5-P-activated PP2A plays in liver.
In addition to Xu 5-P, another
factor is required to completely activate the PP2A induced by high
glucose administration in liver(6) . The nature of this factor
is unknown at present but may involve a covalent modification of the
PP2A. There is evidence that the free catalytic subunit or AC form of
PP2A can be phosphorylated in vitro by tyrosine kinases such
as pp60, the epidermal growth factor, and the insulin
receptor(36) . It is possible that Xu 5-P-activated PP2A is
also regulated by phosphorylation/dephosphorylation, and this
possibility requires further investigation.