(Received for publication, August 1, 1995; and in revised form, September 29, 1995)
From the
Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
Catalytic (C) ()subunit of cAMP-dependent protein
kinase when isolated from animal tissues is phosphorylated at two
sites, Thr-197 and Ser-338. The recombinant protein expressed in Escherichia coli is phosphorylated at these sites and, in
addition, at Ser-10 and Ser-139 (1) . Phosphorylation at
Thr-197 slows the mobility of C subunit in SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and ``activates'' the protein by
reducing its K
values for both ATP and
peptide substrates(2) . Thr-197 falls in the ``loop''
region between subdomains VII and VIII(3) , which also is
associated with activating phosphorylation sites in a number of other
protein kinases, including CDC2 kinase, MAP kinase (or ERK), MAP kinase
kinase (or MEK), and many protein tyrosine
kinases(3, 4, 5) .
In the crystal structure of C subunit, the Thr-197 phosphate appears to stabilize the active configuration of the catalytic cleft through ionic and hydrogen-bonding interactions with the side chains of His-87, Arg-165, Lys-189, and Thr-195(6) . Comparisons of the structures of inactive forms of the CDC2 and MAP kinases with that of the active C subunit suggest that phosphorylation of sites in this ``activation loop'' region is responsible for a structural rearrangement of the catalytic cleft that promotes substrate binding and catalysis(4, 7) . Nevertheless, while phosphorylation of the activation loop in CDC2 kinase, MAP kinase, MAP kinase kinase, and many of the protein tyrosine kinases is regulated by the antagonistic actions of specific protein kinases and protein phosphatases, Thr-197 phosphate in C subunit has been thought to be metabolically stable(8, 9, 10) .
While undertaking experiments to optimize conditions for immunoprecipitation of C subunit from extracts of S49 mouse lymphoma cells, we noted that incubation of radiolabeled, recombinant C subunit with cell extract resulted in apparent dephosphorylation manifested by an increase in SDS-PAGE mobility. Although this reaction was most dramatic in buffers containing SDS or urea, there was also reactivity in the absence of these agents. This report describes experiments that identify the cellular activity as a protein phosphatase with properties similar to those of a multimeric form of protein phosphatase-2A (PP-2A) and characterize its reaction on C subunit.
For anion-exchange purification, cell
suspensions as above were thawed and centrifuged at 4 °C for 1 h at
100,000 g. The supernatant fractions were then loaded
onto columns of Accell Plus QMA (500 A,
Waters/Millipore) equilibrated with QMA buffer; about 1 ml of packed
resin was used for each 7 mg of cell protein. Columns were washed with
five volumes of QMA buffer and then eluted with 13.5 column volumes of
a linear 0-0.5 M gradient of sodium chloride in QMA
buffer. Fractions containing protein phosphatase activity against C
subunit were pooled and concentrated to about 12-15 mg/ml using
Centriplus and Centricon concentrators (Amicon), and small aliquots
were frozen and stored at -70 °C.
Figure 2:
The S49 cell activity causes a
time-dependent, fluoride-sensitive inactivation of C subunit.
Recombinant C subunit at 0.25 mg/ml was incubated at 37 °C with 4
mg/ml S49 cell extract in the absence () or presence of 100 mM sodium fluoride (
). A parallel sample was incubated under
the same conditions, but without extract (
). At various
intervals, samples were diluted into fluoride-containing buffer and
assayed for protein kinase activity. Data are expressed as the fraction
of the initial activity remaining after incubation; those for samples
incubated without fluoride are means (± S.E.) of results from
three independent experiments. Samples from one of the experiments
without fluoride also were analyzed by SDS-PAGE and densitometry as
described under ``Experimental Procedures,'' and the
proportion of total C subunit in the Thr-197 phosphorylated form was
plotted on the same scale (
, dotted
line).
Figure 9:
Ionic strengths approaching the
physiological range decrease the rate of C subunit dephosphorylation by
the S49 cell phosphatase. C subunits at 6 µg/ml were incubated at
37 °C for up to 4 h with 6 mg/ml S49 cell extract protein in
phosphatase buffer with 0, 50, 100, 150, or 200 mM sodium
chloride. At intervals, samples were diluted with buffer containing
okadaic acid and assayed for protein kinase activity. Inactivation
half-times (t) were determined
graphically.
Figure 3:
The S49 cell activity can remove
phosphothreonine from autophosphorylated C subunit. Recombinant C
subunit was phosphorylated in the presence of
[-
P]ATP and dialyzed to remove the
unreacted ATP as described under ``Experimental Procedures.''
Equal portions of the labeled material were then incubated for 1.5 h at
37 °C without (lane a) or with 7 mg/ml of partially
purified S49 cell phosphatase (lane b) or 4 mg/ml of a
gel-filtered S49 cell extract (lane c). The samples were then
subjected to SDS-PAGE, and the labeled C subunits excised from gels for
hydrolysis and phosphoamino acid analysis. A shows
autoradiographic patterns from analytical SDS-PAGE of the samples
before hydrolysis, and B shows thin layer electrophoresis
patterns of the hydrolyzed samples. Positions of the two forms of C
subunit are indicated in A as for Fig. 1; positions of
internal phosphoserine (Ser-P) and phosphothreonine (Thr-P) standards are shown for B.
Figure 1:
An
activity in S49 cell extracts that increases the SDS-PAGE mobility of C
subunit is sensitive to inhibition by fluoride and okadaic acid.
Purified recombinant C subunit (lane b) was mixed with a
gel-filtered extract of S49 kinase-negative cells (lane a) to
give concentrations of 0.2 mg/ml C subunit and/or 4 mg/ml extract.
Mixtures were incubated for 3 h at 37 °C without (lane d)
or with 10 mM EDTA (lane e), 10 mM EGTA (lane f), 100 mM sodium fluoride (lane g),
100 mM sodium chloride (lane h), or 1 µM okadaic acid (lane i). (Lane c shows the mixture
without incubation.) Portions of the samples containing 2.5 µg
extract protein and/or 125 ng of C subunit were subjected to SDS-PAGE
under conditions that resolve the Thr-197-phosphorylated (C) from the nonphosphorylated form (C
) of C subunit(2) , and the C
subunit species were visualized by silver
staining.
The mobility shifts illustrated in Fig. 1were consistent with dephosphorylation at Thr-197. The ``shifted'' form co-migrated with purified ``fast-form'' C subunit, which is not phosphorylated at Thr-197(2) , and the fast-form preparation was unaffected by incubation with cell extract (data not shown). Since phosphorylation at Thr-197 has a dramatic effect on C subunit activity, we next tested whether or not the putative dephosphorylation was accompanied by a decrease in enzymatic activity. Fig. 2shows that incubation with cell extract led to a time-dependent inactivation of C subunit. SDS-PAGE analysis showed that inactivation was paralleled by the mobility shift illustrated in Fig. 1. Sodium fluoride inhibited the extract-dependent inactivation, but there was a slow extract-independent inactivation that was not affected significantly by the inhibitor. For these and other experiments described in this report, we used extracts from a kinase-negative mutant of S49 cells to avoid the complication of C subunit endogenous to the cell extract. We established in early experiments, however, that wild-type S49 cells contain the same phosphatase activity at about the same level (data not shown).
The experiment of Fig. 3demonstrated that the S49
cell activity indeed removed phosphate from Thr-197 of C subunit.
Incubation of fast form C subunit with
[-
P]ATP in the presence of trace amounts of
``slow form'' C subunit results in phosphorylation of the C
subunit on Thr-197 and several serine residues (2) . Fig. 3A shows gel patterns from such a preparation
incubated without or with cell extract or a partially purified
phosphatase fraction from S49 cells (below). The radiolabeled C subunit
was mostly the slow form species, consistent with efficient
phosphorylation at Thr-197. Treatment with either of the phosphatase
preparations reduced the amount of label in C subunit and shifted the
labeled protein to the position of fast form C subunit. Fig. 3B shows that the untreated, labeled C subunit had
radioactivity in both phosphoserine and phosphothreonine but that the C
subunit treated with cell extract or phosphatase had radioactivity only
in phosphoserine.
Figure 4: Anion-exchange chromatography of S49 cell extracts shows that the C subunit phosphatase activity co-elutes with a portion of the phosphatase active on glycogen phosphorylase. The soluble proteins from an extract of kinase-negative S49 cells were fractionated on a column of Accell-Plus QMA as described under ``Experimental Procedures.'' Fractions were assayed for absorbance at 280 nm (thick solid line), sodium chloride concentration (by conductivity thin straightline), and protein phosphatase activity on glycogen phosphorylase a (dotted line) or C subunit (dashed line). To enhance sensitivity of the gel-shift assay for C subunit, a preparation of C subunit was used that had increased susceptibility to phosphatase (``Experimental Procedures''). For purposes of scaling, the phosphatase data were normalized to values for peak fractions.
Figure 5:
Okadaic acid sensitivities of the protein
phosphatase activities of S49 cells are intermediate between those of
the catalytic subunits of PP-1 and PP-2A. An S49 cell extract (),
anion exchange-purified S49 cell protein phosphatase (
), purified
PP-2A catalytic subunit (
), the AC complex of PP-2A (
),
or the recombinant catalytic subunit of PP-1 (
) was incubated
with either glycogen phosphorylase a (A) or recombinant C
subunit (B) for 2.5 h at 37 °C in the absence or presence
of okadaic acid at various concentrations. Phosphatase activity was
monitored for A by loss of phosphorylase activity
(``Experimental Procedures'') and for B by
conversion of C subunit to the faster migrating, Thr-197
dephosphorylated form (Fig. 1). For the reactions of A,
phosphorylase was at 1.5 mg/ml, and phosphatase preparations were at
about 1.3 mg/ml, 0.3 mg/ml, 0.3 µg/ml, 0.5 mg/ml, and 0.4
µg/ml, respectively, for the S49 extract, partially purified S49
phosphatase, PP-2A catalytic subunit, AC complex, and PP-1. For the
reactions of B, C subunit was at 0.2 mg/ml, and phosphatases
were at about 1.3 mg/ml, 2 mg/ml, 4 µg/ml, and 1 mg/ml,
respectively, for the S49 extract, partially purified S49 phosphatase,
PP-2A catalytic subunit, and AC complex. Because PP-1 had virtually no
activity on native C subunit, its okadaic acid sensitivity with the C
subunit substrate was not measured.
Figure 7:
The S49 cell phosphatase has greater
activity against native C subunit relative to its activity on glycogen
phosphorylase than does monomeric PP-2A. Concentration dependences of
the protein phosphatase activities of S49 cell extract (),
partially purified S49 cell protein phosphatase (
), purified PP-2A
catalytic subunit (
), the AC complex of PP-2A (
), and the
recombinant catalytic subunit of PP-1 (
) were determined using
both recombinant C subunit and glycogen phosphorylase substrates. The
data for each preparation were normalized to the activity against
phosphorylase to compare activities against C subunit. The activities
against C subunit were monitored for 1.5 h at 37 °C and scaled to
the maximum dephosphorylation observed under these
conditions.
Fig. 5A shows that, when measured using glycogen phosphorylase as substrate, crude and partially purified S49 cell phosphatase activities had nearly identical sensitivities to okadaic acid that were intermediate between the more sensitive catalytic subunit of PP-2A and the more resistant recombinant catalytic subunit of PP-1. The sensitivity of the S49 cell activities was similar to that observed with a purified AC complex of PP-2A. Fig. 5B shows results from a similar experiment using C subunit as a substrate. Again the okadaic acid sensitivities for crude and partially purified S49 cell activities were similar to each other and of about the same order of magnitude as obtained using phosphorylase as a substrate. The purified catalytic subunit and AC complex of PP-2A gave somewhat steeper inhibition curves, and the activity of the PP-2A catalytic subunit on C subunit was apparently more resistant to okadaic acid than that on phosphorylase. This difference is probably an artifact attributable to higher concentrations of the purified subunit required to achieve efficient C subunit dephosphorylation (see Fig. 7, below). Using phosphorylase as substrate, the recombinant PP-1 was clearly sensitive to inhibitor-2, but neither purified PP-2A nor the S49 cell activity showed any sensitivity to this inhibitor (data not shown).
The
okadaic acid sensitivity of the reaction of PP-1 on C subunit could not
be tested in the experiment of Fig. 5because, as shown in Fig. 6, lanes c-e, recombinant PP-1 had no
detectable activity on native C subunit. In the presence of 3 M urea, however, PP-1 was able to remove the phosphate from Thr-197
of C subunit (Fig. 6, lanes f-h). Neither potato
acid phosphatase nor calf intestinal alkaline phosphatase were able to
dephosphorylate C subunit in the presence or absence of urea. ()
Figure 6: PP-1 can dephosphorylate Thr-197 of C subunit in the presence of urea, but not in its absence. C subunit at 0.25 mg/ml (lane a) was incubated for 3 h at 37 °C with PP-1 in the absence (lanes c-e) or presence of 3 M urea (lanes f-h) and then analyzed by SDS-PAGE and silver-staining as for Fig. 1. PP-1 concentrations were 6.3 (lanes c and f), 13 (lanes d and g), or 25 µg/ml (lanes e and h). PP-1 alone was diluted to give an amount identical to that from the 25 µg/ml samples (lane b).
Fig. 7compares the activities of PP-1, PP-2A, and the crude and partially purified S49 cell preparations on C subunit. The phosphatase concentrations were all expressed in terms of activity against glycogen phosphorylase. As noted above, PP-1 had no detectable activity on native C subunit. When normalized in this way, the S49 cell preparations were about 10 times more effective than the catalytic subunit of PP-2A as C subunit phosphatases. The AC complex of PP-2A appeared to be somewhat more effective on C subunit than the free catalytic subunit, but we had insufficient material to extend the curve to the higher concentrations required to achieve substantial C subunit dephosphorylation.
Figure 8:
Urea at low concentrations stimulates the
dephosphorylation of C subunit by S49 cell extracts. For A, C
subunit was incubated for 2 h at 30 °C with 1.6 mg/ml S49 cell
extract protein in phosphatase buffer with 0.15 M sodium
chloride and urea to give the concentrations indicated. C subunit
dephosphorylation was monitored by SDS-PAGE analysis and is expressed
as the percent of C subunit in the dephosphorylated form. For B, reactions were carried out as for A, but using
fixed concentrations of 0 (), 1.5 (
), or 3 M urea
(
) and various concentrations of S49 cell
protein.
Several studies had suggested that the conformation of C subunit is sensitive to ionic strengths around the physiological range(19, 20) , and C subunit stability is enhanced by ionic strengths above 100 mM(21) . The experiment of Fig. 9investigated the effect of salt concentration on C subunit dephosphorylation using the inactivation assay of Fig. 2. The rate of dephosphorylation was slowed progressively by almost 10-fold as the salt concentration was increased from 0 to 200 mM. Salt concentrations in this range had very little effect on the phosphatase activity against glycogen phosphorylase (data not shown).
The studies described to this point used recombinant C subunit that lacked the N-terminal myristoyl group that is present in the enzyme from mammalian tissues. To assess the effect of this group on the phosphatase reaction, the experiment of Fig. 10compared dephosphorylation of nonmyristoylated C subunit with that of C subunit purified from bacteria that were co-expressing the yeast N-myristoyltransferase. The reactions again were monitored by measuring loss of kinase activity. With either 50 or 150 mM sodium chloride, dephosphorylation of the myristoylated C subunit was about 3-times slower than that of the nonmyristoylated subunit. The myristoylated form also appeared to be more resistant to phosphatase-independent inactivation as shown by its complete stability to incubation in the presence of okadaic acid.
Figure 10:
Myristoylated C subunit is more resistant
to dephosphorylation than is the nonmyristoylated enzyme.
Nonmyristoylated (open symbols) or myristoylated C subunits (filled symbols) at about 10 µg/ml were incubated at 37
°C with 6 mg/ml S49 cell extract in phosphatase buffer with 50
(,
) or 150 mM sodium chloride (
,
),
or with 50 mM sodium chloride plus 1 µM okadaic
acid (
,
). At intervals, samples were diluted and assayed
for protein kinase activity as for Fig. 9. Data are expressed as
for Fig. 2.
Our results indicate that the Thr-197 phosphate of native,
recombinant C subunit can be removed by a protein phosphatase activity
found in extracts of S49 mouse lymphoma cells. This dephosphorylation
could be monitored by either a mobility shift in SDS-PAGE or a
reduction in protein kinase activity, and it resulted in loss of
labeled phosphothreonine from C subunit autophosphorylated in the
presence of [-
P]ATP. Initial
characterization suggested that the cellular activity was related to
PP-1 and/or PP-2A. It was sensitive to fluoride ion and okadaic acid,
which inhibit PP-1 and PP-2A, but resistant to the chelators EDTA and
EGTA, which would have inhibited the Ca
-dependent
PP-2B or the Mg
-dependent PP-2C (Fig. 1).
Vanadate ions, which inhibit the dual-specific protein tyrosine
phosphatases implicated in dephosphorylation of the MAP
kinases(22) , had no effect on the cellular phosphatase active
against C subunit. Further analysis using okadaic acid at
concentrations that can distinguish between PP-1 and PP-2A and
inhibitor-2, which is selective for PP-1, suggested that the cellular
activity was either a form of PP-2A or a closely related enzyme (Fig. 5, and data not shown). Consistent with these results, the
catalytic subunit of PP-2A could dephosphorylate native C subunit but
that of PP-1 showed no activity against this protein ( Fig. 6and Fig. 7). Fractionation of S49 cell extracts by anion-exchange
chromatography resolved multiple peaks of protein phosphatase active
against glycogen phosphorylase a, all of which were inhibited by a
concentration of okadaic acid that had no effect on PP-1. These
probably represent different multimeric complexes of
PP-2A(23) . Only the later eluting peaks had significant
activity against C subunit, suggesting that not all complexes of PP-2A
were equally active on C subunit (Fig. 4). The okadaic
acid-sensitivity of the S49 cell activity was more similar to that of
an AC complex of PP-2A than to that of the PP-2A catalytic subunit (Fig. 5). Furthermore, when normalized for activity against
phosphorylase a, the S49 cell phosphatase was about 10-fold more active
against C subunit than was the purified catalytic subunit of PP-2A (Fig. 7). We conclude from these observations that the S49 cell
activity is probably a multimeric complex of PP-2A (AC or
ABC(23) ), although we cannot rule out the possibility that it
is a novel enzyme with properties very similar to PP-2A. The cell
extracts used in these studies were gel filtered to remove an inhibitor
(or inhibitors) of the phosphorylase phosphatase activity. The activity
against C subunit was unaffected by gel filtration (data not shown),
suggesting another distinction between the phosphorylase and C subunit
phosphatase activities.
Treatment of C subunit with urea promoted dephosphorylation by the S49 cell activity (Fig. 8) and purified PP-2A (data not shown) and enabled the protein to be dephosphorylated by PP-1 (Fig. 4). On the other hand, neither potato acid phosphatase nor calf intestine alkaline phosphatase were able to dephosphorylate C subunit at Thr-197 with or without the chaotropic agent. These latter results might account for the widespread belief that the phosphate on Thr-197 was inaccessible for dephosphorylation, since experiments attempting to assess the role of phosphorylation in activity of C subunit used these broad spectrum phosphatases(8, 9) . The reaction was slowed markedly by physiological salt concentrations and by myristoylation ( Fig. 9and Fig. 10), raising doubts as to its physiological relevance. On the other hand, our in vitro reactions were all carried out at subphysiological concentrations of phosphatase, and dephosphorylation rates increased with increasing phosphatase concentrations (Fig. 8B, and data not shown).
The first published crystal structure of C subunit was a
ternary complex with ATP and inhibitor peptide in which the phosphate
on Thr-197 was closely associated with the functional groups of His-87,
Arg-165, Lys-189, and Thr-195(6) . Structures of the apoenzyme
and binary complexes of C subunit with an iodinated inhibitor peptide
now have been reported and differ from the original structure primarily
by rotation of the smaller of the protein's two lobes to give a
more open conformation around the catalytic
cleft(24, 25) . Where His-87 interacts with the
Thr-197 phosphate in the closed ternary complex conformation, it is
nearly 6 Å away in the open conformation. We suspect that this
opening of the cleft and disruption of the interaction between His-87
and the Thr-197 phosphate is responsible for making the phosphate
accessible to removal by PP-2A. The inhibitory effect of elevated salt
on C subunit dephosphorylation (Fig. 9), then, would suggest
that salt concentrations in the physiological range shift the dynamic
equilibrium toward the closed conformation. Treatment of C subunit with
concentrations of urea below 2 M had relatively small effects
on the subsequent activity of C subunit, while increasing
concentrations between 2 and 4 M led progressively to
irreversible inactivation. ()The enhanced dephosphorylation
of C subunit at low concentrations of urea (Fig. 8), therefore,
probably reflected a loosening or opening of the structure around
Thr-197 rather than complete denaturation of the protein. A similar
enhancement without urea was observed in a C subunit preparation that
had been partially inactivated by storage in buffer without phosphate
(see ``Experimental Procedures''). We attempted to assess the
effect of Mg
-ATP or Mg
-ATP
S on
the dephosphorylation reaction, since these compounds favor the closed
conformation of C subunit, but, unfortunately, these compounds were
also strong inhibitors of PP-2A (data not shown).
We conclude that
native C subunit can assume a conformation in which its critical
phosphate on Thr-197 is susceptible to dephosphorylation by PP-2A and
that this susceptibility is reduced by physiological ionic strength and
the presence of an amino-terminal myristoyl group, two factors reported
to stabilize C subunit structure(21, 25) . The
reaction in vitro on native, myristoylated C subunit under
near physiological conditions of temperature, pH, and ionic strength
was sufficiently slow (e.g. t > 2 h in the
experiment of Fig. 10) to question whether there is significant
turnover of the Thr-197 phosphate in vivo. On the other hand,
if dephosphorylation rates are linear with phosphatase concentration
and all of the active phosphatase in our extracts is active in intact
cells, the intracellular t
for turnover of the
Thr-197 phosphate could be as short as about 20 min.