From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Binding of cyclic nucleotide to or
autophosphorylation of cGMP-dependent protein kinase (PKG)
activates this kinase, but the molecular mechanism of activation for
either process is unknown. Activation of PKG by cGMP binding produces a
conformational change in the enzyme (Chu, D.-M., Corbin, J. D.,
Grimes, K. A., and Francis, S. H. (1997) J. Biol.
Chem. 272, 31922-31928; Zhao, J., Trewhella, J., Corbin, J.,
Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997)
J. Biol. Chem. 272, 39129-31936). In the present
studies, activation of type I PKG by either autophosphorylation or
cGMP-binding alone causes (i) an electronegative charge shift on ion
exchange chromatography, (ii) a similar increase (~3.5 Å) in the
Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of
PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein phosphorylation plays key roles in regulating protein function in myriad biological processes. Activation of the protein kinases that catalyze these protein phosphorylations is certainly one of the major mechanisms by which cellular functions are controlled. A particular protein kinase not only phosphorylates one or more cellular proteins (heterophosphorylation),1 but it commonly phosphorylates itself as well, a process termed autophosphorylation. Protein kinase autophosphorylation is functionally important, since it frequently alters kinase function, e.g. by increasing the catalytic activity, increasing the affinity for allosteric ligand binding, or increasing the kinase binding to cellular proteins such as those containing SH2 domains. Many protein kinases are activated by either allosteric ligand binding or autophosphorylation (1). In some cases, ligand binding stimulates the rates of both autophosphorylation and heterophosphorylation. Furthermore, autophosphorylation of some protein kinases increases both the binding affinity for regulatory ligand(s) and the kinase catalytic activity. Therefore, in these instances ligand binding and autophosphorylation act in concert to produce an enhanced activation. The mechanisms of activation of protein kinases by these two processes are still unknown. Although these processes seem quite different, it seems reasonable that similar molecular perturbations may be involved to produce the final activation state for each process.
Recently, activation of cGMP-dependent protein kinase
(PKG)2 by cGMP binding was
shown to cause a conformational change in the enzyme (2, 3). The
results show that an increased net negative surface charge and
elongation of the enzyme occurs when PKG binds cGMP. These effects are
apparently associated with a conformational change that relieves the
interaction of the autoinhibitory domain with the catalytic site,
thereby activating the protein kinase. Like many other protein kinases,
PKG and type II cAMP-dependent protein kinase (PKA) undergo
autophosphorylation, and this process affects the kinetic properties of
each enzyme (4-18). The type I or type I
PKGs are
autophosphorylated at sites in or near their autoinhibitory domains,
and this modification of the PKGs increases the kinase activity (minus
cyclic nucleotide) (15, 18). However, the mechanism whereby
autophosphorylation activates the cyclic nucleotide-dependent
protein kinases, as well as other protein kinases, is not known (19,
20). Since the autophosphorylation sites in type I PKGs are located in
the autoinhibitory domain (12, 17, 18), it is thought that
autophosphorylation, like cGMP binding, may induce a conformational
change that disrupts the autoinhibition, thus activating these protein
kinases. Whether or not autophosphorylation and allosteric ligand
binding could activate the protein kinases by producing a similar
conformational change has not been studied. Autophosphorylation of the
intracellular tyrosine kinase domain of the epidermal growth factor
receptor causes a 3-5-Å increase in the apparent Stokes radius of
this enzyme (21). Studies of the crystal structure of glycogen
phosphorylase reveals that activation by phosphorylation or by the
ligand activator, adenosine 5'-monophosphate, causes the same overall
conformational change in this protein (22). Although the phosphate is
not introduced by autophosphorylation in the case of phosphorylase, the
same guiding principle will be applied for the present studies,
i.e. that activation of PKG by a phosphorylation event,
i.e. autophosphorylation, or cyclic nucleotide binding
produces a similar conformational change in this enzyme.
To assess possible conformational changes of type I PKG produced by
cGMP binding and/or autophosphorylation, four different techniques have
been used: (i) determining the PKG elution position on ion exchange
columns to detect potential difference in surface charge of the enzyme,
(ii) determining the elution position of the PKG on gel filtration
columns to detect potential difference in mass and shape of the enzyme,
(iii) determining the PKG mobility on native gel electrophoresis to
detect potential differences in surface charge and shape of the enzyme,
and (iv) determining the sensitivity of autophosphorylated PKG to
phosphoprotein phosphatase-1 action. The present studies provide the
first evidence for a similar molecular activation of a protein kinase
by ligand binding and autophosphorylation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of PKG and Protein Kinase Assay--
Bovine aorta
type I PKG was purified to homogeneity as described by Francis
et al. (23). The heterophosphorylation kinase activity of
PKG was determined by the phosphocellulose paper assay using
heptapeptide substrate (RKRSRAE) as described previously (16).
Preparation and [3H]cAMP Binding Assay of Cyclic
Nucleotide-free Regulatory Subunit of Type II PKA--
The dimeric
regulatory subunit of bovine heart type II
PKA (RII
) was purified
according to the method of Corbin et al. (24). The binding
activity of RII
was measured by [3H]cAMP binding assay
as described previously (25). Urea denaturation of RII
to remove
cyclic nucleotides was performed as described by Poteet-Smith et
al. (26). Like native RII
, this urea-treated cAMP-free RII
exhibited two active cAMP-binding sites, inhibited the catalytic
subunit of PKA stoichiometrically, and had a dimeric structure as
verified by determination of the sedimentation coefficient.
Measurement of cAMP and cGMP-- The cyclic nucleotide contents of the purified proteins were determined using the modified version of the cyclic nucleotide assay previously described by Corbin et al. (27) and Chu et al. (2).
Preparation and Purification of Phosphorylated PKG and
RII--
Autophosphorylated PKG was prepared by incubating purified
type I
PKG (38 µg/ml) with 4.8 mM magnesium acetate,
100 µM [
-32P]ATP, and 50 µM cAMP at 30 °C for various times. To assess
32P incorporation, an aliquot (10 µl) of this reaction
mixture was spotted onto phosphocellulose paper, washed with four
changes of 75 mM phosphoric acid, dried, and counted. After
incubation, 10 mM EDTA was added to stop the reaction. To
remove [
-32P]ATP and cAMP, the sample was
chromatographed at 4 °C either on a Sephadex G-25 (superfine) column
(0.9 × 11 cm) equilibrated in 10 mM potassium
phosphate, pH 6.8, 1 mM EDTA, and 0.1 M NaCl or
on a Sephacryl S-200 column (0.9 × 56 cm) equilibrated in 10 mM potassium phosphate, pH 6.8, 1 mM EDTA, 25 mM
-mercaptoethanol (KPEM), and 40 mM NaCl.
Fractions of 0.5 ml were collected. Aliquots (10 µl) of each fraction
were counted in 1 ml of aqueous scintillant to determine the
radiolabeled phosphate content, and the protein content of each
fraction was measured by absorbance at 280 nm. Fractions containing the
peak [32P]PKG were pooled for the experiments. To prepare
autophosphorylated type II regulatory subunit of PKA (RII
), the
protein (10 µM) was incubated with 3.5 mM
magnesium acetate, 35 µM [
-32P]ATP, and
10 µM cAMP at 30 °C for 20 min. The same procedures were performed as described above to remove reaction components and to
determine 32P incorporation and purification of
autophosphorylated RII
. Purified RII
contains a trace
contamination of catalytic subunit of PKA, which is sufficient to
obtain partially autophosphorylated RII
at Ser-95 (6-8). Under the
conditions used, the phosphate incorporation was ~0.75 mol of
32P/mol of RII
subunit.
Ion Exchange Chromatography--
A mixture of purified
unphosphorylated and 32P-labeled autophosphorylated PKG
that had been preincubated in the absence or presence of cGMP was
applied to a DEAE-Sephacel column (0.9 × 10 cm) equilibrated in
KPEM at 4 °C and analyzed as described earlier (2). Aliquots of each
fraction were counted to determine the phosphate content of the kinase.
The unphosphorylated PKG was used as an internal control to normalize
the elution position of 32P-autophosphorylated PKG or
cGMP-bound PKG. For experiments utilizing cGMP-bound PKG, the column
was pre-equilibrated with KPEM containing 100 µM cGMP,
and the KPEM elution buffer also contained 100 µM cGMP to
assure that the cGMP-binding sites of the enzyme remained saturated
with cGMP during chromatography. The cGMP concentration in the KPEM
buffer was determined by measuring absorbance (molar extinction
coefficient for cGMP, 252 = 13,700).
Gel Filtration Chromatography-- Purified unphosphorylated or 32P-labeled autophosphorylated PKG that had been preincubated in the absence or presence of 100 µM cGMP was combined with crystalline catalase (4 mg) in a total volume of 500 µl. The mixture was shaken gently to dissolve the catalase and then loaded onto a Sephacryl S-300 column (0.9 × 168 cm) equilibrated with KPEM buffer containing 0.1 M NaCl at 4 °C. The catalase served as an internal standard for all of the gel chromatographies. Fractions were collected and analyzed as described earlier (2). To determine the phosphate content of the enzyme, the radiolabeled phosphate was measured in aliquots of each fraction by scintillation counting. In experiments in which PKG was presaturated with cGMP, the column was pre-equilibrated with 100 µM cGMP, and the column running buffer also contained 100 µM cGMP. Because cGMP in the column buffer interfered with the absorbance at 280 nm, absorbance at 400 nm was used to determine the elution position of catalase for those experiments involving cGMP-bound PKG. To compare the effect of different wavelength absorbances on the apparent elution position of catalase, absorbances at both 280 and 400 nm were compared for several purified PKG experiments, and the elution position of the catalase peak was the same by either technique.
Determination of Stokes Radius, Sedimentation Coefficient, and Molecular Weight-- Apparent Stokes radii of purified unphosphorylated and autophosphorylated PKG in the presence and absence of cGMP were determined by the method described previously (2, 16). The sedimentation coefficients of the enzymes were determined as described earlier (2). The apparent molecular weights, frictional ratios, and axial ratios were calculated according to the method of Siegel and Monty (28), together with the procedures of Cohn and Edsall (29).
Dephosphorylation of Type I PKG by Phosphoprotein
Phosphatase-1--
Autophosphorylated PKG was produced by incubating
130 µl of 0.1 mg/ml type I
PKG in 10 mM potassium
phosphate, 2 mM EDTA, and 25 mM
-mercaptoethanol in the presence of 40 µM cAMP, 5 mM magnesium acetate, and 100 µM
[32P]ATP at 30 °C for 7 min (partially
autophosphorylated) or 2 h (highly autophosphorylated). Partially
autophosphorylated enzyme contained nearly stoichiometric phosphate at
Ser-63, whereas highly autophosphorylated enzyme contained
approximately stoichiometric amounts of phosphate at Ser-63 and was
nearly saturated at Ser-79. The autophosphorylated PKG was
chromatographed on a Sephacryl S-200 column (0.9 × 48 cm)
equilibrated in 40 mM Tris (pH 7.5), 25 mM
-mercaptoethanol, and 2 mg/ml bovine serum albumin, and the peak
fractions of activity were pooled for use in the phosphoprotein phosphatase reaction. 0.025 unit/ml phosphoprotein phosphatase-1 catalytic subunit (Promega) was preincubated at 4 °C for 15 min in
400 µl of 20 mM Tris (pH 7.5), 2 mg/ml bovine serum
albumin, in the absence or presence of 15 µM cGMP or 50 µM cAMP. 192 µl of a final concentration of 1 µM partially or highly autophosphorylated PKG were added,
and the mixture was incubated at 30 °C for varying amounts of time.
15-µl aliquots of the incubation mixture were removed at certain time
points and placed in a tube containing a final concentration of 1 mg/ml
bovine serum albumin and 10% trichloroacetic acid in a total volume of
540 µl. The tubes were vortexed, incubated at 4 °C for 1 h,
and centrifuged for 10 min at 10,000 × g. 450-µl aliquots
of the supernatant were counted in the scintillation counter to
determine the total amount of free phosphate present. 15-µl aliquots
of the incubation mixture were counted at each time point to determine
the total amount of phosphate present. The amount of free phosphate was
divided by the total amount of phosphate to determine the
percentage of phosphate released.
Native Gel Electrophoresis-- The enzymes were electrophoresed on a 9.5% polyacrylamide gel and 4% stacking gel without sodium dodecyl sulfate at 4 °C using constant current (~10 mA) for 5 h as previously described (2). The proteins were detected by Coomassie Brilliant Blue staining.
Materials-- All materials were obtained as described earlier (2).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic Nucleotide-free and cGMP-bound PKG--
The cAMP and cGMP
contents of the purified type I PKG were measured as described
earlier (2). The cyclic nucleotide occupancy of cGMP-binding sites of
purified PKG used for the following experiments was less than 3%.
Previous studies suggest that the purified PKGs are not significantly
phosphorylated at the autophosphorylation sites that alter catalytic
activity or cyclic nucleotide-binding affinity (18). The purified PKG
also had a very low basal kinase activity ratio (
/+ cGMP ~0.05).
Therefore, the purified type I
PKG was considered to be cyclic
nucleotide-free and not autophosphorylated. To prepare cGMP-bound PKG,
the enzyme was incubated with unlabeled cGMP (~500-fold excess of
cGMP-binding sites) at 4 °C overnight, and equal occupation of the
two intrasubunit cGMP-binding sites was verified as described earlier
(2). For native gel electrophoresis experiments, purified PKG was
incubated with unlabeled cGMP or cGMP analog, 8-Br-PET-cGMP, as
described above to obtain cyclic nucleotide-bound PKG.
Autophosphorylation of PKG--
Autophosphorylated type I PKG
was prepared by incubating purified enzyme in the presence of cAMP,
magnesium acetate, and [
-32P]ATP followed by gel
filtration on a Sephacryl S-200 column to remove cAMP and other
reactants as described under "Experimental Procedures." After
overnight incubation (highly autophosphorylated), the enzyme contained
~2 mol of 32P/mol of subunit with equal distribution of
the phosphate between Ser-63 and Ser-79. The stoichiometry of 2 mol of
phosphate incorporated per mol of subunit indicated that these sites
were largely in the dephosphorylated state in the purified enzyme
before autophosphorylation. The increased phosphate content of the
enzyme increased the basal heterophosphorylation activity in the
absence of cGMP by ~5-fold. When the PKG was incubated for only 7 min
(partially autophosphorylated), the phosphate incorporation was ~0.5
mol of 32P/mol of subunit, and basal heterophosphorylation
activity did not increase. The results confirmed that
autophosphorylation of type I
PKG at Ser-79, which is slowly
autophosphorylated, causes the increase in kinase activity, whereas the
rapid autophosphorylation at Ser-63 does not (18). Since
autophosphorylation of PKG was performed in the presence of cAMP, it
was necessary to verify that cyclic nucleotide was removed by the gel
filtration step. The measured cAMP and cGMP contents of
autophosphorylated enzyme were calculated to be less than 10%
occupancy of cGMP-binding sites of the protein so that the
autophosphorylated PKG I
used for subsequent experiments was
essentially cyclic nucleotide-free unless cGMP was purposely
included.
DEAE-Sephacel Anion Exchange Chromatography of Unphosphorylated,
Autophosphorylated, and cGMP-bound PKG--
When a mixture of
predominantly purified unphosphorylated PKG and a trace amount of
purified 32P-radiolabeled autophosphorylated type I PKG
was chromatographed on a DEAE-Sephacel column as described under
"Experimental Procedures," the peak of autophosphorylated enzyme
activity eluted at higher ionic strength and was partially separated
from the bulk of the unphosphorylated enzyme activity (Fig.
1). Fractions containing the
32P-labeled enzyme showed a higher kinase activity ratio
(
/+ cGMP ~0.4) than did fractions containing unphosphorylatec
enzyme (
/+ cGMP ~ 0.1). The 32P-labeled enzyme
eluting at the highest ionic strength had the highest phosphate content
(1.5 mol/subunit) and also had the highest kinase activity ratio (Fig.
1).
|
|
Sephacryl S-300 Gel Filtration Chromatography of Unphosphorylated,
Autophosphorylated, and cGMP-bound PKG--
Unphosphorylated and
highly autophosphorylated type I PKGs were chromatographed on a
Sephacryl S-300 gel filtration column in the presence of the internal
standard catalase as described under "Experimental Procedures." The
highly autophosphorylated PKG eluted earlier from the column than did
the unphosphorylated form of this enzyme (Fig.
3, A and B). When
the enzyme was preincubated with cGMP and chromatographed in the
presence of buffer containing 100 µM cGMP, the kinase
again eluted earlier than did the control, and there was no significant
difference in elution position of this enzyme compared with the
respective autophosphorylated enzyme (Fig. 3, compare B and
C). There was no additional shift in elution position of the
autophosphorylated PKG when chromatographed in the presence of cGMP
(data not shown). Again, partial autophosphorylation (7-min incubation)
of PKG did not produce a detectable shift in elution position on the
S-300 column (data not shown). The results using gel filtration
indicated that activation of PKG by autophosphorylation and/or cGMP
binding produces a similar shift in elution position of the enzyme. As
was shown for DEAE anion-exchange chromatography (see above), the
combination of autophosphorylation and cGMP binding does not produce an
additive shift. These results are consistent with in vitro
kinase activation studies which indicate that in the presence of
saturating concentration of cyclic nucleotides, autophosphorylation of
PKG does not increase the kinase catalytic activity (18).
|
Physical Parameters of the PKG--
The Stokes radii of different
forms of type I PKG were calculated from the results of gel
filtration as described earlier (2). As can be seen in Table
I, the Stokes radii of autophosphorylated and cGMP-bound PKGs were ~3.5 Å larger than the Stokes radii of the
unphosphorylated enzyme, and there was no significant difference in the
sedimentation coefficients for these autophosphorylated and
unphosphorylated enzymes. The calculated apparent molecular weights
indicated that the shifts in elution position of the PKG were not large
enough to be due to oligomerization of the dimeric enzyme. Furthermore,
the added mass of either the four bound cGMP molecules (367 Da each) or
four phosphate groups (80 Da each) to the dimeric PKG would be
insufficient to produce a size shift of this magnitude. The axial
ratios (Table I) suggested that the autophosphorylated and cGMP-bound
forms are more elongated proteins when compared with the
unphosphorylated or cyclic nucleotide-free enzyme, and this elongation
is reflected in an increase in the apparent Stokes radius. The findings
from both anion exchange and gel filtration chromatography indicated
that activation of type I
PKG by autophosphorylation or cGMP binding
produces a similar apparent conformational change with elongation of
the enzymes and increased net negative surface charge.
|
Native Gel Electrophoresis--
Native gel electrophoresis was
also used to detect differences in molecular parameters of
unphosphorylated, autophosphorylated, and cyclic nucleotide-bound type
I PKG according to the method described by Chu et al.
(2). Both highly autophosphorylated and 8-Br-PET-cGMP-bound PKGs
exhibited the same mobility on native gels, and this mobility was less
than that of the unphosphorylated form of the enzyme (Fig.
4A). PKG has a high affinity
for 8-Br-PET-cGMP, and this analog has been shown to remain bound to
the enzyme during native gel electrophoresis (2). When the enzymes were
presaturated with cGMP (or 8-Br-PET-cGMP) and then electrophoresed in
the presence of 100 µM cGMP, the mobility of the PKGs was
the same irrespective of each treatment (Fig. 4B). This
mobility was also the same as that obtained for either the
autophosphorylated or cGMP analog-bound PKG in Fig. 4A. Both
autophosphorylation and cGMP analog together did not produce an
additive effect on mobility (Fig. 4B). The apparent increase
in the surface electronegativity of autophosphorylated or
cGMP-saturated PKG using DEAE-Sephacel chromatography would be
predicted to increase the mobility of the enzyme toward the anode using
native gel electrophoresis. However, the mobility of the type I
PKG
in the gel is decreased and is consistent with a conformational change
that increases the apparent size of the protein upon activation by
either autophosphorylation or cyclic nucleotide binding. These results
confirm the interpretation of the previous results from gel filtration
analysis.
|
Effect of cAMP on the Regulatory Subunit of PKA--
As described
earlier (2), the cAMP-free RII of PKA was used as a control for each
of the three approaches described above. The cAMP-free,
autophosphorylated RII
and the cAMP-bound RII
were prepared as
described under "Experimental Procedures" and then subjected to
DEAE ion exchange chromatography, gel filtration chromatography, as
well as native gel electrophoresis. Neither autophosphorylation nor
cAMP binding produced a detectable electronegative charge shift on DEAE
chromatography (data not shown). There was also no shift in elution
position of autophosphorylated or cAMP-bound RII
on gel filtration
chromatography, and no mobility shift on native gel electrophoresis.
The results indicated that introduction of extra charge or mass by
addition of the phosphates or four cAMP molecules is not sufficient to
change the surface charge or increase the mass to produce a shift of
the RII
using these procedures. A similar conformational change
caused by autophosphorylation or cGMP binding is a more reasonable
explanation for the findings using PKG.
Effect of Cyclic Nucleotide and Full Autophosphorylation on
Dephosphorylation of PKG--
Type I PKG contains two
autophosphorylation sites (Ser-63 and Ser-79) (17, 18).
Autophosphorylation of Ser-63 occurs first, but significant enzyme
activation occurs only after subsequent autophosphorylation of Ser-79
(18). Dephosphorylation of partially autophosphorylated (Ser-63
only) and highly autophosphorylated (Ser-63 and Ser-79) type I
PKG
by phosphoprotein phosphatase-1 in the presence and absence of cyclic
nucleotide was compared. It can be seen that both cGMP and cAMP sharply
increased the rate of dephosphorylation of partially autophosphorylated
(Ser-63 only) enzyme (Fig.
5A), but neither cGMP nor cAMP
significantly altered dephosphorylation of the highly
autophosphorylated enzyme, i.e. enzyme that was
autophosphorylated at both Ser-63 and Ser-79 (Fig. 5B). The
dephosphorylation of PKG that was autophosphorylated only at Ser-63 was
stimulated 3-5 fold in 0.5-1.5 min. (Fig. 5A). Therefore,
using highly autophosphorylated PKG (Fig. 5B), it is unlikely that the stimulatory effect of cGMP on phospho-serine 63 would
be masked by the absence of an effect of cGMP on dephosphorylation of
phospho-serine 79. When highly autophosphorylated PKG was used as
substrate, the relative rates of dephosphorylation of phospho-serine 63 and phospho-serine 79 by phosphoprotein phosphatase-1 were comparable
as assessed by thin layer chromatography of the phosphopeptides (not
shown). It is of interest that the dephosphorylation rate appears to be
greater when using the partially autophosphorylated PKG as substrate,
but whether this is due to an inhibitory effect of phospho-serine 79 on
the dephosphorylation rate requires further study. The data suggest
that activation of the PKG by ligand binding (cGMP or cAMP) or by
autophosphorylation (both Ser-63 and Ser-79) produces a similar
conformational change. Thus, an effect of either cGMP or cAMP on
dephosphorylation would be expected only when using the enzyme that is
in the inactive conformation (phosphorylated at Ser-63 only). These
results using a predominantly enzymatic approach are consistent with
those using the chromatographic and electrophoretic approaches.
|
Concluding Remarks-- It should be emphasized that the techniques developed here to resolve cGMP-bound and -free PKG, or phosphorylated and unphosphorylated PKG, could be used for crude systems as well as for purified PKG. The approach of measuring changes in the protein phosphatase sensitivity of the PKG that has been activated by different processes is also novel. Therefore, the techniques offer new approaches for studies of interconversion of these forms of PKG in intact tissues treated with various modulators. These techniques may also be useful in studying other proteins (33), including homologous protein kinases that are activated by ligands or autophosphorylation. In the present studies, autophosphorylation of PKG caused an apparent conformational change that is similar to the elongation of PKG that is produced by cGMP binding (2, 3); this has been demonstrated using each of three separation procedures. The finding that cGMP does not enhance the protein phosphoprotein phosphatase-1 sensitivity of the highly autophosphorylated PKG is consistent with this conclusion.
Either autophosphorylation or cyclic nucleotide binding, or a combination of these processes, can activate catalysis in cyclic nucleotide-dependent protein kinases (4-18), and the two processes appear to produce a similar conformational change in the PKG. This induced structural change in the PKG is associated with conversion of the enzyme from a more compact inactive conformation to a more elongated active conformation and may represent the classical interconversion of enzymes between two states, i.e. an inactive T state that has low affinity for substrates and the active R state that has high affinity for substrates (34). Phosphorylase provides one example of the conversion of an enzyme from the T state to the R state, and this is effected by either a phosphorylation event or by ligand binding, i.e. 5'-AMP (35). The active conformation that is produced by either process is essentially the same. It is suggested that either cGMP binding or autophosphorylation produces a similar perturbation to cause activation in each monomer of dimeric PKG. This perturbation within the monomers results in an elongation of the dimeric structure. The results of the present study are consistent with such an interconversion in PKG. Since many protein kinases are activated by both ligands and autophosphorylation, these findings could be relevant to the activation mechanism for some of these enzymes as well. ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Kennard Grimes for assistance in purifying PKG, Dr. Jeffrey A. Smith for helping to autophosphorylate PKG, Alfreda Beasley for assistance in some of the experiments, and Tina Beck for assistance in preparing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK40029 and GM41269 (to J. D. C.) and by the Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China (to D.-M. C.).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.
To whom correspondence should be addressed: Dept. of Molecular
Physiology and Biophysics, Vanderbilt University School of Medicine,
Nashville, TN 37232-0615. Tel.: 615-322-4382; Fax: 615-343-3794.
1 The term heterophosphorylation is derived in part from the Greek heteros (other, different), which is compared with the Greek autos (self). Thus, heterophosphorylation refers to phosphorylation of proteins, peptides, or substances other than the kinase itself or a subunit(s) of the kinase itself. The terms heterophosphorylation and autophosphorylation are related in the same way as are heterophagy and autophagy.
2
The abbreviations used are: PKG,
cGMP-dependent protein kinase; PKA,
cAMP-dependent protein kinase; RII, regulatory subunit of type II
PKA; 8-Br-PET-cGMP,
-phenyl-1,N2
etheno-8-bromo-cGMP.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|