(Received for publication, August 1, 1995)
From the
The type I cGMP-dependent protein kinases (cGK I and
I
) form homodimers (subunit M
76,000),
presumably through conserved, amino-terminal leucine zipper motifs.
Type II cGMP-dependent protein kinase (cGK II) has been reported to be
monomeric (M
86,000), but recent cloning and
sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif
near its amino terminus. In the present study, recombinant mouse brain
cGK II was expressed, purified, and characterized. Sucrose gradient
centrifugation and gel filtration chromatography were used to determine M
values for holoenzymes of cGK I
(168,000)
and cGK II (152,500), which suggest that both are dimers. Native cGK
I
possessed significantly lower K
values for cGMP (8-fold) and
-phenyl-1,N
-etheno-cGMP (300-fold) than did
recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3`,5`-cyclic
monophosphorothioate demonstrated selectivity toward cGK II in assays
of kinase activation or inhibition, respectively. A peptide substrate
derived from histone f
had a 20-fold greater V
/K
ratio for cGK
I
than for cGK II, whereas a peptide based upon a cAMP response
element binding protein phosphorylation site exhibited a greater V
/K
ratio for cGK
II. Finally, gel filtration of extracts of mouse intestine partially
resolved two cGK activities, one of which had properties similar to
those demonstrated by recombinant cGK II. The combined results show
that both cGK I and cGK II form homodimers but possess distinct cyclic
nucleotide and substrate specificities.
Appreciation of cGMP as a distinct intracellular second
messenger was closely followed by an intensive search for effector
proteins in various organisms. Subsequently, a cGMP-dependent protein
kinase (cGK) ()was discovered in arthropods(1) ,
leading to the eventual isolation of cGK from mammalian
tissues(2, 3) . More recently, additional families of
cGMP receptors have been described which include phosphodiesterases (4, 5) and ion channels(6, 7) . The
existence of numerous intracellular cGMP receptors, as well as the
restricted tissue distribution of cGK and the lack of well
characterized physiological substrates, has hindered attempts to
clearly define the physiological roles of cGK(8) . However,
increases in cGMP have been shown to occur in response to physiological
stimuli such as nitric oxide and natriuretic
peptides(9, 10) . In certain tissues and cell types,
elevation of cGMP is associated with a decrease in intracellular
calcium levels, which can cause profound biological effects, including
smooth muscle relaxation (11, 12) and inhibition of
platelet aggregation(13) . Cyclic GMP-regulated processes have
also been implicated in olfaction(7) , pancreatic enzyme and
hormone secretion(14, 15) , intestinal chloride
secretion(16) , and long-term potentiation in hippocampal
neurons(17) .
Investigation of the cellular consequences of
cGK activation is further complicated by the presence of multiple forms
of the enzyme. The type I cGK (cGK I) is predominantly cytosolic and is
present in relatively high concentrations in smooth
muscle(18) , lung(19) , platelets(20) , and
cerebellum(21) . Two isoforms of cGK I, cGK I and cGK
I
, have been purified and their amino acid sequences have been
determined(3, 22) . These isoforms differ only in
their amino termini and both form homodimers composed of
76-kDa
subunits. Analyses of partial cDNA sequences of bovine cGK I
and
I
(23) and the gene encoding human cGK I
(24) provide further evidence that cGK I
and I
arise
from an alternative mRNA splicing event(25) .
The type II
cGK (cGK II) was first identified in rat and pig intestinal microvilli
as an 86-kDa monomer associated with membrane fractions (26) . Molecular cloning of cGK II from a mouse brain (27) and rat intestine (28) cDNA library predicted a
protein with 66 and 45% amino acid identity to the catalytic and cGMP
binding domains of cGK I, respectively. In addition to intestine and
brain, cGK II mRNA was found in mouse lung and kidney(27) ,
suggesting that cGK II may have diverse functions in multiple tissues.
The limited homology between cGK I and II, along with their distinct
mRNA tissue distributions, raised the possibility that these isoforms
have different physical and biochemical properties, which may in turn
result in functional differences.
Although initial studies suggested
that cGK II is monomeric(26) , cGK II possesses an
amino-terminal leucine zipper motif that is also found in the dimeric
cGK I isoforms(8, 28) . Since physical interactions
such as dimerization could influence the properties of
cGK(29) , we sought to determine whether recombinant cGK II is
a dimer using sucrose gradient centrifugation and gel filtration
chromatography. In addition, comparisons of cGMP analog and peptide
substrate specificities of cGK II and cGK I were performed. The
results from these in vitro studies were then used to devise
procedures to differentiate cGK II and cGK I activities from partially
purified extracts of mouse intestine.
For transient transfection, 15-cm plates of COS-1 or HEK-293 cells at 40% confluency were transfected using a calcium phosphate transfection method as described previously(27) . In later experiments, stably expressing HEK-293 cells were clonally isolated after selection with G-418 (750 µg/ml) (Life Technologies, Inc.).
For isolation of cGK II
protein from transfected cells, homogenization buffer (500 µl) (27) containing 1 mM phenylmethylsulfonyl fluoride, 5
µg/ml leupeptin, and 1 µg/ml pepstatin A (Boehringer Mannheim)
(Buffer A) was added to each 15-cm plate of
pCMV.HiscGKII-transfected COS-1 or HEK 293 cells. The cells
were scraped into separate tubes and sonicated for 5 s. After
centrifuging the samples for 10 min, the supernatant was collected and
imidazole (Sigma) was added to a final concentration of 10 mM.
The samples were centrifuged once more and the supernatants containing
His
-cGK II from COS-1 or HEK 293 cells were bound to
nickel-affinity resin (Qiagen), washed with 8 column volumes of Buffer
A containing 10 mM imidazole, and eluted with a continuous
gradient of Buffer A containing 10-250 mM imidazole.
In addition, cGK I and cGK II
were subjected to gel filtration chromatography using a standardized
Sephacryl S-300 column (0.9
55.5 cm) (Pharmacia) equilibrated
in KPEM buffer containing 0.1 M NaCl. Samples (225 µl)
containing cGK I
(3.4 µg) or cGK II (13 µg) and 3 mg of
catalase (Stokes radius = 50 Å) (Sigma) were applied to
the column and fractions (1 ml) were collected. Fractions were assayed
for cGK activity (3) and absorbance at 280 nm. Stokes radii
were determined as described by Wolfe et al.(3) . In
another experiment (not shown), cGK II eluted at the same elution
volume when thyroglobulin was used as an internal standard instead of
catalase. The equation (34) used to calculate the molecular
weights was:
where = viscosity of the medium; N = Avagadro's number; a = Stokes
radius; S = sedimentation coefficient;
= partial
specific volume (0.738 for cGK II and 0.737 for cGK I
);
= density. The equation used to calculate the frictional ratios
was:
where f = frictional coefficient of the sample
and f = frictional coefficient of a sphere
(= 1.0).
In contrast to cGK I, little is known about the biochemical properties and physiological role of cGK II. In intestinal epithelial cells, cGK II may participate in the regulation of chloride absorption and secretion(40) . However, cGK II mRNA is also present in mouse brain, lung and, to a lesser extent, kidney(27) . The primary amino acid sequences of the rat intestine and mouse brain proteins are greater than 99% identical, with the slight difference likely representing species variation(27, 28) . Nonetheless, the physiological role of cGK II in these tissues is likely to differ. In the present study, critical physical and biochemical properties of cGK II and cGK I have been compared in vitro to determine whether differences exist that might suggest a distinct role for cGK II. Results from these experiments were then used to discriminate endogenous cGK activities in mouse tissue extracts.
Figure 1:
Silver-stained SDS-PAGE of
recombinant mouse brain cGK II and bovine lung cGK I. Recombinant
cGK II was purified from HEK 293, COS-1, and Sf9 cells as described
under ``Materials and Methods.'' The cGK II lane shown here
was loaded with 300 ng of cGK II purified from Sf9 cells. The cGK
I
lane was loaded with 300 ng of bovine lung cGK I
purified
as described previously (33) . The gel was stained using the
Bio-Rad Silver Stain Plus Kit. Protein standard molecular masses in kDa
are indicated to the left.
Using histone f as substrate
and a Mg
level of 30 mM, a substrate V
of 1.6 µmol/min
mg was obtained for
cGK II isolated from HEK 293 and Sf9 cells, which is nearly identical
to the value obtained for cGK I
purified from bovine lung (V
= 1.8 µmol/min
mg) (data not
shown). Under similar conditions, de Jonge (26) obtained a V
of 1.5-2.0 µmol/min
mg for cGK
II purified from rat intestinal brush-borders and a V
of 2.5-3.0 µmol/min
mg for bovine lung cGK I.
These results suggest that the recombinant and native cGK II enzymes
possess similar activities. Preparations of cGK II from both HEK 293
and Sf9 cells were used to determine kinetic constants in this study.
Figure 2:
Sucrose density gradient centrifugation of
cGK I and cGK II. A, a mixture containing 3.8 µg of
cGK I
(
) and 140 µg of hemoglobin (
) or B, 10 µg of cGK II (
) and 140 µg of hemoglobin
(
) was applied to a linear sucrose gradient (5-20%) and
centrifuged for 23 h at 37,000 rpm. One-ml fractions were removed
beginning at the bottom of each gradient tube and assayed for cGK
activity and absorbance at 280 nm as described under ``Materials
and Methods.'' In a separate experiment (not shown), phosphorylase b was also included with hemoglobin and cGK I
or cGK II
to facilitate calculation of sedimentation coefficients (Table 1). The results shown are representative of three
experiments.
On a
standardized Sephacryl S-300 gel filtration column, cGK I eluted
virtually identically with the internal standard catalase (Fig. 3A), but recombinant cGK II eluted from the same
column at a significantly higher volume (Fig. 3B). This
is apparently not due to an artifact of the catalase internal standard
since cGK II eluted at a similar position when thyroglobulin was used
as an internal standard (data not shown). Proteins of known Stokes
radii were used to construct a standard curve (not shown), which
indicated that the Stokes radius of cGK II (40 Å) is smaller than
that of cGK I
(50 Å) (Table 1), even though the
subunit M
of cGK II (86,000) is greater than that
of cGK I
(76,000). The differences in behavior of cGK I
and
cGK II by gel filtration is explained by a relatively lower frictional
ratio calculated for cGK II (f/f
=
1.08) compared to cGK I
(f/f
= 1.42). Despite the difference in Stokes radii between
cGK I
and cGK II, the calculated M
of cGK II
(152,500) is similar to the calculated M
of cGK
I
(168,000), which predicts that both isoforms form homodimers.
This dimerization likely occurs via interchain interactions involving
an amino-terminal region which contains a consensus leucine zipper
motif(43) . As both the regulatory and catalytic domains of cGK
II are present on a single subunit, such protein-protein interactions
involving the amino terminus are likely to influence biochemical
properties such as cyclic nucleotide binding and
activation(8, 29) .
Figure 3:
Gel filtration chromatography of cGK
I and cGK II. A, a mixture of cGK I
(3.4 µg)
(
) and catalase (3 mg) (
) or B, cGK II (13
µg) (
) and catalase (3 mg) (
) was chromatographed on a
Sephacryl S-300 gel filtration column. Fractions of 1 ml were collected
and cGK activity and absorbance at 280 nm were measured as described
under ``Materials and Methods.'' The Stokes radii of cGK
I
and cGK II (Table 1) were determined from a standard curve
(not shown) of proteins with known Stokes radii (apoferritin, 59
Å; phosphorylase b, 55 Å; aldolase, 52 Å;
catalase, 50 Å; bovine serum albumin, 47.5
Å).
Figure 4:
[H]cGMP dissociation
behavior of cGK I
and cGK II. A final concentration of 0.03 mg/ml
cGK I
(
) or 0.045 mg/ml cGK II (
) was incubated in a
[
H]cGMP binding mixture with 1.0 µM [
H]cGMP for 1 h at 30 °C to allow
saturation of cGMP binding sites with [
H]cGMP.
After 50-µl aliquots were removed for total binding (B
) determination, the tubes were placed
on ice, a 100-fold excess of unlabeled cGMP was added, and 50-µl
aliquots were taken at different time points for binding (B)
determination (see ``Materials and Methods''). Dissociation t
values obtained from these experiments are
discussed in the text.
Figure 5:
Activation or inhibition of cGK I and
cGK II by cyclic nucleotides and cyclic nucleotide analogs. The
activities of cGK I
(1.3 nM) (open symbols) and
cGK II (5.8 nM) (filled symbols) were measured in the
presence of increasing concentrations of cGMP (
,
) or
1,N
-PET-cGMP (
,
) (A).
Likewise, activation of cGK I
(
) and cGK II (
) by Sp-8-pCPT-cGMPS was examined (B). Activity was
determined as described under ``Materials and Methods'' and
expressed as the percentage of the highest cGK activity obtained.
Inhibition of cGK I
(
) and cGK II (
) by Rp-8-pCPT-cGMPS was also determined (see ``Materials and
Methods'') (C). Activity in these experiments was
expressed as the percentage of cGK activity (1 µM cGMP) in
the absence of inhibitor. The experiments were performed three to seven
times for each cyclic nucleotide. Average K
or IC
values and measurements of error for each
cyclic nucleotide are reported in Table 2.
In this study, the cGMP analog most
selective for cGK I was
-phenyl-1,N
-etheno-cGMP
(1,N
-PET-cGMP), which exhibited a K
value (0.016 µM) 300-fold lower for cGK I
than
for cGK II (K
= 4.7 µM) (Fig. 5A, Table 2). Previous studies have shown
that 1,N
-PET-cGMP is also a potent activator of
cGK I
(K
= 20
nM)(46) , suggesting that this cGMP analog is
generally selective for the cGK I isoforms versus cGK II.
Other cGMP analogs that preferentially activated cGK I
over cGK II
include 8-iodo-1,N
-PET-cGMP (200-fold) and
8-(2,4-dihydroxyphenylthio)-cGMP (19-fold) (data not shown). However,
these analogs are substantially less effective activators of cGK I
than cGK I
(46) and would be less likely to selectively
distinguish between cGK II and cGK I
.
Only one cyclic
nucleotide analog examined, the Sp-isomer of
8-(4-chlorophenylthio)-guanosine-3`,5`-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS), yielded a lower (5-fold) K value for cGK II than for cGK I
(Fig. 5B, Table 2). The corresponding Rp-isomer of 8-pCPT-cGMPS
was tested for its ability to inhibit activation of cGK II and cGK
I
by cGMP. The utility of this analog was demonstrated in a
previous report which showed that Rp-8-pCPT-cGMPS could
selectively inhibit cGK activity in intact human
platelets(47) . In our study, the IC
value of Rp-8-pCPT-cGMPS for cGK II was 114-fold lower than that for
cGK I
in the presence of 1 µM cGMP (Fig. 5C, Table 2). In the presence of 10
µM cGMP, the IC
values of Rp-8-pCPT-cGMPS for cGK I
and cGK II increased 3- and
11-fold, respectively (data not shown). Furthermore, millimolar
concentrations of Rp-8-pCPT-cGMPS fully activated both enzymes
(data not shown), a phenomenon that has been observed previously for
cGK II(48) . Since substitutions at the 8-position of the
guanine ring are poorly tolerated by cGK I
(46) , these
results suggest that the Sp- or Rp-isomer of
8-pCPT-cGMPS may be a selective activator or inhibitor, respectively,
of cGK II compared to the cGK I isoforms.
Even though the cyclic
nucleotide binding sites of the cGK I isoforms are relatively specific
for cGMP, cAMP has been shown to cross-activate cGK in intact pig
coronary arteries(49) . Therefore, the cAMP activation
constants of cGK II and cGK I were compared to determine whether
cGK II might also be a candidate for cross-activation in vivo.
The K
value for cGK I
was
6.4-fold lower than that for cGK II (Table 2), suggesting that
cyclic nucleotide cross-activation of cGK II would require
significantly higher cAMP levels in vivo.
Figure 6:
Phosphorylation of peptide substrates by
cGK I and cGK II. The activities of cGK I
(1.3 nM) (open symbols) and cGK II (5.8 nM) (filled
symbols) were measured in the presence of increasing
concentrations of the synthetic peptide substrates H2Btide (
,
) and CREBtide (
,
) (A). Additional assays
were performed to examine the kinetics of phosphorylation of
IP
Rtide (
), Kemptide (
) and BPDEtide (
) by
cGK I
(B) or cGK II (C). Activity was determined
as described under ``Materials and Methods'' and expressed as
the percentage of the highest cGK activity obtained. The experiments
were performed three to six times for each peptide substrate. Average K
and V
values and
measurements of error for each peptide substrate are reported in Table 3.
A second protein that has been investigated as a
potential substrate for cGK is the cAMP response element binding
protein (CREB)(39) . CREBtide (KRREILSRRPSYR) corresponds to an
amino acid sequence in CREB that contains a site rapidly phosphorylated
by cAK. In contrast, cGK I phosphorylated CREBtide at a much slower
rate than did cAK, although they possessed similar K values(39) . In our study, cGK II had a 2.3-fold lower K
value for CREBtide than did cGK I
, while
the corresponding V
values for both enzymes were
identical (Fig. 6A, Table 3). However, with a cGK
II/cGK I
specificity index of 2.3, CREBtide was the most selective
substrate for cGK II tested and therefore a substrate of choice for
detecting cGK II activity.
Three additional synthetic peptide
substrates, IPRtide, BPDEtide, and Kemptide, were also
analyzed for their relative selectivities for cGK I
or cGK II (Fig. 6, B and C, Table 3).
IP
Rtide (GRRESLTSFG) is derived from a sequence in the
inositol 1,4,5-trisphosphate receptor (IP
R) which is
phosphorylated on the same serine residue in vitro by both cGK
I and cAK(51) . It has been suggested that phosphorylation of
IP
R may mediate some of the effects on intracellular
calcium observed with cGK stimulation(51) . Comparison of
IP
Rtide as a substrate for cGK I
or cGK II revealed a
4.8-fold lower K
value and a 4.4-fold lower V
value for cGK II, suggesting that
IP
Rtide is an equivalent substrate for both enzymes.
BPDEtide (RKISASEFDRPLR) is derived from the sequence of the bovine
lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) that was
determined to be selective for cGK as compared to cAK(52) .
Kemptide (LRRASLG) (53) is derived from pyruvate kinase and is
commonly employed for cAK measurement, although it can also be
phosphorylated by cGK I. A 1.9-fold lower K
value
for BPDEtide was obtained with cGK II compared to cGK I
, whereas
the K
value for Kemptide was not significantly
different between the cGK isoforms. However, the lower cGK II V
values found using these substrates results in
cGK II/cGK I
specificity indices that favor cGK I
. Overall,
the rank order of selectivity for cGK II using these substrates was
CREBtide > IP
Rtide > BPDEtide > Kemptide H2Btide.
Therefore, assays of cGK activity using impure enzyme preparations and
H2Btide as the phosphoacceptor peptide may fail to detect cGK II
activity. Results from these experiments suggest that, unlike the cGK I
isoforms, cGK I
and cGK II interact with substrates differently,
which is likely due to amino acid differences within the catalytic
domains of cGK I
and cGK II.
Kinase assays were conducted in the presence of
protein kinase inhibitor peptide and H2Btide substrate using the mouse
lung and intestine fractions obtained from the gel filtration column (Fig. 7A). Assay of the lung fractions revealed a
single peak of kinase activity at an elution volume consistent with
purified cGK I (see Fig. 3A). Assay of the
intestine fractions, on the other hand, revealed a single peak of
kinase activity that had a prominent shoulder of activity on the
leading edge. The elution position of the peak kinase activity from
intestine corresponds to the elution position of purified recombinant
cGK II (Fig. 3B), whereas the shoulder arose at a
position comparable to that of cGK I
. Autophosphorylation
experiments performed on the intestine fractions confirmed the
existence of a predominant 76-kDa species (which corresponds with the
SDS-PAGE mobility of cGK I
) in the early shoulder fractions (data
not shown).
Figure 7:
Apparent separation and identification of
cGK I and cGK II enzyme activities from extracts of mouse
intestine. A, extracts containing solubilized membrane
proteins from mouse whole intestine (
) or lung (
) were
chromatographed on the same Sephacryl S-300 gel filtration column used
for Fig. 3. The gel filtration chromatography was repeated using
a second, independent sample of mouse intestine (not shown). Fractions
(1.2 ml) collected from the column were assayed at least twice for
kinase activity using the heptapeptide substrate H2Btide in the
presence of the protein kinase inhibitor peptide (see ``Materials
and Methods''). Activity for each fraction was expressed as the
percentage of maximal kinase activity obtained. The hatched bar spans the elution volume where the majority of the purified cGK
I
activity was found in Fig. 3. The open bar spans
the elution volume where the majority of the purified recombinant cGK
II activity was found in Fig. 3. The asterisk and star demarcate intestine fractions 18 and 21, respectively,
which were further assayed in B. B, mouse intestine
fractions 18 (
) and 21 (
) (see A), which correspond
to the elution profiles of purified cGK I
and purified recombinant
cGK II, respectively, were assayed in the presence of increasing
concentrations of 1,N
-PET-cGMP as described under
``Materials and Methods.'' This experiment was repeated with
similar results. C, the same fractions from the intestinal
extract obtained in A were reassayed using either H2Btide
(
) or CREBtide (
) as phosphoacceptors (see
``Materials and Methods''). This experiment was performed
three times with similar results. Note that again, activity is
expressed as a percentage of maximal kinase activity rather than as
absolute kinase activity in order to better discriminate the elution
profile obtained with H2Btide.
To further establish the identity of the kinases
responsible for the intestine peak and shoulder activities, samples
from a shoulder fraction (fraction 18) and the peak fraction (fraction
21) were assayed separately in the presence of increasing
concentrations of 1,N-PET-cGMP (Fig. 7B). Fraction 18 exhibited a K
value of 0.01 µM, similar to the K
value of purified cGK I
(0.016 µM, Table 2), whereas fraction 21 displayed a K
value of 1.0 µM, which approaches the value obtained
with purified recombinant cGK II (4.7 µM, Table 2).
Last, the intestine column fractions used in Fig. 7A were assayed again using either 100 µM H2Btide or 100
µM CREBtide as substrate. According to results shown in Fig. 6A and listed in Table 3, CREBtide should be
a significantly better substrate than H2Btide for cGK II at this
concentration. The peak cGK activity was 9.6-fold higher in the
presence of CREBtide than H2Btide, whereas the shoulder activity was
essentially eliminated (Fig. 7C). It should be noted
that Fig. 7C is plotted as a percentage of control
kinase activity in order to display the results obtained using both
H2Btide and CREBtide. Data from these studies indicate that cGK I
and cGK II activity can be distinguished using selected cyclic
nucleotide analogs and substrates. Together, these results suggest that
endogenous dimeric cGK II and cGK I are responsible for the observed
intestine peak and shoulder activities, respectively.
In an earlier study, cGK II isolated from intestine appeared to be particulate and monomeric(26) . However, cloning of the cGK II cDNA failed to reveal divergent regions containing significant hydrophobicity to account for the observed membrane association(28) . In addition, we found cGK II expressed in COS-1, HEK 293, and Sf9 cells to be predominantly soluble. Nonetheless, Jarchau et al.(28) reported that recombinant cGK II expressed in HEK 293 cells partitioned to the particulate fraction. The discrepancy in cGK II subcellular localization may be explained by differences in expression or extraction conditions. Also, the possibility exists that cGK II associates with membranes by interacting with specific anchoring protein(s) which may not be present in some cell lines(27) .
Previous reports have shown that cGK I
and cGK I
, which differ only in their amino termini, display
distinct cyclic nucleotide analog specificities, K
values and Hill coefficients,
which may in turn produce physiological
consequences(3, 8) . In addition, monomerization of
cGK I
by proteolysis of the dimerization domain resulted in a
cGMP-dependent enzyme with an increased K
and altered cGMP dissociation
rates(29) . Therefore, conformational changes that occur upon
dimerization are likely to influence cGK I
activity(8, 54) . This consideration of the possible
consequences of protein-protein binding, as well as the presence of a
conserved leucine zipper motif in cGK II, led us to investigate whether
cGK II dimerizes prior to embarking on kinetic studies of the enzyme.
It should be noted that the Stokes radius determined with the
recombinant brain cGK II is significantly different from that reported
previously for intestinal cGK II(26) . It is possible that high
expression of recombinant cGK II results in artifactual dimerization.
However, both recombinant and native intestinal cGK II exhibited
identical gel filtration properties in this study. An even greater
discrepancy is noted for the sedimentation coefficient of cGK II, which
was reported earlier to be 5.1 S (26) compared with our value
of 8.8 S. Values for cGK I
of 6.9 S (26) and 7.8 S (this
study) are in better agreement. The finding of a lower frictional ratio
for cGK II than for cGK I
is indicative of a more globular form
for cGK II. Our conclusion that cGK II is dimeric has direct bearing on
the interpretation of subsequent results described in this study. Wolfe et al.(45) found that occupation of only one cyclic
nucleotide binding site by cGMP per cGK I dimer failed to significantly
activate the enzyme, whereas binding of two cGMP molecules to identical
sites in each polypeptide resulted in 50% activation. Therefore, it is
clear that important interchain effects can occur in dimeric cGK
proteins.
Since the cGK I isoforms and cGK II possess relatively
similar K values, more selective
cGMP analogs are needed to facilitate in vivo studies of
isoform function. Our results demonstrate that
1,N
-PET-cGMP is strongly selective for cGK I,
whereas Sp-8-pCPT-cGMPS preferentially activates cGK II. Based
on these data, 1,N
-PET-cGMP is likely to
discriminate between cGK I
or I
and cGK II in crude cell or
tissue extracts and in intact tissues. Conveniently, both analogs are
membrane permeant and Sp-8-pCPT-cGMPS is known to be resistant
to mammalian cyclic nucleotide phosphodiesterases, although it is also
a potent activator of cAMP-dependent protein
kinase(55, 56) .
Cyclic GMP may not be the only
cyclic nucleotide that activates cGK enzymes in
vivo(49) . Even so, our data indicate that cAMP is a
significantly less potent activator of cGK II compared to cGK I,
implying that cGK II would require much higher cAMP levels for
cross-activation. Whether such cAMP concentrations exist transiently in
certain cellular compartments has not been determined. However, since
autophosphorylation of cGK I can increase cAMP affinity 6- to 10-fold (57, 58) , more thorough studies of the effects of cGK
II autophosphorylation are required to determine the likelihood of
cross-activation.
Results from the substrate phosphorylation
experiments indicate that CREBtide is the most selective substrate for
detecting cGK II activity relative to cGK I. Conversely, H2Btide
is by far the least selective substrate of cGK II. From these studies,
it is possible to make inferences about the preferred substrate
phosphorylation sequence of cGK II. Previous reports have established
the importance of a series of basic residues located amino-terminal to
the phosphorylation (p) site in cGK I substrates(50) . In
addition, a basic residue located at the substrate p+1 position (50) and a phenylalanine at the p+4 position appear to
provide selectivity for cGK I relative to cAMP-dependent protein
kinase(59) . From our studies, cGK II seems to favor a typical
cAMP-dependent protein kinase consensus phosphorylation sequence
(Arg-Arg-X-Ser/Thr). Alternatively, our results could be
explained if a basic residue positioned at the p+1 site acts as a
negative determinant for cGK II phosphorylation.
The preceding
studies provide evidence that cGK I and cGK II are dimeric and
distinct in both their regulation by cyclic nucleotides and their
recognition of substrates. Using the criteria determined herein,
extracts of whole mouse intestine were shown to contain two separable
cGK activities. The fractions containing these cGK activities have
enzymatic properties consistent with those of cGK I and cGK II,
respectively. These kinase activities were not inhibited by the
cAMP-dependent protein kinase-specific protein kinase inhibitor peptide
or protein, and they eluted from a standardized gel filtration column
at positions corresponding to purified dimeric cGK I
and cGK II.
Identification of the cGK I and cGK II activities was accomplished
in part through assays of cGMP-dependent phosphorylation using either
100 µM H2Btide or 100 µM CREBtide. At this
concentration, cGK I should maximally phosphorylate both substrates,
although the lower V value of cGK I
for
CREBtide resulted in diminished overall activity. Conversely, cGK II
phosphorylated 100 µM CREBtide to a much greater extent
than 100 µM H2Btide, as predicted from the previous
experiments (Fig. 6A). This relative selectivity may be
further enhanced by using the cGMP analogs that preferentially activate
either cGK I (1,N
-PET-cGMP) or cGK II (Sp-8-pCPT-cGMPS), or possibly by using Rp-8-pCPT-cGMPS, which is a more selective inhibitor of cGK
II. These combinations of cyclic nucleotide analog and peptide
substrate should be useful in the characterization of cGK activities in
other tissues, including lung and brain. Indeed, a recent study
demonstrated the presence of over 40 selective substrates of cGK in rat
brain extracts(60) . Since rat brain contains both cGK I and
cGK II mRNA(61) , it would be of interest to further
characterize such substrates in terms of their isoform selectivity
using the cyclic nucleotide analogs described herein. Ultimately, these
tools may prove helpful in the investigation of the in vivo functions of the cGK isoforms as well.