From the
Department of Biochemistry, Erasmus
University Medical Center Rotterdam 3000 DR, The Netherlands, the
Department of Biochemistry and Cell Biology,
Faculty of Veterinary Medicine, P. O. Box 80.176, 3508 TD Utrecht, The
Netherlands, the ||Institut für Pharmakologie
und Toxikologie, Technische Universität, München 80802, Germany, and
the **Department of Biochemistry, Faculty of Medicine
and Health Sciences, Ghent University and Flanders Interuniversity Institute
of Biotechnology, B-9000 Ghent, Belgium
Received for publication, April 9, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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Sequence comparison and biochemical analysis revealed a large degree of similarity in the structural organization of cGKs I and II (13, 9, 10). Both isotypes possess two cGMP-binding domains on one polypeptide chain that is covalently coupled to a catalytic domain. Their N terminus contains an autoinhibitory region and a leucine zipper motif, and the cGK isoforms are devoid of hydrophobic trans-membrane domains and form homo-dimers. Despite these similarities, cGK II was shown to differ from soluble type I cGK in that it behaved as a membrane- and cytoskeleton-associated protein (4). N-terminal myristoylation of cGK II was found to be responsible for the different subcellular localization of the cGK isotypes (11).
Furthermore, both cGK I and I
and cGK II are known to be
autophosphorylated in vitro in response to both cAMP and cGMP
(4,
12,
13). The major
autophosphorylation sites of cGK I were mapped and found to be present in the
N-terminal autoinhibitory domain
(12,
14). The cyclic
nucleotide-stimulated autophosphorylation caused a shift in the affinity of
cGK I
for cAMP (15) and
both a shift in the EC50 for cAMP and cGMP in combination with an
increase in basal activity of cGK I
(13). Although cGK II was
initially discovered as a major (auto)phosphorylated protein in intestinal
brush-border membranes, the location of the autophosphorylation sites and the
consequences of the autophosphorylation for cGK II function are not known.
We here report that cGK II is autophosphorylated in vitro on multiple sites that are located close to the pseudosubstrate region in the autoinhibitory domain and on one site close to the catalytic domain. The relatively slow autophosphorylation of presumably Ser126 in the pseudosubstrate region had a major effect on the activity of cGK II, whereas autophosphorylation of the other sites had only minor effects on the kinetic parameters of cGK II. Autophosphorylation of cGK II may be physiological relevant because it was also observed in intact cells.
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EXPERIMENTAL PROCEDURES |
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Recombinant baculovirus was obtained by transfection of Sf9 cells (Spodoptera frugiperda), which had been propagated as monolayers at 27 °C in TC-100 medium (Biochrom) containing 10% fetal calf serum (Invitrogen), 4 mM L-glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), and 0.1 mg/ml streptomycin (Invitrogen). Transfection with recombinant bacmid DNA was performed by the lipofection method using the CellFectin Reagent (Invitrogen) according to the instructions of the manufacturer. Positive viral clones were identified by their ability to direct the expression of the appropriate protein as revealed by immunoblotting of whole cell extracts of transfected Sf9 cells, which had been harvested 34 days post-transfection. Recombinant virus was amplified without further purification and viral titer estimated by end-point dilution.
For expression of cGK II, Sf9 cells (1.0 x 1010 cells/6.2
liters) maintained as suspension cultures, were infected with the recombinant
baculovirus at a multiplicity of infection of >10. After 67 h, cells were
harvested by centrifugation at 1,000 x g for 10 min, washed
twice with phosphate-buffered saline, resuspended in 50 ml of chilled buffer A
(10 mM imidazole, 100 mM NaCl, 10 mM
2-mercaptoethanol, 2.5 mM benzamidine, 0.25 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, pH 7.5), and stored at
80 °C. For purification of cGK II, cells were lysed by
freeze/thawing and homogenized by a glass-Teflon homogenizer followed by
centrifugation at 25,000 x g for 20 min. The pellet was
resuspended in 50 ml of buffer A and centrifuged at 25,000 x g
for 10 min. The combined supernatants containing the hexahistidine-tagged cGK
II were loaded onto a column (inner diameter = 1 cm, flow rate = 1.0 ml/min)
packed with 7 ml of a nickel-affinity resin (Qiagen), which had been
equilibrated with 10 volumes of buffer A. The column was sequentially washed
with 70 ml of buffer A and 20 ml of buffer B (100 mM imidazole, 100
mM NaCl, 10 mM 2-mercaptoethanol, 2.5 mM
benzamidine, 0.25 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, pH 7.0) prior to elution of bound cGK II with 30 ml of buffer C
(250 mM imidazole, 100 mM NaCl, 10 mM
2-mercaptoethanol, 2.5 mM benzamidine, 0.25 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, pH 7.0). To reduce the
concentration of imidazole, the eluate (2 mg/ml cGK II) was diluted 1:4
with 20 mM Tris/HCl buffer (pH 7.4) containing 100 mM
NaCl, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.1
mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin and
reconcentrated using Macrosep-10 concentrators (Pall Filtron). This procedure
was repeated twice prior to concentration of cGK II and addition of 50% (v/v)
glycerol. Protein preparations were stored at 20 °C.
Construction and Expression of Mutants of cGK IIThe triple mutation in rat cGK II of Ser110, Ser114, and Ser445 to Ala (3SA) was made directly from the pRc/CMV-cGK II plasmid (10) using the TransformerTM site-directed mutagenesis kit (Clontech), with two mutagenic primers, one containing the Ser110 and Ser114 mutations and another containing the Ser445 mutation, and a selection primer targeted to the unique XbaI site (11). The single Ser to Ala and Ser to Glu mutations were made directly from the pRc/CMV-cGK II plasmid, and the multiple Ser to Ala mutations were made from the 3SA mutant with the QuikChangeTM Site-directed mutagenesis kit (Stratagene). All mutations were confirmed by dideoxy sequencing of the mutant plasmids using ABI Prism® BigDyeTM Terminator Cycle Sequencing kit and an ABI Prism® 377 DNA sequencer (Applied Biosystems).
cDNA encoding rat cGK II or various mutants thereof in a pRc/CMV expression vector was transfected into COS-1 cells 1 day after subculturing at 8090% confluency, by means of a 20-min incubation at 25 °C with 0.5 mg/ml DEAE-dextran in phosphate-buffered saline containing 1 µg of vector DNA per 106 cells. Cells were harvested 2 days after transfection, suspended in buffer A (150 mM NaCl, 10 mM NaPO4, pH 7.4, 1 mM EDTA, 100 µg/ml trypsin inhibitor, and 20 µg/ml leupeptin), frozen in liquid N2, and stored at 80 °C. After thawing, cells were homogenized by brief sonication (three bursts of 3 s, peak-to-peak amplitude 1520 µm), and membranes were isolated by a 10-min centrifugation at 20,000 x g, and resuspended in buffer A to a concentration of 0.41 mg of protein/ml. Subsequently, cGK II was solubilized by addition of 1% Triton X-100 and 0.5 M NaCl, and insoluble material was removed by centrifugation.
Determination of AutophosphorylationTo achieve
autophosphorylation, purified His-cGK II (58 µg/ml) was incubated
for various time periods at 30 °C in 25 mM Tris/HCl, pH 7.4, 10
mM MgCl2, 90 mM NaCl, 5 mM
dithiothreitol, 0.1% Triton X-100, 0.1% bovine serum albumin, and 1
mM [-32P]ATP (0.3 µCi/nmol). Transiently
expressed rat cGK II and cGK II mutant proteins solubilized from COS cell
membranes (0.41 mg of protein/ml) were incubated for 2 min at 30 °C
in 25 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 5
mM
-mercaptoethanol, 0.1 mM
3-isobutyl-1-methylxanthine, 25 mM sodium
-glycerophosphate,
1% Triton X-100, 0.15 M NaCl, and 10 µM
[
-32P]ATP (3 µCi/nmol). Reactions were stopped by
addition of SDS-sample buffer and boiling (3 min). Subsequently, 10-µl
aliquots were separated on 7.5% SDS-PAGE, and, after drying of the gels, the
amount of label incorporated in cGK II was quantitated with the Molecular
Imaging System GS-363 (Bio-Rad).
Identification of the Autophosphorylation
SitesAffinity-purified recombinant His-cGK II (10 µg) was
incubated for 30 min at 30 °C in 25 mM Tris/HCl, pH 7.4, 10
mM MgCl2, 5 mM -mercaptoethanol, 0.25
mM [
-32P]ATP (2.5 µCi/nmol), and 10
µM cGMP. cGK II was denatured and precipitated by boiling for 5
min. Non-incorporated label was removed by washing with 25 mM
Tris/HCl, pH 7.4. The protein pellet was resuspended in 100 µl of 0.5%
NH4HCO3 buffer, and 1 µg of trypsin was added (1
µl of a 1 mg/ml solution). The digestion was continued at 37 °C for 8 h
and terminated by the addition of trifluoroacetic acid (final concentration
0.5%). The resulting peptides were loaded onto a C18 reversed phase-HPLC
column (Vydac, Separation Group, column dimension = 2.1 x 25 mm) and
eluted with a linear gradient of 1% of solvent B (70% acetonitrile in 0.09%
trifluoroacetic acid) per minute. Solvent A consisted of 0.1% trifluoroacetic
acid. The gradient was controlled with an Applied Biosystems A140 gradient
programmer delivering a flow rate of 80 µl/min. Peptides eluting from the
column were recorded by UV adsorption and measured at 214 nm. All UV-absorbing
peaks were manually collected in Eppendorf tubes, and the 32P
radioactivity was counted. The sum of the radioactivity present in all
positive fractions was compared with the total radioactivity present in the
mixture before application on the column. One-fifth of the eluting solvent was
directly deviated into the ion source of the quadrupole time-of-flight mass
spectrometer (Micromass, UK) for determinations of peptide masses. Fractions
containing 32P label were analyzed in detail for the presence of
peptides with 80 or 98 additional atomic mass units. Phosphorylated peptides
with double or triple charges were selected and subjected to fragmentation by
collision with argon gas, using a collision energy of 2035 eV. The
fragmentation spectra were analyzed with MassLynx Micromass software. Peptides
of interest were subjected to automated Edman degradation, using a 477A
Applied Biosystems Inc. sequencer operated according to the manufacturer's
instructions.
Two-dimensional ElectrophoresisPurified recombinant mouse
His-cGK II was autophosphorylated in the presence of 1 mM
[-32P]ATP for 30 min as described above in the presence or
absence of 10 µM cGMP. COS-1 cells transiently expressing cGK II
were grown in 12-well plates and subsequently incubated at 37 °C in
culture medium with or without 50 µM 8-pCPT-cGMP (Biolog,
Bremen, Germany) for 60 min and subsequently washed three times with ice-cold
phosphate-buffered saline. Stripped mouse jejunum mounted in an Ussing chamber
(16) was incubated for 15 min
with or without 0.3 µM heat-stable enterotoxin STa, added to the
mucosal compartment. After the incubations, the samples were suspended in 2%
SDS, 5%
-mercaptoethanol, and 10% glycerol and boiled for 3 min. After
addition of urea (9.5 M), Nonidet P-40 (3%), and the ampholytes
Bio-lyte, pH 68 and pH 310, in 4:1 ratio (2%, Bio-Rad), samples
were subjected to two-dimensional gel electrophoresis using a Mini-PROTEAN II
two-dimensional cell system (Bio-Rad) according to the instructions of the
manufacturer. After blotting the separated proteins, 32P label was
visualized using direct autoradiography, and cGK II was detected with a
specific antibody by the enhanced chemiluminescence method as described
previously (16).
Determination of Protein Kinase ActivityTo assess the
effect of autophosphorylation on the activity of cGK II, purified recombinant
His-cGK II (0.1 mg/ml) was incubated for various time periods at 30 °C in
25 mM Tris/HCl, pH 7.4, 50 mM NaCl, 12 mM
MgCl2, 4 mM dithiothreitol, 0.1% Triton X-100, 0.1%
bovine serum albumin, and 10 µM cGMP with 1 mM ATP
(autophosphorylated) or 1 mM AMP-PNP (control). Reactions were
stopped, and cyclic nucleotides were removed by a 250-fold dilution in the
same buffer without ATP or cyclic nucleotides (resulting in a final 1000-fold
dilution in the assay). Protein kinase activity was determined by incubation
of the samples (4 ng of control or autophosphorylated His-cGK II or 410
µg of COS membrane protein in case of transiently expressed rat cGK II or
mutants thereof) at 30 °C for 4 min in 40 µlof20mM Tris/HCl,
pH 7.4, 0.15 M NaCl, 10 mM MgCl2, 5
mM -mercaptoethanol, 0.1 mM
3-isobutyl-1-methylxanthine, 25 mM sodium
-glycerophosphate,
0.25% Triton X-100, 200 nM protein kinase A inhibitor, 0.1 mg/ml of
a cGK substrate peptide 2A3 (RRKVSKQE
(17)), 0.3 mM
[
-32P]ATP (0.10.2 µCi/nmol), and various
concentrations of cGMP or cAMP as indicated. The reaction was started by
addition of 10-µl aliquots of the cGK II preparations to 30 µl of
prewarmed incubation buffer and quenched by addition of 10 µl of 0.5
M EDTA. The amount of label incorporated into 2A3 was quantified as
described previously (17).
Effect of Prolonged Activation of cGK II on Short-circuit Current in Rat Intestinal MucosaA 1-cm-long segment of rat jejunum was removed under light diethylether anesthesia. The muscle layers were stripped off by blunt dissection, and the mucosa was mounted in an Ussing chamber (0.3-cm2 area exposed) for measurements of short-circuit current (Isc) across the tissue as described (16). To obtain a maximal cGK II-mediated Isc response (16), 50 µM 8-pCPT-cGMP was added to the serosal site. Either 30 min after addition of the cGMP analog, when a maximal Isc was reached, or 3 h after the addition, allowing a more extensive autophosphorylation of cGK II, the 8-pCPTcGMP was removed by three consecutive changes of the serosal bathing solution. After removal of the cGMP analogue the decay of the ISC back to basal levels was monitored.
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RESULTS |
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Mapping of Autophosphorylation Sites in cGK IITo identify
the major autophosphorylation sites, purified mouse recombinant cGK II was
incubated for 30 min with cGMP and -[32P]ATP, denatured by
boiling for 5 min, and cleaved with trypsin. Peptides were separated by
reversed-phase HPLC and collected manually, guided by the UV absorbance
elution profile measured at 214 nm. We also measured the radioactivity of each
fraction and used it as a guide for the degree of in vitro
phosphorylation. The sum of 32P counts of each fraction was 47% of
the total amount initially loaded on the reversed-phase column. Because
conventional HPLC separations tend to produce about 50% recoveries, we
concluded that a loss of major phosphorylated peptides was very unlikely. All
fractions containing a significant amount of radiolabel were subjected to
Edman degradation, and the masses of their constitutive peptides were
determined (see Table I). There
were two fractions (28 and 69) in which both microsequencing and mass
spectrometry identified a single phosphopeptide. Fraction 28 contained a
doubly phosphorylated version of peptide 109118 (numbering is according
to the sequence published by Uhler
(9), including the first
methionine to make it comparable to the rat cGK II sequence published by
Jarchau et al. (10)).
Microsequencing revealed no modification at Thr109 and
Ser117. In contrast, the phenylthiohydantoin (PTH) residues of
Ser110 and Ser114 were clearly absent, strongly
suggesting phosphorylation at the latter residues. The corresponding singly
phosphorylated peptide 109118 was found in peak fractions 29 and 30,
where it was contaminated with at least three additional non-phosphorylated
tryptic peptides. Differential analysis of the PTH residues after each Edman
cycle identified Ser110 as the sole phosphorylated site. Additional
radiolabeled fractions eluting in front of and behind fractions 28, 29, and 30
are likely to contain additional variants of peptides containing residues 110
and 114. Their heterogeneous appearance could be explained by partial tryptic
cleavage at both the N- and C-terminal boundaries, each consisting of dibasic
sequences, as well as by their appearance as their mono- and biphosphorylated
forms. Analyses carried out on these fractions containing the remainder of the
radiolabel were not sufficiently conclusive to justify incorporation into this
study. Mass spectrometric analysis of recombinant mouse and human cGK II prior
to cGMP-dependent autophosphorylation revealed that
010% of
peptide 109118 was unphosphorylated, whereas 90100% was
monophosphorylated, suggesting that at least Ser110 or possibly
also Ser114 (but not both within one cGK II molecule) might be
already prephosphorylated to some extent in the insect cells.
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Peak 11 contained multiple peptides, one of which corresponded to a singly
phosphorylated form of peptide AGVSA-EPTTR (123132). This peptide peak
was collected, dried, and redissolved in 2 µl of 0.05% formic acid in a
2/98 acetonitrile/water mixture. An aliquot was used for nano-liquid
chromatography-tandem mass spectrometry. The fragmentation spectrum allowed
unambiguous assignment of Ser126 as the only phosphorylated moiety.
Because this site is equivalent to the slowly autophosphorylating
Ser79 in cGKI and Ser64 in cGKI
, which
were shown to affect the activity of cGK I (Refs.
12,
13, and
18 and see
Fig. 2), we tried to quantify
the incorporation of phosphate into Ser126 under conditions similar
to those used for the kinetic analysis of autophosphorylated human cGK II as
described below. To this end, the same peptide peak was then taken from HPLC
runs corresponding with the control and the 30-min and 3-h autophosphorylation
samples for a semi-quantitative analysis. In addition to the phosphorylated
and nonphosphorylated species of peptide 123132, this peak also
contained three additional ions derived from non-phosphorylated peptides. The
latter were used as internal standards, and the quantitative phosphorylation
data were related to them. We measured 10 and 40% decreases of the
non-phosphorylated peptide (123132) at time points 30 min and 3 h,
respectively. On the other hand, we measured twice as much phosphopeptide
after 3 h compared with 30-min activation. It is not possible to directly
compare the levels of the non-phosphopeptide with those of the phosphopeptide
(because they ionize differently), our quantitative estimation was therefore
based on the disappearance of the non-phosphopeptide, indicating that
10
and 40% amounts of the enzyme are phosphorylated at Ser126 during
the 30-min and 3-h incubations, respectively. This value is in agreement with
the level of activation of cGK II observed during the autophosphorylation
experiment described below.
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To obtain an alternative view on potential (other) phosphorylation sites, we repeated the reversed-phase HPLC separation with a second batch of 32P-labeled tryptic peptides, this time obtained by limited trypsin cleavage. Whereas the same labeled peptides were found as in the previous experiment, we noticed strong radiolabel in peaks corresponding with fraction 44 of our first separation. Mass spectrometric data revealed the predominant presence of peptides 82107 and 82118 in multiple phosphorylated forms. These peptides were combined and further subdigested with endoproteinase C and purified using the same HPLC conditions as before. Now, every peptide was found to be homogenous as judged from our mass spectrometric analyses. Table II lists the subpeptides resulting from the secondary digest with their masses and calculated numbers of phosphorylation sites. We unambiguously separated and identified peptide 82100 in its unphosphorylated and singly and doubly phosphorylated forms. An aliquot of the singly phosphorylated peptide was loaded in a gold-coated nanospray capillary, and the triply charged ion (690.5 m/z) was selected for tandem mass spectrometric analysis in a quadrupole time-of-flight instrument. The spectra allowed the assignment of phosphoserine at position Ser97. This peptide was recovered in high yields and showed little incorporated 32P label, suggesting that Ser97 was already endogenously phosphorylated in the insect cells. The singly and doubly phosphorylated forms of this peptide could be separated from each other and from the unphosphorylated form. They are present in the chromatogram in a ratio 0P:1P:2P of 5:20:1 (measured by UV absorbance) and 0:1:20 (32P label). This observation is in line with Ser97 as the dominant phosphorylated site and Ser92 as a poorly phosphorylated site. The peptide containing residues 101118 segregated into two forms, one of which was doubly and another one of which was triply phosphorylated. Both forms were recovered in low amounts in the same ratio but showed the highest 32P incorporation. These data are in agreement with our previous assignments of Ser110 and Ser114 as major phosphorylation sites. They also reveal the presence of a minor additional phosphorylation site, which could be either Thr109 or Ser117.
To verify that Ser110, Ser114, and Ser445 are major phosphorylated residues, we mutated these three serines to alanines (3SA) and studied the autophosphorylation of this mutant after transient expression in COS-1 cells. Due to the limited amount of recombinant cGK II, autophosphorylation was performed in the presence of relatively low levels of ATP (10 µM, cf. Ref. 17) that are generally employed in autophosphorylation studies, because these require a high specific radioactivity of ATP. Under these conditions cGMP stimulated the autophosphorylation of rat recombinant cGK II expressed in COS-1 cells only 50%, because cGK II was already autophosphorylated to a considerable level in the absence of cGMP. As expected for the role of Ser110, Ser114, and Ser445 as major phosphoacceptors, the 3SA mutant showed a large (60%) reduction in the amount of 32P incorporation in comparison to wild type cGK II in the presence of cGMP (Fig. 3). Substitution of three more phosphorylatable residues, resulting in a total of six altered sites (Ser97, Ser110, Ser114, Ser117, Ser126, and Ser445; 6SA) caused a further decrease in the cGMP-stimulated and basal autophosphorylation (Fig. 3). The various Ser to Ala mutants showed kinetic parameters toward an exogenous substrate that were not significantly different from wild type cGK II (results not shown), except for the Ser126 to Ala mutation (see below).
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Effect of Autophosphorylation on cGK II FunctionTo assess the possible function of the extensive cyclic nucleotide-stimulated autophosphorylation of cGK II, we determined the effect of autophosphorylation on the kinetic parameters of purified recombinant cGK II. As shown in Fig. 5, preincubation of cGK II in vitro in the presence of cGMP and MgATP resulted in a gradual slow increase in its basal kinase activity when compared with cGK II preincubated in the presence of cGMP and MgAMP-PNP. The addition of MgAMP-PNP during the preincubation was required to stabilize the enzyme in the presence of cGMP, whereas it cannot serve as a phosphate donor for protein phosphorylations. After a 3-h incubation with Mg-ATP and cGMP a 3040% increase in activity was reached, which was increased only slightly after prolonged autophosphorylation (Fig. 5). As shown in Fig 5B, autophosphorylation had only minor effects on the cGMP concentration needed for half-maximal activation.
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Analysis of Autophosphorylation-deficient cGK II MutantsTo determine which of the multiple autophosphorylated residues in cGK II are responsible for the kinetic changes observed, we mutated the various serines into negatively charged glutamic acid residues, which are thought to mimic phosphorylation. As shown in Fig. 6, mutation of the major phosphorylated residues Ser110 and Ser114 into Glu (S110E/S114E) did not affect the kinetic parameters. Likewise, the kinase activity of the Ser445 to Glu mutant was indistinguishable from that of wild type cGK II. However, mutation of the slowly autophosphorylated site Ser126 into Glu resulted in a constitutively active kinase. The critical role of Ser126 as a determinant of cGK II conformation and function was corroborated by the observation that mutation of this Ser126 into Ala also affected the kinetic parameters of cGK II, by lowering the EC50 for cGMP and cAMP (see Fig. 6).
Effect of Prolonged Activation of cGK II on Its Function in Intact
Intestinal EpitheliumFrom the effect of the autophosphorylation on
cGK II activity in vitro and from the phosphorylation-mimicking Ser
to Glu mutation studies, it appears that the relatively slow phosphorylation
of Ser126 in the pseudosubstrate domain may cause cGK II activity
to become independent of cGMP. To assess whether an extensive
autophosphorylation of cGK II actually causes a prolonged activation of cGK II
independently of cGMP in intact tissue, we determined the effect of cGMP
removal on the activity of the CFTR chloride channel in intact tissue. CFTR
activation in intestinal epithelium by a cell-permeable cGMP analogue was
shown to be mediated solely by cGK II and can be probed by measuring the
ISC across the tissue
(16). When 8-pCPT-cGMP was
removed 30 min after addition, just after the chloride secretory response
(ISC) had reached its maximum, the ISC rapidly and
monoexponentially returned to basal levels
(Fig. 7). However, when
8-pCPT-cGMP was removed after a 3-h incubation, the ISC rapidly
declined from the maximal level, at which it had remained constant for the
whole incubation period, to a new and stable level that was 30% (33
± 6%; n = 4) of the maximal ISC, i.e.
still clearly above the basal level. This residual cGMP-independent chloride
secretion showed a similar time course as the "slow" in
vitro autophosphorylation of cGK II (cf.
Fig. 5B) and most
plausibly reflects the contribution of autophosphorylated, constitutively
active cGK II to CFTR phosphorylation. Incubations with 8-pCPT-cGMP
considerably beyond this time period were not possible because of a diminished
viability of the tissue after 45 h.
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DISCUSSION |
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The other major phosphorylated residue in cGK II, Ser445, has a rather aberrant localization in comparison with the other major phosphorylation sites, i.e. it is located in a region connecting the cGMP-binding domains to the catalytic domain. This region is poorly conserved between cGK II and cGK I. In cGK II it contains many serine residues and is extended considerably in comparison with cGK I. In the catalytic subunit of cAK an N-terminal serine was also shown to be autophosphorylated (Ser10 (21)). Phosphorylation of Ser10 in cAK (21) and of Ser445 in cGK II (this study) failed to exert a major impact on the kinetic properties of the kinases, implying that the functional consequences of these phosphorylation events remain unclear. Interestingly Ser445 is missing in a recently described splice variant of cGK II, which was claimed to inhibit normal cGK II (22).
Autophosphorylation of cGK II could also be stimulated by cAMP albeit at a 100-fold higher concentration (4). In the presence of physiological concentrations of ATP, cAMP stimulated the phosphorylation of much of the same sites as cGMP and could, like cGMP, induce a slow constitutive activation of cGK II.2 However, at low ATP concentrations (micromolar), cAMP was more potent than cGMP in stimulating cGK II autophosphorylation (19)2 and induced phosphorylation of sites not phosphorylated in the presence of cGMP.2
In contrast to endogenously expressed cGK II, the purified recombinant mouse and human GK II, used for the in vitro autophosphorylation analysis, was non-myristoylated. However, it is unlikely that the presence of a myristoyl group affects autophosphorylation, because the lipid moiety extends from the N terminus and is mainly involved in membrane anchoring and does not interfere with the kinetic parameters of cGK II (11).
Because the cGMP-stimulated autophosphorylation caused a clear shift in the
iso-electric point of cGK II, we were able to assess that autophosphorylation
is not merely an in vitro artifact but also occurs in intact cells or
tissue upon activation by a (patho)physiological stimulus. This finding
supports a possible role of autophosphorylation as a determinant of cGK II
functions in vivo. The slow increase in basal activity observed
in vitro is caused presumably by the slow phosphorylation of
Ser126 located in the pseudosubstrate region, because the degree of
phosphorylation after 30 min and 3 h correlated well with the degree of
phosphorylation of this site as estimated from the mass spectrometry data.
Recently, autophosphorylation of cGK II was found not to alter the basal
activity (19). However, the
concentration of ATP used in that study (100 µM, whereas the
Km of cGK II for ATP is 0.4 mM
(17)), may have been too low
to cause a substantial degree of phosphorylation of Ser126. The
slow cGMP-induced phosphorylation of this site in our studies, using 1
mM ATP, leading to a cGMP-independent activation of cGK II, may
serve as a memory function, as noted in the case of prolonged
autophosphorylation of cGK I
(13). From studies of cGK
II-dependent chloride secretion in intact intestinal epithelium, we could
estimate that the maximal fraction of the functionally active pool of cGK II
converted into a cGMP-independent form upon continuous (3 h) exposure of the
enzyme to cGMP in intact epithelial cells was
30%. This slow rate of
cGMP-triggered autophosphorylation and conversion into an autonomously active
enzyme is in clear contrast to the fast and stoichiometric conversion into a
constitutively active form observed upon autophosphorylation of other classes
of protein kinase, e.g. Ca2+-calmodulin kinase
II (23). In the latter case,
secondary autophosphorylation occurring only after translocation of the enzyme
to postsynaptic sites does not further affect its catalytic activity but
induces its subsequent dissociation from the substrate site
(24). In analogy it is
conceivable therefore that autophosphorylation of cGK II is not primarily
involved in the regulation of its catalytic activity, but rather in the
regulation of other enzyme functions, e.g. the interaction of cGK II
with its physiological substrates (e.g. CFTR
(25)) or with anchoring
proteins (26). The
availability of the various autophosphorylation mutants described here may
help to delineate such a putative role of cGK II autophosphorylation in future
studies.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 31-30-253-5378; Fax: 31-30-253-5492; E-mail: A.B.Vaandrager{at}vet.uu.nl.
1 The abbreviations used are: cGK, cGMP-dependent protein kinase; cAK,
cAMP-dependent protein kinase; AMP-PNP, adenosine
5'-(,
-imido)triphosphate; 8-pCPT-cGMP,
8-(4-chlorophenylthio)-cGMP; PTH, phenylthiohydantoin; STa, Escherichia
coli heat-stable enterotoxin; CFTR, cystic fibrosis transmambrane
conductance regulator.
2 A. B. Vaandrager, B. M. Hogema, M. Edixhoven, C. M. M. van den Burg, A. G.
M. Bot, P. Klatt, P. Ruth, F. Hofmann, J. Van Damme, J. Vandekerckhove, and H.
R. de Jonge, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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