Autophosphorylation of cGMP-dependent Protein Kinase Type II*

Arie B. Vaandrager {ddagger} § , Boris M. Hogema {ddagger}, Marcel Edixhoven {ddagger}, Caroline M. M. van den Burg {ddagger}, Alice G. M. Bot {ddagger}, Peter Klatt ||, Peter Ruth ||, Franz Hofmann ||, Jozef Van Damme **, Joel Vandekerckhove ** and Hugo R. de Jonge {ddagger}

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40–50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclic GMP-dependent protein kinases (cGKs)1 play an important role in the signaling of various cGMP-linked hormones and neurotransmitters, including nitric oxide (NO), natriuretic peptides, and guanylin (13). In mammalian tissues two types of cGK have been identified. Type I cGK, which has {alpha} and {beta} isoforms, is more ubiquitously expressed and acts as a key regulator of cardiovascular homeostasis (13). In contrast, type II cGK was described originally as an intestine-specific form (4) and is now known to be involved in various physiological processes, including intestinal fluid homeostasis, skeletal bone growth, renin secretion, and renal Ca2+ reabsorption (57). Furthermore, its widespread distribution in various areas of the brain suggests an important role of cGK II in NO/cGMP signaling in the central nervous system (8).

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{alpha} and I{beta} 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{alpha} 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{beta} (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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Recombinant His-cGK II—A 2.3-kb insert, coding for mouse or human cGK II fused to an N-terminal hexahistidine tag, was cloned into the BamHI site of pFastBac1 (Invitrogen), yielding the cGK II bacmid transfer vector pFB1His6cGK II. The cGK II transfer vector was transformed into Epicurian coli XL2-Blue ultracompetent cells, and correct transformants were identified by restriction site analysis and dideoxy sequencing of the plasmid DNA. Transformation of pFB1cGK II into Max Efficiency DH10Bac cells (Invitrogen), identification of recombinant clones and isolation of the recombinant baculovirus shuttle vector DNA (bacmid) were performed according to the instructions of the Bac-to-Bac baculovirus expression kit purchased from Invitrogen.

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 3–4 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 II—The 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 80–90% 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 15–20 µm), and membranes were isolated by a 10-min centrifugation at 20,000 x g, and resuspended in buffer A to a concentration of 0.4–1 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 Autophosphorylation—To achieve autophosphorylation, purified His-cGK II (5–8 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 [{gamma}-32P]ATP (0.3 µCi/nmol). Transiently expressed rat cGK II and cGK II mutant proteins solubilized from COS cell membranes (0.4–1 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 {beta}-mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 25 mM sodium {beta}-glycerophosphate, 1% Triton X-100, 0.15 M NaCl, and 10 µM [{gamma}-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 Sites—Affinity-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 {beta}-mercaptoethanol, 0.25 mM [{gamma}-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 20–35 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 Electrophoresis—Purified recombinant mouse His-cGK II was autophosphorylated in the presence of 1 mM [{gamma}-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% {beta}-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 6–8 and pH 3–10, 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 Activity—To 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 4–10 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 {beta}-mercaptoethanol, 0.1 mM 3-isobutyl-1-methylxanthine, 25 mM sodium {beta}-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 [{gamma}-32P]ATP (0.1–0.2 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 Mucosa—A 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autophosphorylation of Recombinant cGK II—Although autophosphorylation of cGK II is easily detectable in intestinal brush-border membranes, it proved unpractical to obtain sufficient native rat or pig cGK II purified in an intact and active form to permit detailed analysis of its autophosphorylation. Instead we used purified His-tagged, non-myristoylated, recombinant cGK II expressed in Sf9 insect cells for our studies. As shown in Fig. 1, cGMP stimulated the incorporation of phosphate into recombinant cGK II, similar to their stimulation of the autophosphorylation of native cGK II in rat brush-border membranes observed previously (4). The incorporation of phosphate into cGK II was due to intramolecular autophosphorylation, because it was minimally affected by a 10-fold dilution of the enzyme or by immobilization of the His-tagged cGK II on a nickel column (data not shown). The cyclic nucleotide-stimulated autophosphorylation followed a biphasic time course: the first 2–3 mol of phosphate was incorporated within 15–30 min, whereas higher stoichiometries were reached more slowly thereafter (Fig. 1).



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FIG. 1.
Time course of cGK II autophosphorylation. Recombinant human cGK II purified from Sf9 cells was incubated for various time periods in the presence of 1 mM [{gamma}-32P]ATP and in the absence (open circles, control) or presence of 10 µM cGMP (filled circles, cGMP). The amount of phosphate incorporated was expressed per mole of cGK II monomer.

 

Mapping of Autophosphorylation Sites in cGK II—To identify the major autophosphorylation sites, purified mouse recombinant cGK II was incubated for 30 min with cGMP and {gamma}-[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 109–118 (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 109–118 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 ~0–10% of peptide 109–118 was unphosphorylated, whereas 90–100% 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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TABLE I
Analysis of autophosphorylation sites in cGK II

Mouse recombinant cGK II purified from Sf9 cells was incubated for 30 min with 0.25 mM [{gamma}-32P]ATP and 10 µM cGMP. Trypsin fragments were separated on HPLC, and the peptides that had incorporated significantly more radioactivity than average were analyzed with mass spectroscopy and microsequencing. The identity and number of phosphorylated residues in each fragment was deduced from its mass. Those underlined and in boldface are the serines that were shown to actually contain a phosphate as assessed by microsequencing of the peptides by Edman degradation or by MS/MS analysis in a Q-TOF instrument.

 


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TABLE II
Identification of autophosphorylation sites in cGK II; analysis of peak 44

Mouse recombinant cGK II purified from Sf9 cells was incubated for 30 min with 0.25 mM [{gamma}-32P]ATP and 10 µM cGMP. Trypsin fragments were separated on HPLC and fraction 44, containing multiple phosphopeptides was redigested with endopeptidase C and rerun on HPLC. Fractions of this second run were analyzed with mass spectroscopy. The identity and number of phosphorylated residues in each fragment were deduced from its mass. Underlined are the potentially phosphorylated residues, and those in boldface are the serines that were shown to actually contain a phosphate as assessed by MS/MS analysis in a Q-TOF instrument.

 
A clear result was obtained with the peptide present in peak 69. Its abundance allowed Edman microsequencing leading to its assignment in the region 444–466. Its determined mass (2477.98 Da) corresponds with the mass of the monophosphorylated tryptic peptide 444–466. Microsequence data showed normal recoveries of PTH-amino acid derivatives, with the exception of Ser-445, which was completely missing, suggesting that the single phosphoryl group in the peptide resides at Ser445.

Peak 11 contained multiple peptides, one of which corresponded to a singly phosphorylated form of peptide AGVSA-EPTTR (123–132). 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 {beta} and Ser64 in cGKI {alpha}, 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 123–132, 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 (123–132) 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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FIG. 2.
Autophosphorylation sites in cGKs are located in close vicinity of the "pseudosubstrate" domain. Comparison of autoinhibitory domains of mouse cGK II with bovine cGK I{beta} and cGK {alpha}. Residues shown to be autophosphorylated in response to cGMP or in basal conditions are shown underlined and in boldface. Not shown is the position of phosphorylated residue Ser445 of cGK II, which is located in the loop between the regulatory and the catalytic domain.

 

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 82–107 and 82–118 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 82–100 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 101–118 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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FIG. 3.
Mutation of the autophosphorylated residues in cGK II inhibits cGMP-stimulated autophosphorylation. Recombinant rat cGK II (wt, open bars), a cGK II mutant in which the three major autophosphorylated serines (Ser110, Ser114, and Ser445) were changed to Ala (3SA, hatched bars), or a mutant of 3SA in which three additional minor sites (Ser97, Ser117, and Ser126) were mutated into Ala (6SA, cross-hatched bars), were expressed in COS-1 cells. Solubilized membranes were incubated for 2 min with 10 µM [32P]ATP without (cont) or with 10 µM cGMP. 32P incorporated into cGK II or the mutant cGK II was quantitated and corrected for the amount of cGK II activity present in the preparations as determined in parallel by substrate (2A3) phosphorylation at saturating levels of cGMP (40 µM). The kinetic properties of the 3SA mutant was similar to that of wild type cGK II, whereas the 6SA mutant had a 3- to 5-fold lower EC50 for cGMP and 10-fold lower EC50 for cAMP, similar to a single Ser126 to Ala mutant (S126A, cf. Fig. 6). Data shown are mean ± S.E. of three experiments.

 



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FIG. 6.
Mutation of Ser126 in the "pseudosubstrate" domain, which mimics its phosphorylation, activates cGK II. Mutation of other phosphorylatable serines has no effect. Recombinant rat cGK II mutants in which Ser126 was changed into Ala (S126A, open triangle) or into Glu (S126E, closed circle) or mutants of cGK II in which the major autophosphorylated serines were mutated into Glu (Ser110 in combination with Ser114, S110E/S114E, closed triangles and Ser445, S445E, diamonds), were expressed in COS cells (non-mutated cGK II, open circles). Kinase activity of solubilized COS membranes was assayed in the presence of 0.3 mM ATP and various concentrations of cGMP or cAMP with the synthetic peptide 2A3 as a substrate. Kinase activities were expressed relative to the maximal activity in the presence of 40 µM cGMP. Data shown are mean ± S.E. of two to three experiments.

 
Autophosphorylation of cGK II in Intact Cells—The cGMP-stimulated autophosphorylation of mouse recombinant cGK II could be detected by two-dimensional electrophoresis, because it caused the appearance of multiple, more acidic forms of cGK II, as can be expected after incorporation of one to four additional phosphate groups (Fig. 4). Labeling with [{gamma}-32P]ATP confirmed that the more acidic forms had incorporated the major amount of phosphate (see Fig. 4). To investigate whether the cGMP-stimulated autophosphorylation of cGK II is an in vitro artifact, or occurs also in intact cells, we incubated cGK II-transfected COS-1 cells with a membrane-permeable cGMP analogue and performed two-dimensional electrophoresis on COS cell lysates. As shown in Fig. 4, incubation with 8-pCPT-cGMP induced a conversion of cGK II into more acidic forms, similar to the shift in isoelectric points observed after autophosphorylation of cGK II in vitro. When we repeated the experiment with a mutant in which the three major autophosphorylated serines (at 110, 114, and 445) were changed to alanine (3SA), this shift in isoelectric point upon addition of 8-pCPT-cGMP was no longer observed (data not shown). This indicates that the more acidic forms of cGK II indeed represent phosphorylated forms and constitute the major autophosphorylation sites in vivo. These more acidic, autophosphorylated forms of cGK II also appeared on two-dimensional phosphoprotein maps of rat intestinal mucosa after preincubation with the cGMP-linked agonist STa (Fig. 4). Taken together, these data indicate that cGK II can be autophosphorylated to a considerable extent under physiological conditions.



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FIG. 4.
Autophosphorylation of cGK II can be detected by isoelectric focusing. A–D, mouse recombinant cGK II purified from Sf9 cells was incubated for 30 min with 1 mM [{gamma}-32P]ATP without (A and C) or with 10 µM cGMP (B and D). After two-dimensional electrophoresis the 32P incorporated was visualized by autoradiography (A and B), and cGK II was detected by immunoblotting (C and D). E–H, COS cells, transiently expressing cGK II, were incubated for 60 min without (E) or with 100 µM 8-pCPT-cGMP (F). Stripped mouse jejunum, mounted in an Ussing chamber, was incubated for 15 min without (G) or with 0.3 µM heat-stable enterotoxin STa, added to the mucosal compartment (H). After lysis, and two-dimensional electrophoresis, cGK II was visualized by immunoblotting. The data shown are representative for two to three independent experiments.

 

Effect of Autophosphorylation on cGK II Function—To 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 30–40% 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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FIG. 5.
Autophosphorylation of cGK II in vitro resulted in a slow increase in its basal kinase activity. A, human recombinant cGK II purified from Sf9 cells was preincubated for various time periods with 10 µM cGMP in the presence of 1 mM ATP (filled circles, autophosphorylated) or 1 mM AMP-PNP (open circles, control). After removal of cGMP and nucleotides by a 1000x dilution, kinase activity was assayed in the presence of 0.3 mM ATP and 10 µM cGMP using the synthetic peptide 2A3 as a substrate. Kinase activities in the absence of cGMP (basal activity) were expressed relative to the maximal activity in the presence of 10 µM cGMP. B, human recombinant cGK II purified from Sf9 cells was preincubated for 0.5 h (closed triangles) and 3 h (closed circles) with 10 µM cGMP in the presence of 1 mM ATP or for 3 h with 10 µM cGMP and 1 mM AMP-PNP (open circles, control). After removal of cGMP and nucleotides by a 1000x dilution, kinase activity was assayed as under A in the presence of various concentrations of cGMP. Kinase activities are expressed as a percentage of the maximal activity obtained in the presence of 10 µM cGMP. Maximal activities did not differ substantially for the various preincubations. Data shown are mean ± S.D. of two experiments.

 

Analysis of Autophosphorylation-deficient cGK II Mutants—To 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 Epithelium—From 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 4–5 h.



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FIG. 7.
Time-dependent induction of cGMP-independent, cGK II-mediated chloride secretion in mouse intestinal epithelium. Mouse jejunal mucosa was mounted in Ussing chambers, and the short-circuit current (Isc) response to 8-pCPT-cGMP (50 µM, reflecting cGK II activation of the CFTR chloride channel (16)) was monitored. The higher than basal current remaining after rapid washout of 8-pCPT-cGMP (block arrows) most plausibly reflects the activity of autophosphorylated, autonomously active cGK II.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Type II cGK was found to be autophosphorylated in the presence of cGMP and physiological levels of ATP on three major sites (Ser110, Ser114, and Ser445), and at least three minor sites (Ser92, Ser126, and Thr109 or Ser117). The major sites, Ser110 and Ser114, are located 11 and 7 amino acids N-terminally form the putative pseudosubstrate sequence, respectively (Refs. 3 and 14 and Fig. 2). The major autophosphorylated residues in cGK I {alpha} and {beta} were also found around the pseudosubstrate domain (see Fig. 2). This suggests a similar preference for autophosphorylation sites among the cGKs. Interestingly, the Ser114 in cGK II does not possess the putative consensus sequence for phosphorylation by cGKs, i.e. two basic residues at positions P-3 and P-2 (13). A similar lack of a consensus sequence for phosphorylation was observed in the case of the autophosphorylation sites in cGK I {beta} and {alpha} (1214). On the other hand, the presence of such a consensus sequence in the case of Ser110 might explain the preferential autophosphorylation of this residue in cGK II, occurring for a large part even under basal conditions. The pseudosubstrate region is relatively well conserved among the cGKs and cAMP-dependent protein kinases and is thought to be critical for autoinhibition of the catalytic activity of cAK and cGK in the basal state (3, 14, 19). The observation, that a Glu substitution for Ser126 in the middle of the pseudosubstrate site renders cGK II constitutively active, strongly suggests that the pseudosubstrate sequence in cGK II and cGKI have very similar functions. A similar activation was observed when the equivalent serine in the pseudosubstrate region of cGK I{beta} (Ser79) and cGK 1{alpha} (Ser64) were changed into an Asp (18, 20). The effect of mutating Ser126 in cGK II to Ala on cGMP affinity is also similar to the effect of the equivalent mutations in cGK I{alpha} and in cGK I{beta} (18). The preferential autophosphorylation of residues in the vicinity of the pseudosubstrate site also suggests that this region is in close contact with the catalytic head.

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 {beta} (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.


    FOOTNOTES
 
* This study was supported by a grant from the Netherlands Organization for Scientific Research. The work in Belgium was supported by the Fund for Scientific Research-Flanders and by the Interuniversity Attraction Poles of the Belgian Prime Ministers Office (Grant IUAP-P4/23). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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'-({beta},{gamma}-imido)triphosphate; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; PTH, phenylthiohydantoin; STa, Escherichia coli heat-stable enterotoxin; CFTR, cystic fibrosis transmambrane conductance regulator. Back

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. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Karine Smans (Janssen Pharmaceutica, Beerse) for donating batches of purified human cGK II.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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