Autoinhibition and Isoform-specific Dominant Negative Inhibition of the Type II cGMP-dependent Protein Kinase*

Construction of cGKII and cGKIβ Mutants

Plasmid constructs encoding alanine substitutions for candidate residues responsible for regulation of cGKII autoinhibition (R118A/R119A, K122, V125A, S126D, and R118A/R119A/V125A) were constructed using a PCR site-directed mutagenesis protocol as previously described (16). The construction of the truncated cGKII regulatory domain mutant denoted IIRΔ417–762 was performed using primers and a cGKII template in a one-step PCR site-directed mutagenesis method as described (11). The IIRΔ417–762 construct contained an amino-terminal hexahistidine tag (N-terminal His-tag) and was truncated at Lys⁴¹⁷ with COOH-terminal addition of either a FLAG epitope sequence (DYKDDDDK) or the last five amino acids of the RIα subunit of cAK (VSLSV). The primers for the amino-terminal truncation of the cGKII regulatory domain were designed with a BamHI site at the 5′ end that was used to ligate the fragment into pcDNA3.0 (Invitrogen). Mutations of the autoinhibitory region were introduced by subcloning a fragment of the R118A/R119A/V125A (called^{R118A/R119A/V125A}IIRΔ417–762) construct into vectors that contained the 5′ end of the IIRΔ417–762 construct.

Construction of the His-tagged cGKIβ truncation mutant, IβRΔ341–686, was carried out using a sense primer that encoded an NH₂-terminal hexahistadine tag for Iβ and an antisense primer that contained the BamHI site after the codon for Gly³⁴⁰ to allow the COOH-terminal fusion to either the triple-FLAG epitope encoded by the pFLAG-CMV-14 vector (Sigma) or the c-Myc epitope encoded by pCMVTag5a (Stratagene). The University of Michigan Biomedical Research Core Facilities synthesized the primers used for this approach and sequenced all final plasmid constructs.

Expression and Purification of cGKII Mutants

For in vitro studies, hexahistidine-tagged wild type and mutant cGKII proteins were purified from HEK-T cells following transfection (17) unless otherwise indicated. Purification was performed essentially as described (11). Briefly, the cells were harvested and sonicated in Buffer A (10 mm Tris, pH 8.0, 0.1% Triton X-100, 200 mm NaCl) containing Complete^TM EDTA-free protease inhibitors (Roche Molecular Biochemicals) 36 h post-transfection. The solution was centrifuged at 20,000 ×g for 30 min at 4 °C, after which 20 mmimidazole in Buffer A was added to the solution, and the supernatant was decanted into fresh tubes to be spun again for another 30 min. The supernatant was then loaded onto a 500-μl column of nickel affinity resin (Qiagen) and washed with 20 volumes of 20 mmimidazole in Buffer A. The hexahistidine-tagged protein was eluted by stepwise application of 1 ml of 30, 60, and 90 mmimidazole, with the highest concentration of cGKII protein found within the first 500 μl of the 1 ml of 60 mm imidazole elution step. The protein concentration was determined as described (11).

The purification of the previously characterized FLAG-tagged cGKI-C (18) from a 15-cm² plate of transfected HEK-T cells was performed by washing the cells three times with phosphate-buffered saline prior to harvesting in 4 ml of extraction buffer (0.9% phosphate-buffered saline, 10 mm NaPO₄, pH 7.5, 1% Triton X-100, 0.1% Nonidet P-40, 1% Tween 20) and homogenizing with 10 strokes of a Dounce homogenizer. The extract was centrifuged at 13,000 rpm for 10 min and the supernatant was collected into 14-ml Falcon tubes. One-hundred microliters of M2-agarose beads (Sigma) were added to the supernatant and the mixture was allowed to rotate for 1 h at 4 °C. After washing the beads three times with extraction buffer, the enzyme was eluted in Buffer A by adding an excess of FLAG-peptide (Sigma). The units of active cGKI-C were determined using the substrate Kemptide and matched with the activity of the R118A/R119A/V125A constitutively active cGKII in the in vitro kinase assay.

All purified enzymes were stored at −20 °C in a storage solution containing a final concentration of 50% glycerol, 50 mmKPO₄, pH 6.8, 25 mm β-mercaptoethanol, 1 mm EDTA, and 0.1% bovine serum albumin. Prior to the addition of storage solution, all mutant kinases were assayed to be certain that both basal and maximum kinase activity remained linear over all time periods. Additionally, kinase activity assays were performed to ensure that the K _a andV _max of the freshly purified kinase was consistent with kinetics assayed after storage, and all freshly purified truncation mutants were assayed for stoichiometry of [³H]cGMP binding and IC₅₀ against the constitutively active R118A/R119A/V125A full-length cGKII (also called constitutively active cGKII).

Determination of Kinase Activity

Kinase activity was determined by varying the concentration of cyclic nucleotide using the conditions and methods described (11). The reaction mixture consisted of 20 mm Tris, pH 7.5, 0.5 mmisobutylmethylxanthine, 10 mm magnesium acetate, 10 mm dithiothreitol, 0.2 mg/ml bovine serum albumin, 200 μm unlabeled ATP, 11 nm[γ-³²P]ATP (specific activity 200–300 cpm/pmol;Amersham Biosciences), and 100 μm synthetic H2Btide (Arg-Lys-Arg-Ser-Arg-Ala-Glu) as the phosphate acceptor unless otherwise indicated. The K _a value for each kinase was calculated using the Lineweaver-Burk analysis of at least three experiments. For IC₅₀ determination, the stock solution of 5 nm enzyme was incubated on ice with varying concentrations of the regulatory truncation mutant between 3 and 5 min prior to dilution into the kinase reaction mixture. All synthetic peptides were acquired from Research Genetics (Huntsville, AL) or the University of Michigan Biomedical Core Facilities. Single tailed Student's t tests were applied to determine significance in observed differences of kinase activity.

Autophosphorylation Assay

The determination of phosphate (P_i) incorporation into wild type cGKII was performed in a similar manner to that described (8). Briefly, purified cGKII enzyme at a concentration of 1 μm was incubated in Buffer B (5 mm potassium phosphate, pH 6.8, 1 mm EDTA, 25 mm β-mercaptoethanol) in the presence of 100 μm ATP and 4.5 μm magnesium acetate (if indicated), under conditions noted in the figure legends. Because bovine serum albumin was found to be a substrate for cGKII, it was not included in the autophosphorylation mixture after it was determined that the linear increase of kinase activity over time was not affected by its omission. The enzyme was then incubated at 30 °C and the autophosphorylation reaction was terminated at the indicated time by adding 18 μl of the mixture to SDS-PAGE buffer (2% SDS, 12.5% β-mercaptoethanol, 25% glycerol, 0.25 m Tris, pH 6.8, and bromphenol blue). Phosphate incorporation was determined by performing electrophoresis of the reaction mixture on a 10% SDS-polyacrylamide gel and excising the 86-kDa band after Coomassie Blue staining. Analysis of the log of the rate of phosphorylationversus log of the enzyme concentration (van't Hoff analysis) indicated that both the basal and cGMP-stimulated autophosphorylations were intramolecular events.

To determine the effect of autophosphorylation on enzymeK _a or V _max, the autophosphorylation mixture was diluted 50-fold into an ice-cold solution that contained Buffer B, 0.1% bovine serum albumin, and 25 mm dithiothreitol. After incubating on ice for 2 min, this stock solution of autophosphorylated enzyme was used in phosphotransferase reactions carried out as described above.

[³H]cGMP Dissociation Assay

Dissociation rates of [³H]cGMP from wild type and IIRΔ417–762 variants were determined using the cyclic nucleotide dissociation assay as described (11). Briefly, a final concentration of 450 nmof purified enzyme was incubated for 10 min at 30 °C in a mixture of KPEH buffer (5 mm potassium phosphate, pH 6.8, 1 mm EDTA, 25 mm β-mercaptoethanol, and 0.5 mg/ml histone IIA), 50 μm cGMP, and 3.2 μm[³H]cGMP (specific activity, 15.6 Ci/mmol; AmershamBiosciences). The mixture was then chilled for 5 min on ice before 30-μl samples were precipitated in 100% saturated, ice-cold (NH₄)₂SO₄ and filtered over a 0.45-μm nitrocellulose filter (Millipore). Dissociation rates were determined by sampling the mixture in an identical manner after the addition of 100-fold excess of unlabeled cGMP.

In Vivo Assays of cGKII and cGKI Activity

Two assays were used to determine the activity of the kinase in vivo. To study the effect of mutations on the regulation of gene transcription, a luciferase reporter gene construct was used (11). Briefly, expression vectors for the kinase, FLAG-tagged PKIα and a human chorionic gonadotropin luciferase reporter gene containing two cAMP response elements, were co-transfected with vectors encoding the truncated cGKII mutant inhibitor (if indicated) and the Rous sarcoma virus β-galactosidase to control for transfection efficiency. Transfected cells were cultured for 24–36 h in the presence of 10% fetal calf serum, exposed to 8-Br-cGMP at the indicated concentration for 6–8 h (also in the presence of 10% fetal calf serum), and harvested and assayed for luciferase activity as described (11).

To address in vivo activation on a shorter time scale and determine the effects of the mutations on phosphorylation, thevasodilator-A-kinase stimulatedphosphoprotein (VASP) assay was used as described (18). The expression vectors for kinase, as well as FLAG-tagged VASP and FLAG-tagged PKIα, were co-transfected with vectors for enhanced green florescent protein (p-EGFP C1; CLONTECH) and β-galactosidase (Rous sarcoma virus-βgal) to control for transfection efficiency. After 24 h the cells were serum starved for at least 4 h prior to harvesting with ice-cold homogenization buffer. The samples were then immediately sonicated, a sample was taken for protein and β-galactosidase assay determination, and the remainder diluted into RIPA buffer (20 mm Tris-HCl, pH 8.0, 1% HPO₄, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 1 mm EDTA, 50 mm NaF, 5 mm sodium pyrophosphate) followed by storage at −20 °C. SDS loading buffer was added to the samples and electrophoresed on a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose, and blotted with the indicated antibodies. Chemiluminescent intensity was determined using Bio-Rad Fluro-S MaxiImage (Diabetes Center, University of Michigan). Results are expressed as % maximum phospho-VASP, which was calculated as the percent ratio of VASP conversion in the experimental condition over the maximum amount of VASP phosphorylation when the constitutively active kinase was expressed alone. The specific conditions and mass of DNA added to the transfection conditions are similar to that noted (11, 18) for both assays. Variations of these conditions are noted in the figure legends.

Autophosphorylation Does Not Diminish Autoinhibition

For cGKIα and -Iβ, autophosphorylation disrupts autoinhibition, allowing the catalytic domain to phosphorylate substrate in the absence of cGMP. To determine the kinetics of autophosphorylation in cGKII, purified wild type cGKII was incubated with [γ-³²P]ATP and assayed for incorporation of radioactivity. Fig.1 A shows that 5 μm cGMP induced phosphate incorporation at a maximum of 3 P_i/mol of cGKII monomer and that cAMP induced incorporation of an additional mole of P_i/mol of cGKII monomer even though maximum kinase activations in the two conditions were identical (data not shown). However, autophosphorylation of cGKII in the presence of either cAMP or cGMP did not elevate basal kinase activity (Fig.1 B) in contrast to similar experiments with cGKIβ (8) and -Iα (9). Additionally, autophosphorylation in the presence of cGMP did not shift the sensitivity of the kinase for activation (K _a) by cGMP (Fig. 1 C), as has been shown for cGKIβ (8). This was also observed when assayed with Kemptide, which has a lower K _m for cGKII (19), or varying concentrations of ATP (data not shown).

Determination of Residues That Are Necessary for cGKII Autoinhibition

Autophosphorylation in all other cyclic nucleotide-dependent protein kinases results in a moderate release of autoinhibition. The inability of cGKII autophosphorylation to alter the activity of the kinase may be because of unique autoinhibitory interactions between its regulatory and catalytic domains. We set out to determine critical residues for cGKII autoinhibition by examining the alignment of pseudosubstrate inhibitors of the cyclic nucleotide-dependent protein kinases (Fig.2). Many of the determinants of the inhibitory potency of the cAK protein kinase inhibitor (PKI) (20) and the RI (21, 22) and -II (23) subunits of cAK are known. Alignment of RIα, cGKIβ, and cGKIα with cGKII (Fig. 2) revealed that the sequence ¹²¹AKAGV¹²⁵ of cGKII aligns with the RI and PKI pseudosubstrate motif RRXAΨ, where Ψ represents a hydrophobic residue and the underlined residue is the designated “P-site.” Determinants for binding of RIα and the pseudosubstrate inhibitor PKI to the catalytic cleft of cAK consist of several elements, including a P-6 arginine (found in RIIα, RIIβ, and the PKIs), the P-3 and P-2 arginine residues and a P + 1 hydrophobic residue (16, 20, 24). Electrostatic interactions between glutamate residues in the catalytic cleft and the P-3 and P-2 residues of PKI are responsible for their inhibitory potency toward the C subunit. These electrostatic interactions are supplemented by the hydrophobic interactions between the P + 1 residue and a hydrophobic pocket within the substrate-binding site (20, 25). On the basis of these alignments and previous studies, a series of mutants were designed to substitute an alanine for the P-6, P-5, and P-2 basic residues of cGKII, Arg¹¹⁸, Arg¹¹⁹, and Lys¹²², respectively, as well as the P + 1 hydrophobic residue Val¹²⁵ (see Fig. 2 within the gray box).

Our first goal was to determine whether the residue at the P-2 position (Lys¹²², see Fig. 2 in the gray boxunder the P-2 position) was required for autoinhibition of cGKII. If so, we expected to observe an increase of basal activity in the Lys¹²² mutant, indicative of disrupted autoinhibition. However, we observed only a small change in basal activity (10%; TableI). The other conserved basic residues, the P-6 and P-5 arginine residues (Arg¹¹⁸ and Arg¹¹⁹), were also targeted for alanine substitution. The P-6 residue was targeted because the homologous residue in PKI has been shown to be a major determinant of high affinity binding at a highly conserved glutamate in the catalytic cleft. Also, arginine at the P-5 position was targeted because the homologous residue in RIα and cGKIβ contributes to the autoinhibitory potency of their regulatory domains (21, 22). The R118A/R119A cGKII mutant showed a 3-fold increase in the basal activity of the enzyme (Table I), suggesting that residues distal to the predicted pseudosubstrate sequence contribute to cGKII autoinhibition.

In other cyclic nucleotide-dependent protein kinases, disruption of autoinhibition can enhance the ability of cyclic nucleotide binding to activate the enzyme (26), indicated by a decrease in the K _a for a cyclic nucleotide. To determine whether K122A or R118A/R119A demonstrated increased sensitivity to cGMP, the activities of these mutants were monitored in the presence of varying concentrations of cyclic nucleotide. Fig.3 A illustrates the significant increase in the sensitivity of the enzyme to cGMP (4.2-fold for K122A and 7.4-fold for R118A/R119A; Fig. 3 A and Table I). The mutant kinases were also monitored in vivo to ensure that the purification procedure did not introduce an experimental artifact. Similar to our in vitro observations, K122A and R118A/R119A cGKII mutants showed a 5-fold leftward shift in the 8Br-cGMP induction of luciferase expression (Fig. 3 B; EC₅₀ of 10 μm for each mutant, relative to an EC₅₀ of 50 μm for wild type).

Thus, compared with cGKIβ, cGKII autoinhibition is unique because residues that are relatively distant from the conserved serine residue of the putative pseudosubstrate sequence (P-5 and P-6) contribute to autoinhibition as much as residues within the pseudosubstrate sequence (21, 22). Because substitution of Lys¹²² in cGKII abolishes the only basic residue that aligns with the autoinhibitory pseudosubstrate sequence in cAK and cGK, it was expected that this mutant would have a significant impact on autoinhibition, potentially creating a constitutively active kinase. Instead, K122A showed modest activity in the absence of cyclic nucleotides, demonstrating that other components contribute to autoinhibition.

Because mutation of the charged residues Arg¹¹⁸/Arg¹¹⁹ or Lys¹²² did not completely abolish autoinhibition, we also considered the importance of a hydrophobic residue at the P+1 position in cGKII. This residue is highly conserved in cGKIα and -Iβ and cAK inhibitors (see Fig. 2). A hydrophobic residue at the P + 1 position has been shown to participate in autoinhibition of cGKI (27) and has also been shown to be important in the inhibitory potency of peptides designed from the pseudosubstrate sequence in RIα and PKI (22, 24). If the residue homologous to the P + 1 position (Val¹²⁵) contributed to autoinhibition like Arg¹¹⁸/Arg¹¹⁹ or Lys¹²², an alanine substitution at that site would decrease the potency of autoinhibition. Indeed, the V125A mutant of cGKII showed a significant elevation of basal activity relative to wild type cGKII (2.9-fold; Table I), in addition to an increase in sensitivity to cGMP (6.6-fold; Fig. 4 A and TableI). This shows that the P + 1 residue contributes to autoinhibition, indicating that the interaction of the cGKII catalytic domain with the¹²¹AKAGV¹²⁵ pseudosubstrate sequence was analogous to that seen in other cyclic nucleotide-dependent protein kinases.

To determine the contribution of adjacent residues, we targeted Arg¹¹⁸/Arg¹¹⁹ and Val¹²⁵ of the cGKII sequence ¹¹⁸RRGAKAGVS¹²⁶ to test whether a combination of Ala substitutions could produce a constitutively active kinase. If these residues were primary factors contributing to autoinhibition, then combined disruption of these three residues would yield a constitutively active enzyme. A cGKII enzyme with alanine substitutions for Arg¹¹⁸, Arg¹¹⁹, and Val¹²⁵ demonstrated a dramatic increase in basal activity relative to wild type (25-fold; Fig. 4 A and TableI). The increase between the basal and cGMP-stimulated activation for the R118A/R119A/V125A mutant enzyme (from 0.30 to 0.41 μmol/min/mg) may represent residual autoinhibition mediated by Lys¹²² or to additional, less potent inhibitory elements in the regulatory domain. In vivo, these mutants also displayed a pattern of activation that is consistent with that seen in vitro(EC₅₀ of 10 μm for V125A and constitutive activity for R118A/R119A/V125A; Fig. 4 B). Taken together, these data define Arg¹¹⁸, Arg¹¹⁹, and Val¹²⁵ as major determinants of cGKII autoinhibition. The impact of these residues on autoinhibition would not have been predicted from previous models of regulatory and catalytic domain interactions in cyclic nucleotide-dependent protein kinases (22). Our data show that for cGKII, the integrity of these residues is essential for autoinhibition and defines a region from Arg¹¹⁸-Val¹²⁵ that we call the autoinhibitory subdomain (AI subdomain).

The AI Subdomain Is Not Sufficient for cGKII Autoinhibition

To determine whether the AI subdomain would be sufficient to inhibit cGKII, we created a 16-mer COOH-terminal acetylated peptide that included the AI subdomain (SRRGAKAGVSAEPTTR; cGKII AI-peptide) that was based on the sequence of cGKII between Ser¹¹⁷ and Arg¹³². For comparison, we also tested a peptide derived from the corresponding cGKIβ AI sequence (EPRTKRQAISAEPTAF; cGKIβ AI-peptide) to determine whether it was sufficient for inhibition of a constitutively active cGKI mutant that contained only the catalytic domain of the kinase (cGKI-C (18)). Fig 5shows that the cGKII AI-peptide did not suppress the activity of the constitutively active cGKII. Also, the cGKIβ AI-peptide was not able to inhibit the cGKI-C, as expected from previous studies (22).

Because the AI subdomain was not sufficient for inhibition of cGKs, we wanted to determine whether the larger regulatory domain, which includes the AI subdomain as well as the cyclic nucleotide-binding sites, would be sufficient for inhibition as is seen by the R subunits of cAK. Studies of cAK have shown important interactions between the residues near the CNBS of the R and C subunits of cAK (23, 28-30). We anticipated that these secondary interactions may be important for inhibition and constructed the IIRΔ417–762 truncation mutant representing the amino-terminal regulatory domain of cGKII. To determine whether the IIRΔ417–762 truncation mutant inhibited the constitutively active cGKII, the proteins were purified from transfected cells and subjected to in vitro kinase assays. Fig. 5 shows that preliminary in vitro experiments demonstrated that the IIRΔ417–762 protein was a significantly more potent inhibitor of cGKII than the AI peptides.

Characterization of the Inhibitory Activity of the cGKII Regulatory Domain

Detailed analysis of the IIRΔ417–762 inhibition showed suppression of the constitutively active cGKII with an IC₅₀of 4.5 nm (Fig.6 A). The^{R118A/R119A/V125A}IIRΔ417–762 protein (Fig.6 A) did not inhibit the constitutively active cGKII, demonstrating that the residues outside of Arg¹¹⁸, Arg¹¹⁹, and Val¹²⁵ were not sufficient for inhibition by the IIR truncation mutant. In addition, heat denatured IIRΔ417–762 (Fig. 6 A) did not show significant inhibition of the constitutively active kinase. Also, the ability of cyclic nucleotides to abrogate the inhibitory properties of the IIRΔ417–762 fragment was demonstrated by the addition of 5 μm cGMP to the incubation mixture (Fig. 6 A), the amount necessary to fully activate wild type cGKII. The most direct explanation for these results is that cGMP binding to the IIRΔ417–762 truncation mutant altered the conformation of the protein and rendered it incapable of inhibiting full-length cGKII, likely through mechanisms that have been described for cGMP release of inhibition in cGKI (31).

To demonstrate the similarities of cGMP binding kinetics between the wild type kinase and the truncation mutant, and that the loss of inhibition by the ^{R118A/R119A/V125A}IIRΔ417–762 was not because of a disruption of the overall conformation of the regulatory domain, we examined the dissociation of [³H]cGMP from the CNBSs. Both forms of IIRΔ417–762 showed identical stoichiometry of [³H]cGMP binding and similar kinetics of [³H]cGMP dissociation compared with the wild type cGKII, IIRΔ417–762, and ^{R118A/R119A/V125A}IIRΔ417–762 (Fig.6 B). These results demonstrated that the catalytic domain of cGKII was not required for effective cGMP binding and that the R118A/R119A/V125A mutations of the IIRΔ417–762 did not introduce general structural defects that would have compromised its integrity.

Taken together, these data demonstrate a surprising affinity between the regulatory and catalytic domains of cGKII. The similarity of the mechanism of cGKII autoinhibition and inhibition by IIRΔ417–762 was demonstrated by the necessity for the integrity of the AI subdomain in both conditions (compare the effect of the R118A/R119A/V125A mutation on autoinhibition in Fig. 4 A and^{R118A/R119A/V125A}IIRΔ417–762 inhibition of constitutively active cGKII in Fig. 6 A). These data demonstrate that the regulatory domain, which includes the AI subdomain, is sufficient for autoinhibition, and that the AI subdomain is necessary but not sufficient for autoinhibition.

Determination of Isoform Specificity of IIRΔ417–762 and IβRΔ341–686

Because there is significant homology between the catalytic domains of cGKs and cAKs, we tested whether the IIRΔ417–762 could inhibit the catalytic subunit of cAK. Fig.7 A shows that IIRΔ417–762 does not inhibit Cα, even at concentrations that maximally inhibited the constitutively active cGKII (100 nm IIRΔ417–762). However, the Cα is almost completely inhibited by equimolar amounts of PKIα-(5–24) (Fig. 7 A), as demonstrated previously (32).

To determine whether IIRΔ417–762 could inhibit cGKI activity, we tested its ability to inhibit a previously characterized truncation mutant of cGKI (cGKI-C) that possessed only the catalytic domain (18). The catalytic activity of the cGKI-C showed a small but consistent inhibition at the highest concentrations of IIRΔ417–762 (25% inhibition; Fig. 7 B) and this inhibition was not seen in the presence of cGMP (data not shown). The magnitude of inhibition of cGKII by the IIRΔ417–762 was significantly greater (75% inhibition; Fig.7 B). The specificity of IIRΔ417–762 inhibition for the constitutively active cGKII relative to cAK Cα and cGKI-C was also observed using Kemptide as substrate (data not shown).

In Vivo Inhibition of Wild Type and Constitutively Active cGKII by Overexpression of a Regulatory Domain

The in vivoinhibition of cGKII activity by IIRΔ417–762 was examined by co-transfection experiments in HEK-T cells. Fig.8 A shows that overexpression of the IIRΔ417–762 caused a 12-fold rightward shift in the 8-Br-cGMP-dependent induction of gene transcription by cGKII, suggesting that IIRΔ417–762 co-expression inhibited the wild type kinase. Quantitative Western blot analysis demonstrated that the levels of wild type cGKII expression were identical in all conditions (data not shown), which was consistent with the observation that the maximum relative light units in both conditions were equivalent. However, these results did not distinguish between competitive, indirect, or noncompetitive modes of inhibition by IIRΔ417–762. Direct binding and inhibition of the full-length cGKII would mediate competitive inhibition by the IIRΔ417–762 protein. However, instead of competing directly at the catalytic cleft for substrate, co-expression of the IIRΔ417–762 might have indirectly inhibited the activity by simply binding cGMP or co-expression may have reduced cGKII kinase activity in a noncompetitive manner by forming a nonfunctional heterodimer with a full-length cGKII protein. These indirect and noncompetitive modes of inhibition are not likely, because the large increase (12-fold) of the cGKII EC₅₀ in the presence of the IIRΔ417–762 likely exceeded the amount of suppression expected if the two binding sites of the truncation mutant were absorbing cGMP. Second, active monomers of cGKII resulting from endogenous proteolysis have been observed in some preparations (33), suggesting that each monomer of the cGKII dimer is independently capable of catalysis. Additionally, high affinity interactions observed between the two domains in vitro (Fig. 6 A) show that heterodimerization is not required for IIRΔ417–762 inhibition.

It should be noted that the inhibitory efficacy of IIRΔ417–762 toward wild type cGKII in the presence of a cyclic GMP analog is likely because of two factors that are unique to the in vivo assay. First, because IIRΔ417–762 and wild type cGKII are overexpressed, the intracellular concentration of each would be much higher than the IC₅₀ of their interaction, which increases the likelihood of complex formation by the high probability of interaction with a binding partner. Second, there is likely a limited availability of endogenous substrate relative to the IIRΔ417–762 inhibitor, which increases the likelihood of the enzyme remaining bound to inhibitor, rather than forming an enzyme-substrate complex.

To confirm the mode of inhibition by the IIRΔ417–762 mutant in vivo, a second in vivo approach was used. A phosphorylation dependent shift in the SDS-PAGE mobility of the cGK substrate VASP has been used to quantify the changes of cGKIβ activity in vivo (18). We used this in vivophosphorylation assay to determine the inhibitory activity of IIRΔ417–762 by coexpressing it with both a constitutively active cGKII (R118A/R119A/V125A cGKII) and VASP in the absence of cyclic nucleotide.

The Western blot in Fig. 8 B shows that in the absence of transfected cGKII, VASP migrates at the more rapidly migrating dephospho form. Co-transfection with the constitutively active cGKII results in the full conversion to the slowly migrating phosphorylated form of VASP. Co-transfection of the constitutively active cGKII with the IIRΔ417–762 truncation mutant significantly inhibits VASP phosphorylation when an 8-fold excess of IIRΔ417–762 DNA was expressed (79 ± 11.3% inhibition; the ratio of expression of IIRΔ417–762 to constitutively active cGKII was confirmed by quantitative Western blot analysis). The intensity of the upper and lower bands was quantified and the % maximum conversion of VASP to phospho-VASP (% conversion relative to the condition with VASP expressed with the constitutively active cGKII alone) is expressed on the ordinate axis of Fig. 8 C.

To determine whether the Arg¹¹⁸, Arg¹¹⁹, and Val¹²⁵ residues were required for the inhibitory properties of this regulatory domain truncation mutant in vivo, similar experiments were performed with the^{R118A/R119A/V125A}IIRΔ417–762 truncation mutant (Fig.8 B). At levels of expression equivalent to IIRΔ417–762, the ^{R118A/R119A/V125A}IIRΔ417–762 mutant demonstrated a lack of inhibition of the constitutively active cGKII (5 ± 4% inhibition;p < 0.05 when comparing the difference between % inhibition between IIRΔ417–762 and^{R118A/R119A/V125A}IIRΔ417–762).

Taken together, these data show that the IIRΔ417–762 truncation mutant could suppress cGKII kinase activity in vivo even at a low stoichiometry of IIRΔ417–762. In addition, the residues that were necessary for inhibition in vitro are also requiredin vivo, suggesting that inhibition by the IIRΔ417–762 truncation mutant was not simply because of cGMP binding or heterodimerization with cGKII.

Isoform Specificity of IIRΔ417–762 and IβRΔ341–686 in Vivo

The VASP phosphorylation assay was used to assess thein vivo isoform specificity of the cGK regulatory domains. To do so, the cGKIβ regulatory domain truncation mutant (IβRΔ341–686) was co-expressed with the constitutively active cGKII or with a constitutively active form of cGKIβ (S79D cGKIβ) that has been characterized previously (18). Interestingly, it was demonstrated that the IβRΔ341–686 inhibits constitutively active cGKII (59 ± 15% inhibition) to an extent comparable with IIRΔ417–762 (49 ± 10% inhibition; Fig.9, A and B), in addition to inhibiting the constitutively active form of cGKIβ (13 ± 5%, p < 0.05 when compared with activity in the absence of inhibitor). IIRΔ417–762 was exclusive in its inhibitory influence toward the constitutively active cGKII, and did not influence the constitutively active cGKIβ (101 ± 5%; Fig.9 B), matching observations seen in the in vitroassay (Fig. 8 B, closed circles). Thus the cGKII truncation mutant showed isoform-specific inhibition, whereas the corresponding cGKIβ truncation mutant inhibited both cGKII and cGKIβ.

Conclusions

A major goal of this study was to characterize the residues required for autoinhibition in the type II cGMP-dependent protein kinase. Our results showed that cGKII did undergo significant autophosphorylation in the presence of cAMP and cGMP. However, cGKII autoinhibition was distinct from other cyclic nucleotide-dependent protein kinases in that autophosphorylation did not lead to activation of the cGKII kinase. These findings suggested that the AI subdomain of cGKII might involve residues that are not conserved with the cAK and cGKI isoforms.

To identify potentially unique interactions between the regulatory and catalytic domains, we characterized the contribution of several cGKII residues near the AI subdomain identified by sequence comparison of autoinhibitory sequences. It was found that cGKII was unique because autoinhibition was dependent upon residues that reside outside the conserved pseudosubstrate site. Because the presence of basic residues at both P-5 and P-6 is not conserved with cGKI isoforms, these observations may explain the distinct modes of activation between cGKI and II, as is reflected in their differences in the contribution of each CNBS toward kinase activation (11) and their response to autophosphorylation.

However, the residues identified within the AI subdomain of cGKII were not sufficient for inhibition of the enzyme as determined from in vitro studies using synthetic peptides derived from the AI subdomain. However, a truncation mutant of cGKII containing only the regulatory domain was a potent inhibitor of the full-length enzymein vitro and this inhibition required the same residues (Arg¹¹⁸, Arg¹¹⁹, and Val¹²⁵) shown to be required for autoinhibition.

The conclusions concerning cGKII autoinhibition were extended to in vivo experiments with the cGKII truncation mutant. In vivo inhibition of the constitutively active cGKII by this truncation mutant was demonstrated to be a high affinity, isoform-specific interaction that could be disrupted by cGMP or by mutation of residues surrounding the AI subdomain (Fig.10). Although 5 nm cGMP reversed the inhibition of the IIRΔ417–762 protein when used to inhibit low concentrations of cGKII (Fig. 6 A), significantly higher concentrations of 8-Br-cGMP were required to overcome IIRΔ417–762 inhibition in vivo (Fig. 8 A), presumably because of the much higher concentrations of cGKII and IIRΔ417–762 found in vivo. These findings are clearly applicable to the design of isoform-specific inhibitors of cGKs as well as the more detailed elucidation of the activation mechanisms for the cyclic nucleotide-dependent protein kinases. Pharmacological attempts to discriminate cGK contribution to physiological phenomena have been limited by cross-reactivity of inhibitors between cAKs and cGKs (15), and thus far there is no isoform-specific inhibitor of any cGK available. The present studies offer a molecular probe for cGKII inhibition in vivo in an isoform-specific manner, a tool that would be useful in discriminating cGK activity in cells where the two cGK isoforms have been indicated to be co-expressed (34, 35).