Differential Binding of cAMP-dependent Protein Kinase Regulatory Subunit Isoforms Ialpha and IIbeta to the Catalytic Subunit*

Xiaodong ChengDagger §, Christopher PhelpsDagger , and Susan S. TaylorDagger §||

From the § Howard Hughes Medical Institute, Dagger  Department of Chemistry and Biochemistry, School of Medicine, University of California, San Diego, La Jolla, California 92093-0654

Received for publication, July 19, 2000, and in revised form, October 23, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant Delta (1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH2 terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg133 with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH2-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The regulatory (R)1 subunits of cAMP-dependent protein kinase (PKA) are multifunctional proteins that control in a variety of ways the physiological functions of this ubiquitous protein kinase (1, 2). While the R subunits have long been recognized as the primary receptor for cAMP and the major physiological inhibitor for the catalytic (C) subunit in eukaryotic cells, it is now apparent that these highly modular proteins play other important roles as well (3). The R subunits of PKA have a well defined domain structure that consists of a dimerization domain at the NH2 terminus followed by an autoinhibitor site and two-tandem cAMP-binding domains. While the portion of the R subunit COOH-terminal to the inhibition site is responsible for high affinity binding of the C subunit and cAMP, the remaining NH2 terminus serves as an adaptor for binding to kinase anchoring proteins (3) and other adaptor proteins, such as the SH3-containing Grb2 (4). This segment or region of the R subunit is responsible for in vivo subcellular localization and targeting of PKA.

There are two general classes of PKA, designated as type I and type II, due exclusively to differences in the R subunits, RI and RII (5-7). Four different regulatory subunits, RIalpha (8), RIbeta (9), RIIalpha (10), and RIIbeta (11) have been identified. Regulatory isoforms are differentially expressed in tissues (12-14), and their subcellular distribution also appears to be distinct (8-11, 15-18). The existence of a family of protein kinase A anchoring proteins (AKAPs) that tether the type II regulatory subunits to specific subcellular structures has been well documented (3), and AKAPs for both RI and RII (19, 20) as well as an RI-specific AKAP protein (21) have been identified recently. At present the exact physiological functions of RI and RII are not clear. However, growing evidence suggests that they are functionally different. This is supported by the fact that although the ratio of the total R subunits/C subunits in normal tissue was found to be relatively constant around 1:1, the relative amount of RI and RII varies and depends highly on physiological conditions and the hormonal status of the tissue (13, 14, 22, 23). One recent study shows that in RIIbeta gene knockout mice, an increase level of RIalpha compensates the loss of RIIbeta in brown fat cells. The switching of PKA isoform from type II to type I resulted in an increased basal level of PKA activity and increased energy expenditure. The RIIbeta knockout mice are leaner and protected against diet-induced obesity (24). These results demonstrate clearly that RI and RII are functionally distinct.

While both RI and RII contain two tandem and highly conserved cAMP-binding domains at the carboxyl terminus (25), RI and RII differ significantly at their amino terminus, especially at a proteolytically sensitive hinge region that binds to the peptide recognition site of the C subunit. The hinge region of RII subunits contains a serine at the phosphorylation (P) site that can be autophosphorylated by the C subunit (26); whereas RI subunits contain a pseudo phosphorylation site (Arg-Arg-Gly-Ala-Ile) and type I holoenzyme has an essential high affinity-binding site for MgATP (27, 28). When an autophosphorylation site was introduced into the RI subunit by replacing the alanine with a serine, the mutant RI subunit became a good substrate for the C subunit. However, the formation of holoenzyme was no longer facilitated by MgATP, and the affinity of the mutant holoenzyme for cAMP was independent of MgATP (29). In contrast, elimination of the autophosphorylation site by point mutation causes the RII subunit to lose its ability to revert transformed fibroblasts (30).

The amino acid sequence flanking the phosphorylation site of protein kinase substrates has been shown to be important for substrate specificity. Most PKA phosphorylation sites contain Arg at the P-3 and P-2 sites and a hydrophobic amino acid at the P+1 position (31). A close examination of the primary sequence immediately NH2-terminal to the inhibition site of the R subunits reveals that there is significant sequence diversity between RI and RII in this region (Fig. 1). This suggests that recognition of RI and RII by the C subunit at this region could be different. We tested this hypothesis by introducing specific mutations into the NH2-terminal proximal side, an adjacent region that interacts with the sequence NH2-terminal to the inhibition site of R subunit, of the active site cleft of the C subunit. Interaction between mutant C subunits and RIalpha and RIIbeta was then quantitatively examined by kinetic and surface plasmon resonance studies.



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Fig. 1.   Sequence alignment of the hinge of different R subunit isoforms. Amino acid sequences immediately NH2-terminal to the autoinhibitor sites (underlined) in R subunits are compared with that of PKI. P (the phosphorylation site), P-3, P-4, P-5, P-6, P-11, and P+1 sites are marked by arrows.



    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All chemicals used are either reagent grade or molecular biology grade. Kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly, was synthesized at the Peptide and Oligonucleotide Facility at the University of California, San Diego, and further purified by reverse-phase preparative high performance liquid chromatography before use. The concentration of the peptide substrate was determined by turnover with the catalytic subunit under the conditions of limiting peptide. Oligonucleotides were synthesized with a DNA synthesizer (Applied Biosystem Inc., model 380B). Pyruvate kinase from rabbit muscle and lactate dehydrogenase from bovine heart were from Sigma.

Protein Preparation-- Mutations R133A and D328A were introduced into the C subunit by the Kunkel method as described previously (32). The mutant proteins C(R133A) and C(D328A) were expressed in Escherichia coli BL21(DE3) using the pLWS-3 vector (33). The recombinant wild-type and mutant C subunits were purified by phosphocellulose chromatography and FPLC Mono S column (34). Isozyme II was pooled and used for all experiments.

Wild type RIalpha was purified using DE52 anion exchange chromatography followed by FPLC gel filtration on Superdex 200 (Amersham Pharmacia Biotech) as described previously (35). H6-RIalpha was purified on nickel resin (Qiagen) and RIIbeta was isolated by co-purification with H6-C using a protocol similar to that of Hemmer et al. (36). Briefly, equal volumes of E. coli cultures that overexpressed RIIbeta and H6-C were mixed and co-lysed in the presence of 5 mM MgATP. After batch binding the holoenzyme onto Ni2+ resin, the free RII was eluted with 5 mM cAMP. Pooled fractions from the cAMP elution were precipitated with ammonium sulfate, and further purified by FPLC gel filtration on a Superdex 200 column. To obtain cAMP-free R subunits, RIalpha and RIIbeta were dialyzed against 8 M urea to completely remove cAMP. The chemically denatured R subunits were then refolded by dialysis to remove the urea (37), and further purified by passing through a Superdex 200 gel filtration column. All proteins were at least 95% pure judged by SDS-polyacrylamide gel electrophoresis.

Holoenzyme Formation-- PKA holoenzymes were reconstituted from individually purified C and R subunits by mixing freshly isolated R and C subunits in a molar ration of 1:1.2 and dialyzed against buffer A (20 mM potassium phosphate, 100 mM KCl, 2 mM dithiothreitol, 0.1 mM ATP, 1 mM MgCl2, and 5% glycerol, pH 6.5) extensively at 4 °C to remove excess cAMP. The dialyzed sample was assayed for PKA activity first in the absence of cAMP and then in the presence of 100 µM cAMP to confirm the formation of the PKA holoenzyme. PKA holoenzyme complex was further purified on a Superdex 200 FPLC gel filtration column to remove excess C subunit.

Trypsin Digestion of the Holoenzyme-- A typical reaction mixture (12 µl) contained 20 µg of RIalpha 2C2 holoenzyme, 35 ng of trypsin, and with or without 8 mM cAMP. The digestion reaction was started by adding the protease, and 5 µl of reaction mixture was withdrawn and mixed with 5 µl of 2 × SDS sample buffer after incubating at room temperature for 10 or 30 min. The samples were boiled for 2 min and loaded onto a 12% SDS-polyacrylamide electrophoresis gel for analysis of the digestion products.

Sequencing of Cleavage Products-- After electrophoresis, proteins were transblotted onto a polyvinylidene difluoride (0.2 µm, Bio-Rad) membrane. The electroblotting was performed in 10 mM Caps, 10% methanol (pH 11) buffer at 200 mA constant current for 1 h. After rinsing with water (3 times) and methanol, the membrane was stained with 0.1% Coomassie R-250 in 1% acetic acid, 40% methanol for 2 min. The membrane was destained in 50% methanol and patted dry after rinsing with water. The protein bands of interest were excised from the membrane with a razor blade and subjected to NH2-terminal protein sequencing.

Protein Kinase Activity Assay-- The enzymatic activity of the C subunit was measured spectrophotometrically with a coupled enzyme assay (38). In this assay, the formation of the ADP is coupled to the pyruvate kinase and lactate dehydrogenase reactions. The reaction rate is determined by following the decrease in absorbance at 340 nm. Reactions were pre-equilibrated at room temperature and initiated by adding the peptide substrate. The Michaelis-Menten parameters for ATP and Kemptide were determined by fixing one substrate at near saturating concentration while varying the concentration of the other substrate. Inhibition of the catalytic subunit by R or PKI was assayed by mixing varying amounts of inhibitor with 20 nM C subunit and subsequently determining the decrease of phosphotransfer activity.

A radioisotopic method (39) was also used to measure the activity of the C subunit at low concentration, typically around 100 pM. With this assay, the inhibition constants of R and PKI to the catalytic subunit can be accurately determined. The kinase reaction mixture (50 µl) contained 50 mM Mops (pH 7.0), 10 mM MgCl2, 0.25 mg/ml bovine serum albumin, 0.1 mM peptide, and 0.1 mM ATP at 100 cpm/pmol, 100 pM catalytic subunit, and varying amount of inhibitor, RIalpha , RIIbeta , or PKI. The reaction was pre-equilibrated at room temperature for 10 min and initiated by adding the peptide substrate. Following a 10-min incubation, aliquots (45 µl) were withdrawn, spotted onto discs of Whatman P81 paper, and immediately immersed in 75 mM phosphoric acid (10-20 ml/sample) to terminate the reaction. After washing three times in phosphoric acid and once with ethanol, the discs were dried under a heating lamp. Radioactivity was measured by liquid scintillation spectrometry.

Surface Plasmon Resonance-- Surface plasmon resonance was used to measure binding between wild-type/mutant C subunits and R subunits, RIalpha and RIIbeta . C(R133A), C(D328A), and wild-type C subunits were immobilized to a sensor chip (CM dextran surface) by direct amine coupling as described previously (40). All surface plasmon resonance experiments were performed in running buffer containing 20 mM Mops (pH 7.0), 150 mM KCl, 0.1 mM ATP, 1 mM MgCl2, 0.5 mM dithiothreitol, and 0.005% polysorbate 20, a nonionic detergent surfactant, at room temperature. For each run, five injections at different R concentrations between 30 and 500 nM were performed and sensorgrams were collected for each injection. The C subunit surface was regenerated by injection of 10 µl of 10 µM cAMP and 4 mM EDTA in running buffer following each injection of R subunit.

Kinetic constants of binding were obtained using the BIAcore pseudo-first order rate equation,
<FR><NU>dR</NU><DE>dt</DE></FR>=K<SUB><UP>asso</UP></SUB>CR<SUB><UP>max</UP></SUB>−(k<SUB><UP>asso</UP></SUB>C+k<SUB><UP>diss</UP></SUB>R<SUB>t</SUB>) (Eq. 1)
Where kasso is the association rate, kdiss is the dissociation rate, C is the concentration of injected analyte, and R is the response. The dissociation rate, kdiss, was calculated by integrating the rate equation when C = 0, yielding ln(Rt1/Rtn) kdiss(tn - t1). Plots of dR/dt versus Rt have a slope of kdiss. Plotting kdiss against C gives a slope that is equal to the kasso. Affinity constants were calculated from the equation Kd = kdiss/kasso.

Data Analysis-- The Michaelis constant, Km and maximal velocity, Vmax were determined from plots of initial velocity, v versus substrate concentration, [S] according to,


v=<FR><NU>V<SUB><UP>max</UP></SUB>[S]</NU><DE>(K<SUB>m</SUB>+[S])</DE></FR> (Eq. 2)
The maximal velocity was then converted to kcat by dividing Vmax by the total enzyme concentration.

The apparent inhibition constant, Ki,app, was obtained directly fitting the inhibition curves of C subunits by R subunits or PKI according to,
<FR><NU>V</NU><DE>V<SUB>0</SUB></DE></FR>=<FR><NU>K<SUB>i,<UP>app</UP></SUB></NU><DE>K<SUB>i,<UP>app</UP></SUB>+[<UP>I</UP>]</DE></FR> (Eq. 3)
where V and V0 are the measured kinetic rate of the catalytic subunit in the presence or absence of inhibitor, respectively, [I] is the total inhibitor concentration.

Since both PKI and R subunits act as competitive inhibitors of the catalytic subunit when Kemptide is used as a substrate, the inhibition constant, Ka, can be further calculated from the Ki,app using the Morrison equation,
K<SUB>i</SUB>=<FR><NU>K<SUB>i,<UP>app</UP></SUB></NU><DE>1+<FR><NU>[S]</NU><DE>K<SUB>m</SUB></DE></FR></DE></FR> (Eq. 4)
where [S] is the Kemptide concentration and Km is the Michaelis constant of catalytic subunit for Kemptide.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteolytic Digestion of Type I Holoenzyme Complex-- Trypsin digestion of type I holoenzyme resulted in two stable protein fragments that corresponded to Delta (1-72)R (band A, Fig. 2) and Delta (1-8)C (band B, Fig. 2) based on NH2-terminal protein sequencing. Cleavage of the holoenzyme in the presence of cAMP led to further degradation of the regulatory subunit. Between residues 72 and 92 (P-4 Arg) there are two potential trypsin cleavage sites at residues 76 and 90. The apparent protection of these two cleavage sites in the holoenzyme complex indicated that this region of the R subunit (between residues 73 and 91) was also involved in holoenzyme formation.



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Fig. 2.   Trypsin digestion of the type I holoenzyme in the absence and presence of cAMP. Type I holoenzyme was digested by trypsin in the presence and absence of cAMP as described under "Experimental Procedures." Arrows indicate the original position of the R and C subunit, and cleavage products A and B.

Isoform Specific Differences in the P-11 to P-4 Region of RI and RII Subunits-- The proteolysis results led us to focus on potential interactions between the first few residues (P-11 to P-4) that lie NH2-terminal to the consensus peptide of R subunits at the active site cleft of the C subunit. A close comparison of the structure of C:PKI-(5-24) and the isoform-specific differences between the RI and RII subunits (Fig. 1) suggested that there may be key differences in the way that the R subunits in the P-11 to P-4 region NH2-terminal to the consensus site complement the active site of the C subunit. This led us to further probe the interactions between the C subunit and R subunit isoforms by introducing two mutations (R133A and D328A) into the C subunit around the active site cleft.

Characterization of Catalytic Mutants-- Mutants C(R133A) and C(D328A) expressed in E. coli to levels that were similar to the wild-type C and could be purified readily to homogeneity using conventional or co-lysis purification procedures. The purified mutants (isozyme II) phosphorylated at Ser338, Thr197, and Ser10 have atomic masses of 40,597 ± 5 and 40,642 ± 4, respectively, as determined by electron spray mass spectroscopy. These values are in good agreement with the calculated values of 40,597 and 40,638 as expected for the Arg to Ala and Asp to Ala mutations.

The steady-state kinetic properties of the mutants were determined and summarized in Table I. Similar to that of the double mutant C(R133A,R134A) (32), mutant C(R133A) showed a 2-3-fold increase in its Km for both Kemptide and ATP and a 2-fold increase in kcat. Mutant C(D328A) displayed a 2-fold increase in its Km for Kemptide while its kcat and Km for ATP remained unchanged as compared with the wild-type C. 


                              
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Table I
Kinetic parameters for mutant and wild-type C subunit

Inhibition of C(R133A), C(D328A), and Wild Type C Subunits by RIalpha , RIIbeta , and PKI-- Having demonstrated that the kinetic properties of the mutant catalytic subunits were not significantly perturbed, the ability of these mutant C subunits to be inhibited by RIalpha , RIIbeta , and PKI was tested using the spectrophotometric assay. When 20 nM of the wild-type and mutant catalytic subunits were used in the assay, cAMP-free RIalpha titrated the C(R133A) and wild-type C subunits stoichiometrically, indicating that the affinity of RIalpha for these C subunits is much lower than 20 nM. However, mutant C(D328A) required higher concentrations of RIalpha to be inhibited to the same extent as the wild-type C subunit. Quantitative analysis of the inhibition curve gave an apparent inhibition constant of 28 nM (Fig. 3A) and a calculated intrinsic inhibition constant of 2.2 nM (Table II). The opposite was observed when stripped RIIbeta was used. RIIbeta , while it inhibited the wild-type C and C(D328A) readily, showed a decreased affinity toward C(R133A) (Fig. 3B). The measured Ki,app and Ka of RIIbeta to C(R133A) were 21 and 1.9 nM, respectively (Table II). Interaction between PKI and mutant catalytic subunits were also measured. As shown in Fig. 3C, linear inhibition curves were obtained when the wild-type and C(D328A) were titrated with PKI. In contrast, the inhibition constants of PKI to C(R133A) were 888 and 83 nM, for Ki,app and Ka, respectively. These results were in good agreement with the early observation (32).



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Fig. 3.   Inhibition of the activity of wild-type and mutants catalytic subunits by RI, RII, and PKI. The activity of 20 nM wild-type (), C(R133A) (black-triangle), and C(D328A) (black-square) in the presence of varying amounts of RI (A), RII (B), and PKI (C) was measured by the spectrophotometric assay.


                              
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Table II
Intrinsic inhibition constants for mutant and wild-type C subunit

The radioisotopic method was also used to study the tight interaction between catalytic subunits and R subunits and PKI so that the effects of mutations C(R133A) and C(D328A) on interaction with RIalpha , RIIbeta , and PKI could be quantitatively assessed and compared. Since it was possible to measure the PKA activity at very low concentration (pM) with the radioisotopic method, subnanomolar affinity could be easily measured. Inhibition of 100 pM wild-type and C(D328A) catalytic subunits by PKI was measured by 32P-phosphorylation of Kemptide, and the result was summarized in Fig. 4A. It was clear that unlike C(R133A), mutant C(D328A) interacted with PKI with high affinity similar to the wild-type C-subunit. The inhibition constant, Ki, for C(D328A) is 0.49 nM (Table II).



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Fig. 4.   Inhibition of the activity of wild-type and mutants catalytic subunits by RI, RII, and PKI. The activity of 100 pM wild-type (), C(R133A) (black-triangle), and C(D328A) (black-square) in the presence of varying amounts of PKI (A), RI (B), and RII (C) was measured by the radioactive assay.

The inhibition constants for RI to wild-type C and C(R133A) and for RII to wild-type C and C(D328A) were also determined (Fig. 4, B and C). These inhibition constants, summarized in Table II, established clearly that mutation of 133 reduced affinity of the C subunit for RII and PKI but not for RI. In contrast, mutation of 328 affected interaction between C and RI but not RII and PKI.

R-C Interaction Measured by Surface Plasma Resonance-- As mentioned above, interaction between C(R133A) and RIalpha and between C(D328A) and RIIbeta , was measured indirectly by the ability of these mutants to be inhibited by the R subunits. Surface plasmon resonance was used to further confirm these observations and to obtain a direct estimation of the binding affinities of these mutant C subunits to RIalpha and RIIbeta . After immobilizing C, C(R133A), and C(D328A) to a sensor chip by amine coupling, binding of RIalpha and RIIbeta was measured.

The association and dissociation constants for wild-type, C(R133A), and C(D328A) C subunits obtained by surface plasmon resonance measurements were summarized in Table III. Overall, the association and dissociation constants for C subunit to RIalpha were 8 × 105 M-1 s-1 and 2 × 10-4 s-1, respectively. However, C(D328A) showed a slower association constant (4.5 × 105 M-1 s-1) and a faster dissociation constant (5.9 × 10-4 s-1). This led to an apparent Kd of 1.3 nM, a 5-fold increase as compared with that of the wild-type and C(R133A) C subunits. Although the C subunit interacted with the RIIbeta with similar affinity as with RIalpha , with Kd values around 0.2 nM, both association and dissociation constants were six times faster for interaction between C subunits and RIIbeta than for C subunit and RIalpha . In contrast to RIalpha , C(R133A) displayed a Kd of 1 nM RIIbeta , a 5-fold increase as compared with that of the wild-type C subunit, interaction between the C(D328A) and RIIbeta was indistinguishable from wild-type C. This was exactly the opposite of the observation made for interaction between the C subunits and RIalpha , and was consistent with the inhibition data.


                              
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Table III
Apparent binding constants between R and C subunits measured by surface plasmon resonance



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Two major interaction sites between the R and C subunits were defined previously (36, 41, 42). One is the autoinhibitory consensus site in the linker region of R that fills the active site cleft of the C subunit and competes for the substrate-binding site (41). The other site, termed a peripheral recognition site, lies COOH-terminal to the inhibitory consensus site and complements the surface of C that is dominated by phosphothreonine 197 and the activation loop (36, 42, 43). This peripheral recognition site is an essential docking surface for cAMP-binding domain A in the holoenzyme complex. The exact boundaries of R/C interaction at the active site cleft, however, are not clear at present. Using limited trypsin digestion to map the core of the type I holoenzyme complex, the apparent protection of two tryptic cleavage sites, at residues 76 and 90, between residue 72 and the pseudo-substrate inhibition site in the R subunit clearly suggests that the NH2-terminal boundary of the RI subunit interaction site is between residues 72 and 76, indicating that this region that lies NH2-terminal to the inhibition site may also play an important role in R/C interaction. Although all R subunit isoforms share extensive sequence similarity and contain two tandem cAMP-binding domains that are homologous to the cAMP-binding domain of the cAMP receptor protein in E. coli (25), RI and RII show distinct sequence differences that are conserved within each subfamily (44). These sequence differences are most striking in the linker region that extends from the dimerization/docking domain through the consensus inhibitor site. In RII the consensus inhibitor site contains the sequence RRXS, an autophosphorylation site for C (26), whereas the RI has the sequence RRXA that cannot be autophosphorylated but is important for high affinity binding of MgATP (28). However, the differences between RI and RII cannot be accounted for exclusively by the presence or absence of the autophosphorylation site. Given the proteolysis results, coupled with modeling based on the C:PKI crystal structure, we reasoned that the residues that immediately precede the consensus site arginines might be important for holoenzyme formation. This analysis led us to test two specific sites, Arg133 and Asp328, on the surface of the C subunit as potential isoform-specific interaction sites for the P-11 to P-4 regions of RI and RII.

Arg133 and Asp328, located on the NH2-terminal proximal side of the peptide-binding site in the catalytic subunit, define two potential binding sides that lie on opposite sites of the peptide-binding cleft. Arg133 reaches out from the large lobe and is part of the hydrophobic binding pocket where the P-11 Phe in PKI docks, while Asp328, is part of the mobile acidic patch in the COOH-terminal tail that is functionally a part of the small lobe (Fig. 5). Asp328 was identified as a putative recognition site for a basic residue at the P-4 position of peptide substrates when an oriented peptide library was screened to determine the optimal peptide substrate (45). Substitution of Arg177 and Lys178 (the Arg133/Arg134 equivalents) of the yeast C subunit with Ala caused a 4.2-fold decrease in binding affinity for the yeast R subunit, which is more closely related to the mammalian type II R subunit, without impairing the catalytic activity (43). The equivalent mutations in murine C subunit (R133A/R134A) led to a defect in PKI interaction without significantly affecting the interaction with the RI subunit (32). Arg133, together with Tyr235, Pro236, and Phe239 form the hydrophobic P-11 binding pocket that is necessary for tight binding of PKI (32). Based on these observations and on the isoform-specific differences in RI and RII in the P-6 to P-4 region, mutations of R133A and D328A were introduced to dissect interaction between the catalytic subunit and specific regulatory subunit isoforms.



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Fig. 5.   GRASP representation of the crystal structure of the catalytic subunit of cAMP-dependent protein kinase showing the locations of Arg133 and Asp328. The surface of the C subunit that precedes the active site cleft is shown with positions of Asp328 and Arg133 highlighted. Also shown are the residues that contribute to the P-2 site (Glu230 and Glu170) and P-11 site (Tyr236, Pro236, and Phe239). The P-3 to P+1 portion of consensus site peptide from the MnATP·PKI-(5-24)·C subunit ternary complex (1ATP) is also shown. The boxes indicate the two surfaces thought to be complemented by RI and RII, respectively. The figure was generated from the ternary complex where PKI-(5-24) was deleted using the GRASP program.

C(R133A) and C(D328A) exhibited similar kinetic parameters both for Kemptide and ATP as the wild-type C indicating interaction between the mutant C subunits and this substrate is not significantly perturbed by these mutations. On the other hand, a 5-fold decrease in binding affinity between C(D328A) and RIalpha was observed while interaction of C(D328A) with RIIbeta and PKI remained unchanged. Hence, it can be concluded that Arg328 is a RI-specific interaction site. This is consistent with the mutational study showing that equivalent mutation in yeast C subunit does not affect its interaction with the yeast R subunit, which is more closely related to the type II R subunits (43). Based on sequence alignment of R subunits and PKI, only RI subunits contain a conserved Arg at the P-4 position. The likely reason for such selectivity is that a basic residue at P-4 position can interact favorably with Asp328 (45). Manual substitution of the P-4 Gly of PKI with Arg in C:PKI crystal structure model confirms that Asp328 is within hydrogen-bonding distance with the guanidine group of the P-4 Arg.

Mutation R133A significantly impairs the interaction of C with PKI. The measured inhibition constant, Ki, for the mutant C was increased more than 300-fold, in a good agreement with an early study (32). Interaction between C(R133A) and RIIbeta was also affected, but to much less extent with a 5-fold decrease in binding affinity, whereas C(R133A) mutant was identical to the wild-type C subunit in terms of forming holoenzyme with the RIalpha subunit. The interaction of R subunits with C at the NH2-terminal proximal side of the peptide-binding site is thus isoform specific. Furthermore, RII is more like PKI than RI in terms of interacting with C at the NH2-terminal proximal side of the peptide-binding site. This is in contrast to the consensus site peptide itself where PKI more closely resembles RI having: 1) an Ala at the P site and 2) an ATP requirement for tight binding to C. These differences can be attributed to the diversity of the amino acid sequence immediately NH2-terminal to the autoinhibition site in the RI and RII subunits. In this region, while RI and RII differ significantly, sequence similarity between RII and PKI is apparent. Both RII and PKI contain an Arg at the P-6 position and this Arg is important for PKI binding to C (46). Although RII lacks the P-11 Phe, another important amino acid in PKI that is critical for catalytic subunit recognition, modeling of the C:PKI crystal structure by substituting the P-5 threonine with phenylalanine, a conserved amino acid residue in RII, suggests that the aromatic side chain of this P-5 phenylalanine can reach down to occupy the space that is filled by the P-11 Phe in PKI. Thus, the highly conserved P-5 Phe in RII can potentially function in a similar way as the P-11 Phe in PKI. Both side chains could fill the same hydrophobic pocket formed by Arg133, Tyr235, Pro236, and Phe239. The P-6 Arg of RII would then fill the same position as it does in PKI interacting with Glu203.

The fact that RI and RII interact differentially with the C subunit at P-6 to P-4 proximal to the active cleft could also have significant consequences for the overall hydrodynamic features of the type I and type II holoenzymes. If P-6 to P-4 residues direct R subunits in different directions, then the NH2-terminal dimerization domain which is also the docking site for AKAPs could be oriented differently relative to the rest of the protein for RI and RII. This difference in orientation of the NH2 termini of R subunits would eventually lead to different overall structures between type I and type II PKA holoenzymes (Fig. 6). The Stokes radius of the type I holoenzyme (52 Å) versus the type II holoenzyme (57 Å) is indicative of significant differences in overall shape and, therefore, supports our interpretation. Furthermore, the major parameters for determining these differences in Stokes radii are associated with the region that links the dimerization domain to the consensus site peptide in R subunits based on the Stokes radii of RI/RII chimeras where the dimerization domain of RIalpha and RIIbeta are switched (47). These results are consistent with our observations that the P-11 to P-4 linker regions in different R subunit isoforms differentially interact with the C subunit in PKA holoenzyme complexes. As indicated in Fig. 6, the P-5 and P-4 Arg's in RI would direct the peptide chain toward the carboxylate-rich COOH-terminal tail of the C subunit, most likely locking the COOH-terminal tail firmly into a closed conformation. This is totally consistent with the fact that MgATP is required for the formation of type I holoenzyme, and MgATP is essential for the closed conformation of the C subunit (28, 48). It is also consistent with the early observations that the type I holoenzyme is sensitive to dissociation with high salt or histones (28) which both would disrupt the acid-rich tail and lead to the release of ATP and activation of the holoenzyme. In contrast, the same region in RII could interact with the NH2-terminal proximal side of the inhibitor recognition site of C in a similar way as PKI, making contact with C(R133A) and the P-11 hydrophobic pocket. These interactions are exclusively with the large lobe and do not require a closed conformation. In addition, they are hydrophobic and thus would not be as sensitive to salt. The presence of a phosphor-Ser at the P site of the RII subunits further dictates that the conformation of C when it is bound to RII will assume a more open conformation. These interactions will orient the NH2-terminal dimerization domain of RII toward the large lobe (Fig. 6). This diversity in peptide recognition in close proximity to the active site also has implications for peptide substrate specificity and may explain the lack of single consensus sequence for substrates of PKA (31).



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Fig. 6.   Schematic representation of type I and type II PKA holoenzyme formation. Topology of PKA holoenzymes showing the different orientation of the N-terminal dimerization/docking domain of in type I and II holoenzyme complexes. In both holoenzyme complexes, the autoinhibition sequences of the R subunits dock into the active site cleft of the C subunit and the cAMP-binding domain A interact with the peripheral recognition site. However, the P-5 and P-4 Arg's in RI will direct the peptide chain toward the carboxylate-rich COOH-terminal tail of the C subunit, most likely locking the COOH-terminal tail firmly into a closed conformation. In contrast, the same region in RII can interact with the NH2-terminal proximal side of the inhibitor recognition site of C in a similar way as the PKI, making contact with C(R133A) and the P-11 hydrophobic pocket. These interactions are exclusively with the large lobe and do not require a closed conformation.

Of course, differential binding of RIalpha and RIIbeta to the C subunit at the region just NH2-terminal to active site cleft would not necessarily leads to different orientations of the entire NH2-terminal domain of the R subunit in type I and type II holoenzymes. The model presented in Fig. 6 is only the most likely possibility. Additional experimental data are required to validate our proposed model as well as the specific location of the NH2-terminal domain. However, this model is supported by a number of previous findings. In a recent study, Gangal et al. (50) showed that interaction of the RII subunit, but not the RI subunit, with the myristylated C subunit led to an increase in both NH2-terminal backbone flexibility and exposure of the NH2-myristate. In an earlier report, Herberg et al. (51) demonstrated that deletion of the NH2-terminal A helix of the C subunit significantly affected the interaction between the C subunit and RII subunit, but not the RI subunit. Collectively, these data and our current study strongly support a model that the orientation of the NH2 terminus of the R subunit is isoform-specific and that differential interaction between the R subunit isoforms and the C subunit at the region NH2-terminal to the pseudo substrate site maybe responsible for this difference.

The effects of the R133A mutation on the C-PKI interaction versus RI confirm that the region responsible for the high affinity binding of C resides primarily on the NH2-terminal side of the inhibition site (46, 49). On the other hand, perturbation on R-C interaction caused by both R133A and D328A mutations is much milder, indicating that residues important for tight binding of C are mainly located at the COOH-terminal side of the autoinhibitor site in R subunits (46, 49). However, results from this study suggest that although amino acid sequences NH2-terminal to the autoinhibitor site in R does not contribute significantly to the affinity of R-C interaction, this region may be functionally extremely important for distinguishing RI and RII isoforms.

Clearly, PKA isoforms I and II possess different physiological functions in vivo, and RI and RII subunits exert their distinct functions by assuming specific cellular localization and different cAMP sensitivity. The ability of the catalytic subunit to interact differentially with RI and RII subunits provides another mechanism for fine turning the regulation of PKA within cells although the precise nature of such regulation remains to be elucidated.


    ACKNOWLEDGEMENTS

We thank Siv Garrod for excellent technical support on protein sequencing, Teresa Clifford for assistance in protein purification, and Dr. Elzbieta Radzio-Andzelm for preparation of Fig. 5.


    FOOTNOTES

* This work was supported by United States Public Health Service Grant GM34921 (to S. S. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Jeane B. Kempner Scholar and supported by American Cancer Society postdoctoral fellowship PF-4315. Present address: Dept. of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031.

|| To whom correspondence should be addressed. Tel.: 858-534-3677; Fax: 858-534-8193; E-mail: staylor@ucsd.edu.

Published, JBC Papers in Press, November 10, 2000, DOI 10.1074.jbc.M006447200


    ABBREVIATIONS

The abbreviations used are: R, cAMP-dependent protein kinase regulatory subunit; AKAP, protein kinase A anchoring protein; C, cAMP-dependent protein kinase catalytic subunit; Caps, 3-(cyclohexylamino)-1-propanesulfonic acid; Grb2, growth factor receptor-bound protein 2; Mops, 3-(N-morpholino)propanesulfonic acid; PKA, cAMP dependent protein kinase; PKI, heat stable protein kinase inhibitor; SH3, Src homology domain 3; FPLC, fast protein liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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