©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis of the Regulatory Subunit (RII) of cAMP-dependent Protein Kinase II Reveals Hydrophobic Amino Acids That Are Essential for RII Dimerization and/or Anchoring RII to the Cytoskeleton (*)

(Received for publication, August 29, 1994; and in revised form, November 11, 1994)

Ying Li Charles S. Rubin (§)

From the Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In neurons cAMP-dependent protein kinase IIbeta (PKAIIbeta) is sequestered in the dendritic cytoskeleton because the regulatory subunit (RIIbeta) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RIIbeta binding (tethering) site. A key feature of the tethering site is that several amino acids with large aliphatic side chains mediate the high-affinity binding of RIIbeta.

Mutagenesis, recombinant protein expression, and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RIIbeta. Several crucial residues are located in an NH(2)-terminal region that encompasses amino acids 13-36. Substitution of Ala for Leu or Phe generates monomeric RIIbeta subunits that cannot bind AKAP75. The results are not due to general misfolding since mutant RIIbeta monomers bind cAMP and inhibit the catalytic subunit of PKAIIbeta with the same affinity and efficacy as wild-type RIIbeta dimers. Moreover, substitution of Ala for Leu, Val, Leu, Phe, Leu, or Leu and replacement of Leu with Ile or Val did not impair the dimerization reaction. Evidently, large hydrophobic side chains of Leu and Phe play pivotal roles in stabilizing RIIbeta-RIIbeta interactions. A secondary consequence of destabilizing RIIbeta dimers is the loss of intracellular targeting/anchoring capacity because monomers fail to bind AKAP75. Other NH(2)-terminal residues directly modulate the affinity of RIIbeta dimers for the AKAP75 tethering site. Replacement of Val-Leu with Ala-Ala produced a dimeric RIIbeta protein that binds AKAP75 4% as avidly as wild-type RIIbeta. It is possible that the aliphatic side chains of Val and Leu interact with the essential Leu and Ile residues in the AKAP75 tethering region.


INTRODUCTION

Cyclic AMP-dependent protein kinase IIbeta (PKAIIbeta) (^1)is the predominant PKA isoform and principal mediator of cAMP action in the central nervous system (reviewed in (1, 2, 3) ). More than 70% of PKAIIbeta is tethered to specific sites in the cytoskeleton or organelles of neurons via the high-affinity binding of RIIbeta by A Kinase Anchor Proteins (AKAPs)(1, 4, 5, 6) . Bovine AKAP75, human AKAP79, and rat AKAP150 are novel, homologous proteins that contain a conserved 22-residue domain near the carboxyl terminus that mediates the binding of RIIbeta(7, 8, 9) . RIIalpha is also bound at this site, whereas RIalpha and RIbeta are not ligands. Several amino acid residues with long aliphatic side chains (Leu, Leu, Ile, Leu, see (6) for sequence) contribute to a hydrophobic environment that stabilizes AKAP75bulletRIIbeta complexes(9) . Contributions of hydrophobic residues to the RIIbeta binding activity are dependent on both the size of the side chains and their position in the primary sequence(9) .

Recent studies indicate that AKAPs direct signals carried by cAMP to specific effector sites. In rat forebrain neurons AKAP150 and RIIbeta (PKAIIbeta) are enriched and co-localized along microtubules in the cytoskeleton of dendrites(5) . In cultured rat hippocampal neurons an AKAPbulletRIIbeta (PKAIIbeta) complex is necessary for the maintenance of kainate-stimulated ion currents through a glutamate-gated channel (10, 11, 12) . In addition, overexpression of AKAP75 in a model cell system depleted RIIalpha, RIIbeta, and catalytic subunits of PKA from the cytoplasm and sequestered PKAII isoforms in the cytoskeleton(13) .

The cited studies support a modification of a model originally proposed in Glantz et al.(5) and Hirsch et al.(6) : AKAPs place PKAIIbeta in proximity with (a) neurotransmitter-activated adenylate cyclase in the plasma membrane, (b) plasma membrane substrates such as neurotransmitter receptors (for desensitization) and ion channels, and (c) substrates in the cytoskeleton such as microtubule-associated proteins. This arrangement generates target sites for the reception and propagation of signals carried by cAMP. Moreover, the scheme permits the modulation of neuronal function over longer distances via alterations in the cytoskeleton and the electrical properties of synapses.

Residues 1-50 in RIIbeta and RIIalpha subunits are crucial for binding with AKAP75 and microtubule-associated protein 2, an RIIalpha selective binding protein(14, 15, 16) . This NH(2)-terminal region of RII subunits also mediates homodimerization(14, 17) . At present, little is known about individual amino acid residues at the NH(2) terminus of RII isoforms that promote dimerization and/or the tethering of RII subunits with AKAPs. Central questions are: which amino acids are essential for dimerization of RIIbeta or RIIalpha? Which residues are crucial for high-affinity binding with AKAPs? Are the AKAP binding and dimerization regions identical, or are they overlapping domains that may be partially resolved?

These questions were investigated by pursuing the observation that hydrophobic interactions are important factors in establishing stable AKAP75bulletRIIbeta complexes(9) . We now report that certain amino acids with large aliphatic side chains and an aromatic residue (Phe) play prominent roles in linking PKAIIbeta to AKAPs. Several of these residues are also involved in RIIbeta dimerization. However, some mutated RIIbeta dimers bind AKAPs poorly, whereas one monomeric mutant RIIbeta is an effective competitor for the binding site on AKAP75. AKAP binding and RIIbeta dimerization domains may be partially overlapping structures composed of both shared and distinct elements.


EXPERIMENTAL PROCEDURES

Mutagenesis and Protein Expression

cDNA encoding human RIIbeta was obtained from a brain cDNA library (Clontech) in bacteriophage gt10 by screening with a P-labeled 45-mer oligonucleotide that encodes amino acids 36-50 in the RIIbeta sequence(18) . The preparation and labeling of oligonucleotides and conditions for screening cDNA libraries are described in previous publications(19, 20) . A 1.5-kilobase cDNA insert, which contains the open reading frame for RIIbeta as well as 5`- and 3`-untranslated sequences, was excised from the recombinant gt10 vector by digestion with EcoRI. Recessed ends of the insert were made blunt and the cDNA was cloned into EcoRV-digested pGEM5Z (Promega). The sequence preceding and including the initiator ATG in RIIbeta cDNA was modified from AGGATG to CATATG by oligonucleotide-directed mutagenesis to create an NdeI restriction site. This recombinant plasmid was designated pGRIIbeta. Mutagenesis was performed as described previously(9) . A series of synthetic oligonucleotide primers (oligonucleotide facility of the Albert Einstein Cancer Center) was used to introduce the desired mutations in the NH(2)-terminal region of RIIbeta. The sequences of these primers and the corresponding changes in the amino acid sequence of RIIbeta are shown in Table 1. All mutated cDNAs were sequenced as described previously(1, 2) .



Wild-type and mutant cDNAs (474 base pairs) encoding residues 1-158 of RIIbeta (designated RIIbeta-N158) were excised from pGRIIbeta by digestion with NdeI and BamHI and cloned into the expression plasmid pET-14b (Novagen) which was digested with the same restriction enzymes. The partial RIIbeta cDNA is preceded by plasmid DNA that encodes an initiator Met and 19 additional amino acids (MGSSHHHHHHSSGLVPRGSH). Transcription of the chimeric gene is governed by a promoter sequence for bacteriophage T7 RNA polymerase.

Recombinant pET-14b plasmids were introduced into Escherichia coli BL21 (DE3)(2) . The host bacterium contains a chromosomal copy of the phage T7 RNA polymerase gene under the control of the lac promoter(21) . Cultures (200 ml) of transformed E. coli BL21 were grown to A = 0.8. Subsequently, isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 1 mM and the incubation was continued for 2 h at 37 °C. Next, bacteria were harvested by centrifugation at 8,000 times g for 10 min at 4 °C. All further operations were performed at 0-4 °C. Pelleted cells were washed once with 20 ml of 20 mM potassium phosphate buffer, pH 7.0. The bacteria were then resuspended in 3 ml of 20 mM potassium phosphate, pH 7.0, containing lysozyme (0.1 mg/ml), aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), and benzamidine (10 mM). After a 30-min incubation at 0 °C bacteria were disrupted by a combination of freezing in liquid N(2) and thawing at 4 °C (two cycles) and subsequent sonication for 60 s at half-maximal power in a Heat Systems ultrasonic disintegrator. The bacterial lysate was centrifuged at 40,000 times g for 30 min and the resulting supernatant solution was applied to a column (1 ml) of iminodiacetic acid Sepharose 6B (Pharmacia Biotech Inc.) that was charged with Ni(II) by pre-equilibration with 50 mM NiSO(4). The column was washed sequentially with 20 ml of 20 mM Tris-HCl, pH 7.9, containing 0.5 M NaCl and 5 mM imidazole, and then 30 ml of Tris-HCl, pH 7.9, containing 0.5 M NaCl and 60 mM imidazole. Fusion proteins were eluted with 8 ml of Tris-HCl, pH 7.9, 0.5 M NaCl, 1 M imidazole. Purified proteins were dialyzed against 3 changes (one liter each) of 10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl. After a final dialysis against phosphate-buffered saline containing 50% (v/v) glycerol the samples were stored at -20 °C. AKAP75 binding activity and/or physicochemical properties of the expressed proteins were maintained for more than 1 year under these conditions.

We described the cloning of the intron-less gene for AKAP75 into a mammalian expression vector (pAKAP75) in a previous publication(6) . The AKAP75 coding region was released from pAKAP75 by digestion with NotI and ApaI and subcloned into pGEM5Z that was cleaved with the same restriction enzymes. An NdeI site that includes the initiator ATG was created by site-directed mutagenesis as described above. DNA encoding full-length AKAP75 was prepared by cutting with XbaI (a unique XbaI site in the 3`-untranslated sequence precedes the ApaI site used for cloning into pGEM5Z, see (6) ), filling in the recessed terminus with Klenow DNA polymerase I, and then cleaving with NdeI. The resulting DNA fragment was cloned into pET-14b which was digested with BamHI, made blunt with Klenow DNA polymerase I and then cleaved with NdeI. Induction and purification of (His)(6)-AKAP75 were carried out as described above.

Intact, unmodified RIIbeta and several RIIbeta mutants (see ``Results'') were expressed in the baculovirus/Sf9 cell system. The baculovirus transfer vector pVL1392 and baculovirus DNA containing a lethal deletion were obtained from PharMingen (San Diego, CA). A cDNA insert that encodes the entire RIIbeta polypeptide was obtained from the plasmid pGRIIbeta (see above) by digestion with NdeI. After incubation with Klenow DNA polymerase I and dNTPs, the blunt-ended insert was cloned to the SmaI site of the transfer plasmid pVL1392. Recombinant baculovirus was produced and used to infect Sf9 cells according to the manufacturer's (PharMingen) protocols. Three days after infection, Sf9 cells from three T75 flasks were harvested by centrifugation at 1000 times g. After washing twice with phosphate-buffered saline at 4 °C, pelleted Sf9 cells were disrupted as described previously(22) . The cell lysis buffer (22) was modified by (a) substituting 20 mM sodium phosphate, pH 7.4, for 20 mM Tris-HCl, pH 7.5, and (b) omitting non-ionic detergent. Cell homogenates were centrifuged at 40,000 times g for 15 min at 4 °C and the supernatant solution (cytosol) was collected. The cytosol was supplemented with 0.15 M NaCl and 5% (v/v) glycerol prior to gel filtration analysis (see below).

Radiolabeling of RIIbeta, RIIbeta-N158 Proteins, and AKAP75

RIIbeta and RIIbeta-N158 proteins were labeled with P by incubation with [-P]ATP-Mg and the catalytic subunit of protein kinase A as previously reported(1) .

AKAP75 was phosphorylated by employing a similar procedure. Three modifications were made in the reaction mixture(1) : phosphate buffer was replaced with 25 mM Tris-HCl, pH 8.0; the catalytic subunit of PKA was replaced with homogeneous alpha subunit of C. elegans casein kinase II(23) ; and NaCl was added to a final concentration of 0.1 M.

Electrophoresis of Proteins

Samples of AKAP75, wild-type and mutant RIIbeta subunits, and wild-type and mutant NH(2)-terminal segments of RIIbeta were denatured in gel loading buffer and subjected to electrophoresis in polyacrylamide gels containing 0.1% SDS as described previously(1, 2) . Phosphorylase b (M(r) = 97,000), transferrin (78,000), albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), myoglobin (17,000), and cytochrome c (12,000) were used as standards for the estimation of M(r) values. Nondenaturing electrophoresis was performed in a 7.5% polyacrylamide gel at 4 °C as described previously(24) .

Overlay Binding Assays

The methodology and application of the ``overlay assay'' for detecting the binding of soluble P-labeled RIIbeta (or RIIalpha) with immobilized AKAPs has been described in several publications(1, 2, 25) .

In this paper we demonstrate that the binding assay can be executed successfully when the immobilized and soluble ligands are interchanged (see ``Results''). Western blots containing wild-type and mutant RIIbeta-N158 proteins were screened with 0.3 nMP-labeled AKAP75 (70,000 cpm/ml) in the overlay buffer. Conditions of the assay and procedures for the visualization and quantification of bound ligand were identical with those used for P-labeled RIIbeta(1, 2, 9, 25) .

Determination of Stokes Radius

Wild-type and mutant proteins that correspond to an NH(2)-terminal segment (residues 1-158) of RIIbeta were characterized by gel filtration on a column (1 times 51 cm) of Sephacryl S-100 (Pharmacia) that was equilibrated with 20 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1 mM EDTA, 0.2 mM dithiothreitol, and 5% (v/v) glycerol. The flow rate was 5 ml/h and 110 fractions (0.36 ml) were collected. Samples (5 µg) of P-labeled RIIbeta-N158 proteins were mixed with 2 mg of albumin and 2 mg of ovalbumin (to block nonspecific interactions of RIIbeta-N158 polypeptides with the beads) in a 0.4-ml column buffer and then applied to the column. The elution patterns of the RIIbeta-N158 proteins were determined by measuring P radioactivity in aliquots of each fraction in a scintillation counter. Elution patterns were confirmed by probing Western blots of proteins in aliquots (15 µl) of the fractions with anti-RIIbeta IgGs as described previously(13) . Immunoblots were developed by an enhanced chemiluminescence procedure as reported in previous studies(6, 9, 13) . Typically, 85-90% of the radiolabeled protein was recovered between the beginning and end of the elution peak. The column was calibrated with four standards (Sigma): cytochrome c (Stokes radius = a = 1.87 nm), ovalbumin (a = 3.05 nm), albumin (a = 3.55 nm), and transferrin (a = 4.0 nm). The void volume was determined with blue dextran (Pharmacia). Elution data were plotted according to Siegel and Monty (26) to generate a standard curve.

Stokes radii for full-length, wild-type, and mutant RIIbeta subunits were estimated by gel filtration on a column (1.5 times 80 cm) of Sephacryl S-300 (Pharmacia) equilibrated with the column buffer described above. The flow rate was 11 ml/h and 110 fractions (1.25 ml) were collected. The elution patterns of RIIbeta subunits were determined by performing [^3H]cAMP binding assays as described previously(27) . Approximately 80-85% of the applied binding activity was recovered in each case. The column was calibrated with apoferritin (a = 6.10 nm), aldolase (a = 4.81 nm), albumin (a = 3.55 nm), and ovalbumin (a = 3.05 nm). Standards were obtained from Sigma.

Sucrose Density Gradient Centrifugation

Radiolabeled RIIbeta-N158 and individual mutant RIIbeta-N158 proteins (5 µg, 1 times 10^6 cpm) were mixed with 0.5 mg of albumin, 0.5 mg of ovalbumin, 0.5 mg of cytochrome c, and 0.5 mg of bovine pancreatic trypsin inhibitor in 0.3 ml of 20 mM sodium phosphate, pH 7.2, containing 0.2 mM EDTA and 0.2 mM dithiothreitol. The mixtures were layered on top of linear 5-20% (w/v) sucrose gradients (10 ml) that were prepared in the same buffer. Samples were centrifuged at 39,000 rpm for 28 h at 5 °C in a Beckman SW41 rotor. After centrifugation, 50 fractions (0.2 ml) were collected and P radioactivity and RIIbeta-N158 protein were assayed as described above for gel filtration experiments. The distributions of the internal standards in the gradients were established by electrophoresis of aliquots of the fractions in a 0.1% SDS-15% polyacrylamide gel and subsequent staining with Coomassie Blue. Fractions obtained from parallel sucrose gradients that were loaded with 2 mg of a single standard were assayed by absorbance at 280 nm. The distributions of trypsin inhibitor (s(w) = 1.0), cytochrome c (s(w) = 1.85), ovalbumin (s(w) = 3.1), and albumin (s(w) = 4.3) determined by absorbance were in agreement with the results from SDS-polyacrylamide gel electrophoresis. Sedimentation coefficients for wild-type and mutant RIIbeta-N158 proteins were determined from a standard curve as described under ``Results.''

Measurement of the cAMP Binding Activities of RIIbeta and Mutant RIIbeta Subunits

Wild-type and monomeric mutant RIIbeta subunits (see ``Results'') were expressed in Sf9 cells and partially purified by gel filtration on a column of Sephacryl S-300 as described above. The concentrations of the RIIbeta proteins in peak fractions were determined by a quantitative Western immunoblot procedure as outlined in a previous publication(13) . Equilibrium [^3H]cAMP binding assays were carried out according to the procedure of Doskeland and Ogreid(28) .

Inhibition of the Catalytic Subunit of PKAIIbeta by Wild-type and Mutant RIIbeta Subunits

A fixed concentration of purified PKA catalytic subunit (29) was preincubated with various amounts of wild-type or mutant RIIbeta subunits for 1 h at 4 °C in 25 µl of Mops-Na0H buffer, pH 7.0, containing 1 mM dithiothreitol. Subsequently an aliquot (20 µl) was assayed for phosphotransferase activity using Kemptide (LRRASLG, Sigma) as a substrate. Assays were performed as described previously(30) . No inhibition was observed when 5 µM cAMP was added to the assays. Concentrations of the RIIbeta and catalytic subunits are given in Fig. 9.


Figure 9: Cyclic AMP and catalytic subunit binding activities of wild-type RIIbeta and monomeric RIIbeta mutants. A, the concentration dependence of [^3H]cAMP binding activity was determined for RIIbeta (box), Ala-Ala RIIbeta (circle), and Phe-RIIbeta (up triangle) as described under ``Experimental Procedures.'' Assays were performed with 40 nM wild-type RIIbeta; the concentration of mutant RIIbeta subunits was 35 nM. B, the ability of wild-type and mutant monomeric RIIbeta subunits to inhibit the phosphotransferase activity of the catalytic subunit (8 nM) of PKAIIbeta was assayed as described under ``Experimental Procedures.'' Percent maximum enzymic activity is reported as: (P radioactivity incorporated into the synthetic peptide substrate Kemptide in the presence of the indicated concentrations of RIIbeta or RIIbeta mutant) (P radioactivity incorporated into Kemptide in the absence RII subunits) times 100. Since the wild-type and mutant RIIbeta subunits produced very similar plots only a single line is drawn through the data points. The symbols used are the same as those in A. Experiments for A and B were replicated three times. Typical results are shown.




RESULTS

Strategies, Mutagenesis, and Functional Assays

Bulky hydrophobic side chains of several amino acids in the tethering domain of AKAP75 play prominent roles in mediating the high-affinity binding of RIIbeta(9) . In order to further elucidate the mechanism of anchoring of PKAIIbeta, we searched for ``complementary'' hydrophobic amino acid residues near the NH(2) terminus of RIIbeta that could promote complex formation with AKAP75. Boundaries for the range of the search were suggested by the observations that residues 1-50 in both RII subunit isoforms are sufficient to generate an AKAP binding site and a dimerization domain in the absence or presence of contiguous COOH-terminal sequences(14, 15, 17) .

Candidate hydrophobic residues in RIIbeta were substituted with Ala or other amino acids by site-directed mutagenesis, as described under ``Experimental Procedures.'' A list of RIIbeta mutants that were produced and studied is provided in Table 1. Following mutagenesis, cDNAs encoding partial (NH(2)-terminal) and full-length RIIbeta polypeptides were subcloned into the E. coli expression plasmid pET-14b. These constructs direct the synthesis of fusion proteins in which the RIIbeta sequences are preceded by 20 amino acid residues encoded by DNA in the plasmid (see ``Experimental Procedures''). When transformed E. coli were incubated with isopropyl-1-thio-beta-galactopyranoside the highest levels of fully soluble, recombinant proteins were observed for chimeric polypeptides that contained residues 1-158 of RIIbeta (Fig. 1A). Moreover, the presence of the oligopeptide sequence (His)(6), which corresponds to residues 5-10 in the fusion protein, enabled the single-step purification of recombinant RIIbeta (hereafter designated RIIbeta-N158) to near-homogeneity by chromatography on Ni-saturated iminodiacetic acid Sepharose 6B (Fig. 1B). In contrast, full-length RIIbeta fusion protein (438 amino acid residues) was expressed at a lower level and was partially (50%) insoluble. The soluble portion of full-length fusion protein bound the metal chelate resin with very low efficiency, thereby precluding facile purification of the various mutant proteins. Presumably, this is due to interactions between the NH(2)-terminal (His)(6) region and a sequence in the central or COOH-terminal region of RIIbeta.


Figure 1: Expression and purification of NH(2)-terminal segments of RIIbeta. Wild-type and mutant polypeptides that contain 158 residues of the NH(2)-terminal sequence of RIIbeta were expressed in E. coli BL21 as described under ``Experimental Procedures.'' A, samples of total soluble proteins derived from 0.1 ml of an E. coli culture were fractionated in a 0.1% SDS-10% polyacrylamide gel. The transformed bacteria were incubated for 2 h in the absence (odd-numbered lanes) or presence of isopropyl-beta-D-thiogalactopyranoside (even numbered lanes). The recombinant RIIbeta-N158 proteins contained either all wild-type residues (lanes 1 and 2) or Ala substitutions for the following pairs of residues: Leu-Leu (lanes 3 and 4), Val-Leu (lanes 5 and 6), Leu-Leu (lanes 7 and 8), Phe-Leu (lanes 9 and 10), and His-Phe (lanes 11 and 12). The gel was stained with Coomassie Blue. Induced, recombinant polypeptides exhibit an apparent M(r) of 25,000 (calculated M(r) = 19,600). This parallels the overestimation of the M(r) value for full-length RIIbeta under the same conditions(32, 33) . B, RIIbeta-N158 fusion proteins were purified as described under ``Experimental Procedures.'' Purified proteins were analyzed by electrophoresis in a 0.1% SDS-15% polyacrylamide gel and stained with Coomassie Blue. Lane W received the NH(2)-terminal segment of wild-type RIIbeta; lanes 2-8 and 10-15 contained the RIIbeta-N158 mutants listed in Table 1. The number of the lanes corresponds to the numbering of the mutants in Table 1. The protein content of the lanes varied over the range 0.4-1.4 µg.



The availability of purified wild-type and mutant RIIbeta-N158 polypeptides allowed direct characterization of their AKAP binding activities and physical properties in the absence of interfering proteins. To verify the validity of this approach, wild-type RIIbeta-N158, which contains the PKAIIbeta ``autophosphorylation'' site (Ser), was labeled by incubation with [-P]ATP and the PKA catalytic subunit and then incubated with a Western blot containing AKAP75. Immobilized anchor protein bound P-RIIbeta-N158 and intact P-RIIbeta (prepared as described in (24) ) with the same affinity. Typical results are shown in Fig. 2A. PhosphorImager analysis revealed that the molar ratio of bound RIIbeta-N158:bound RIIbeta remained at 1 as the concentration of the two proteins was varied over the range of 0.1-10 nM (data not shown). Binding of full-length and partial RIIbeta polypeptides was blocked by excess nonradioactive RIIbeta-N158 or RIIbeta (Fig. 2A). In competition binding experiments, both RIIbeta-N158 and RIIbeta had IC values of 2 nM. Dimerization of RIIbeta-N158 was demonstrated by gel filtration chromatography, sedimentation analysis, and nondenaturing electrophoresis (see below).


Figure 2: Characterization of recombinant AKAP75 and the AKAP75 binding activity of RIIbeta-N158. A, Western blot that contained 20 ng of recombinant AKAP75 in each of 6 lanes was prepared. Overlay binding assays were performed (see ``Experimental Procedures'') using 0.2 nMP-labeled RIIbeta-N158 (lanes 1, 3, and 5) or 0.2 nMP-labeled intact RIIbeta (lanes 2, 4, and 6). Each lane from the filter was incubated with 3 times 10^5 cpm of probe in 3 ml; probes were labeled to the same specific activity. Lanes 3 and 4 were probed in the presence of 100 nM nonradioactive RIIbeta-N158; lanes 5 and 6 were probed in the presence of 100 nM nonradioactive RIIbeta. An autoradiogram is shown. B, full-length AKAP75 was expressed as a (His)(6) fusion protein and was purified by Ni chelate chromatography. Samples from 3 different preparations (lanes 2-4) were analyzed by electrophoresis in a 0.1% SDS-8.5% polyacrylamide gel. Lanes 2-4 received 0.4, 1, and 2 µg of recombinant protein. Lane 1 contained the marker protein transferrin (M(r) = 78,000). A Coomassie Blue-stained gel is presented. Minor amounts of proteolytic fragments were occasionally observed.



Mutations in RIIbeta that disrupt AKAP binding activity can be detected rapidly and efficiently by reversing the standard RIIbeta binding ``overlay'' procedure (see ``Experimental Procedures''). To perform this screening assay DNA encoding full-length AKAP75 (6) was subcloned into pET-14b and the resulting AKAP75 fusion protein was purified to homogeneity by Ni-chelate chromatography (Fig. 2B). Purified AKAP75 was labeled with P by incubation with casein kinase II and [-P]ATP.

Mutational Analysis of the AKAP-binding/Dimerization Region of RIIbeta

Initial structure-function analysis involved the substitution of pairs of hydrophobic or neutral amino acids with Ala (mutations 1-8, Table 1). When a polyvinylidene difluoride filter containing purified, immobilized RIIbeta variants was probed with P-labeled AKAP75 (see ``Experimental Procedures'') three classes of mutated proteins were evident (Fig. 3). Replacement of Gly-Thr, Leu-Gln, or Glu^4-Ile^5 (not shown) had little effect on sequestration of AKAP75. These results suggest that side chains within a stretch of 33 residues (amino acids 6-38) are critical for the NH(2)-terminal functions of RIIbeta. Substitutions of various internal bulky hydrophobic residues (Val-Leu, Leu-Leu, and Phe-Leu) diminished, but failed to extinguish AKAP75bulletRIIbeta-N158 complex formation. The Leu-Leu to Ala-Ala substitutions produced the maximal decline (90%) in binding affinity in this subclass of RIIbeta mutants (Fig. 3, lane 4). In contrast, substitutions near the NH(2) and COOH termini of the functional domain(s) (Leu-Leu to Ala-Ala; His-Phe to Ala-Ala) abolished interactions between AKAP75 and the NH(2) terminus of RIIbeta (Fig. 3, lanes 2 and 6).


Figure 3: Screening mutant RIIbeta-N158 proteins for AKAP75 binding activity. Samples (25 ng) of wild-type and variant RIIbeta-N158 proteins were electrophoresed in a 0.1% SDS-10% polyacrylamide gel and then transferred to a polyvinylidene difluoride (Immobilon-P) membrane. The membrane was probed with P-labeled AKAP75 as described under ``Experimental Procedures'' and exposed to x-ray film at -70 °C. The resulting autoradiogram is shown. Lane 1 received wild-type RIIbeta-N158. Other proteins assayed were RIIbeta-N158 mutants with Ala substituted for: Leu-Leu (lane 2), Val-Leu (lane 3), Leu-Leu (lane 4), Phe-Leu (lane 5), His-Phe (lane 6), Leu-Gln (lane 7), and Gly-Thr (lane 8).



Properties of the mutated NH(2) termini were characterized further in competition binding assays (Fig. 4). Both intact RIIbeta (not shown) and wild-type RIIbeta-N158 potently inhibited the binding of P-RIIbeta to AKAP75 and yielded IC values of 2 nM under standard assay conditions. Replacement of Leu-Leu and Val-Leu with Ala-Ala diminished the avidity of the NH(2) terminus of RIIbeta for AKAP75 substantially, yielding IC values that increased 20-25-fold. Variants of the RIIbeta NH(2) terminus that contained Ala at positions 12 and 13 or 35 and 36 were unable to compete for the high-affinity binding site on AKAP75. Mutant polypeptides containing Ala-Ala substitutions for Gly-Thr (Fig. 4), Leu-Gln (not shown), and Glu^4-Ile^5 (not shown) exhibited IC values that were only 1.5-2.5-fold higher than that exhibited by the wild-type protein.


Figure 4: Competitive inhibition of the binding of RIIbeta to AKAP75 by wild-type and mutant RIIbeta-N158 proteins. Multiple Western blots, which contained 30 ng of AKAP75 in each lane, were prepared on Immobilon-P membranes. Individual excised lanes were incubated with 0.3 nMP-labeled RIIbeta (2 times 10^5 cpm in 2.5 ml) in the presence or absence of the indicated concentrations of nonradioactive competing polypeptides. The samples were processed according to the standard overlay binding procedure as indicated under ``Experimental Procedures.'' The amounts of P radioactivity bound to AKAP75 were quantified in a PhosphorImager (Molecular Dynamics) as described previously(13) . The data are presented as % maximal P binding activity. This represents: (amount of P bound in the presence of competitor protein the amount P bound in the absence of the competitor) times 100. These studies were replicated three times and yielded very similar results in each case. A typical set of results is shown. In addition, the same results were also obtained when nondenatured AKAP75 was directly applied to Immobilon-P and processed as described above. The competing proteins were: wild-type RIIbeta-N158 (bullet) and RIIbeta-N158 mutants in which Ala replaced Leu and Leu (circle), Val and Leu (box), Leu and Leu (), Gly and Thr (), or His and Phe (up triangle).



Leu and Phe in RIIbeta Play Dominant Roles in Establishing AKAP75 Binding and Subunit Dimerization Activities

Substitution of the long aliphatic side chains of Leu and Leu with Ala should have little or no effect on the net charge and helicity of the RIIbeta-derived polypeptides. It is likely that the principal consequence of the mutations is a reduction in hydrophobic surface area in the folded AKAP-binding region of RIIbeta(31) . In order to evaluate this proposition experimentally and determine whether Leu and Leu contribute equally or differentially to the AKAP binding site, six additional mutants of RIIbeta-N158 were generated (mutants 10-15, Table 1). Replacement of Leu-Leu with pairs of Ile or Val produced partial RIIbeta proteins that avidly complexed AKAP75 (Fig. 5, lanes 4 and 5). Conversion of Leu alone to either Ala or a charged residue (Glu) yielded a protein with AKAP75 binding activity that was indistinguishable from that displayed by the proteins containing Ile-Ile and Val-Val. However, insertion of Ala in place of Leu produced an RIIbeta NH(2)-terminal fragment that had a very low affinity for AKAP75 (Fig. 5, lane 3). Competition binding assays and quantitation via PhosphorImager analysis disclosed that the affinity of Ala RIIbeta-N158 for AKAP75 was reduced by >90% relative to the mutant polypeptides containing Ile-Ile (data not presented). Thus, it appears that a large aliphatic side chain at position 13 in RIIbeta is essential for tethering PKAIIbeta to AKAP75. Moreover, Leu plays a dominant, central role in facilitating complex formation whereas the neighboring Leu side chain contributes in a minimal manner.


Figure 5: AKAP75 binding activity of RIIbeta-N158 proteins that contain mutations at residues 12, 13, and 36. Samples (25 ng) of mutant RIIbeta-N158 proteins were applied to a polyvinylidene difluoride membrane and probed with radiolabeled AKAP75 as described in the legend for Fig. 3. The resulting autoradiogram is presented. The mutated NH(2)-terminal segments of RIIbeta contained the following amino acid substitutions: Glu for Leu (lane 1), Ala for Leu (lane 2), Ala for Leu (lane 3), Ile-Ile for Leu-Leu (lane 4), Val-Val for Leu-Leu (lane 5), and Ala for Phe (lane 6).



Substitution of Phe with Ala (mutant 15, Table 1) in RIIbeta yielded the most striking result. AKAP75 binding activity was totally abrogated (Fig. 5, lane 6). Thus, the bulky aromatic side chain subserves an essential and pivotal role in linking RIIbeta to AKAP75 and hence, the cytoskeleton.

Identification of Hydrophobic Amino Acids That Are Essential For RIIbeta Dimerization

The ability of wild-type and mutant RIIbeta-N158 polypeptides to dimerize was assessed by monitoring their hydrodynamic properties. Since intact R dimers are highly-elongated asymmetric proteins(32, 33) , it is not possible to estimate their molecular weights solely on the basis of either gel filtration experiments or sedimentation analysis(26) . However, if both Stokes radius and sedimentation coefficients are determined then accurate molecular weight values can be calculated as described by Siegel and Monty (26) (also see (32) ). Stokes radii were estimated by gel filtration on a calibrated column of Sephacryl S-100. Typical elution profiles for the wild-type protein and the Ala-Ala, and Ala-Ala, variants are shown in Fig. 6A and the calibration curve is shown in Fig. 6B. The wild-type protein and mutant polypeptides containing Ala-Ala, Ala-Ala, Ala-Ala, Ala-Ala, Ala, Glu, Ile-Ile, and Val-Val eluted between the transferrin and albumin standards and had Stokes radii of 3.85 nm. In contrast, mutant proteins that exhibited little or no AKAP binding activity (Ala-Ala, Ala, Ala mutants) and the Ala-Ala variant emerged after the albumin peak and had Stokes radii of 3.45 nm. Proteins with the larger and smaller Stokes radii were designated ``Group I'' and ``Group II'' proteins, respectively. The Stokes radii of all polypeptides in a given group were essentially the same: Group I, 3.85 nm ± 0.13 (S.E., n = 24); Group II, 3.45 nm ± 0.11 (S.E., n = 10). These two groups of proteins were also resolved by sucrose density gradient centrifugation. Representative fractionation patterns for Ala-Ala RIIbeta-N158 (Group I) and Ala-Ala RIIbeta-N158 (Group II) are shown in Fig. 7, A and B. Proteins in Group I had s(w) values of 2.2 ± 0.1 (Fig. 7B). Group II proteins migrated more slowly in the sucrose gradients and exhibited s(w) values of 1.3 ± 0.1.


Figure 6: Determination of the Stokes radius for wild-type and mutant RIIbeta-N-158 proteins. Samples of wild-type and variant proteins were labeled with P and characterized by gel filtration on a column of Sephacryl S-100 as described under ``Experimental Procedures''. A, superimposed representative elution patterns for wild-type RIIbeta-N158 () and mutants having Ala substituted for either Leu and Leu (bullet) or Val-Leu (). Only the relevant fractions are shown. B, the column was calibrated with standards (open circles) as described under ``Experimental Procedures.'' The data were plotted according to Siegel and Monty(26) . K = (elution volume of protein peak - void volume) (total column volume - void volume). Stokes radii measured for Group I and Group II RIIbeta-N158 proteins (see text) are indicated with solid circles. C, a correlation between oligomerization state and mobility in a nondenaturing polyacrylamide gel was established for RIIbeta-N158 proteins. Samples (1.5 µg of protein) of wild-type (lane 1) and mutant RIIbeta-N158 proteins were electrophoresed in a 7.5% polyacrylamide gel as described under ``Experimental Procedures.'' The variant polypeptides had Ala substituted for Leu and Leu (lane 2), Leu and Leu (lane 3), Val and Leu (lane 4), Phe and Leu (lane 5), Gly and Thr (lane 6), and Phe (lane 8). The mutant RIIbeta-N158 protein in lane 7 contained Ile in place of Leu and Leu. A gel that was stained with Coomassie Blue is shown. Proteins applied to lanes 1 and 4-7 were determined to be dimers (D) on the basis of their hydrodynamic properties; polypeptides in lanes 2, 3, and 8 are monomers (M).




Figure 7: Determination of sedimentation coefficients for wild-type and mutant RIIbeta-N158 proteins. Samples of wild-type and variant proteins were labeled with P, mixed with internal standards, centrifuged in a 5-20% (w/v) sucrose gradient, and assayed as described under ``Experimental Procedures.'' A, distribution of Ala-Ala RIIbeta-N158 after sucrose density gradient centrifugation; B, distribution of Ala-Ala RIIbeta-N158 after centrifugation. Only the relevant fractions are shown for A and B. Fraction 1 is at the top of the gradient. C, determination of s, values for Group I and Group II proteins from the calibration curve.



Molecular weights (M(r)) were then calculated (26) from the equation M(r) = [6N/(1-)] times [abullets], where a = Stokes radius; s = sedimentation coefficient; n = Avogadro's number; = viscosity of water; = density of water; = 0.731, the partial specific volume of wild-type (32) and mutant RIIbeta-N158. (^2)The experimental results for Stokes radius and s(w) yielded molecular weights of 36,000 for Group I proteins and 19,000 for Group II proteins. These values are within 10% of molecular weights established for RIIbeta-N158 dimer (39,200) and monomer (19,600) on the basis of derived amino acid sequence data(18) . Thus, most RIIbeta-N158 polypeptides that retain partial or maximal AKAP75 binding activity undergo dimerization. Conversely, mutant RIIbeta-N158 proteins that are either incapable of binding AKAP75 (Leu-Leu to Ala-Ala, Phe to Ala mutants) or exhibit an extremely low affinity for the anchor protein (Leu to Ala mutant) are monomers. The variant monomeric protein in which both Leu and Leu are replaced with Ala is an exception to these generalizations.

An independent analysis of the dimerization state of the variants was performed by nondenaturing electrophoresis. In this system proteins are resolved on the basis of their overall size and their charge to mass ratio(34) . In RIIbeta variants that contain Ala for Leu or Phe substitutions the charge to mass ratio is expected to be virtually constant. Thus, monomers should be readily separated from dimers by virtue of their higher mobilities. Electrophoretic analysis on a 7.5% acrylamide gel resolved the RIIbeta-N158 mutant proteins into two classes (Fig. 6C). The rapidly migrating group included the polypeptides that behaved as monomers in hydrodynamic experiments; the slowly migrating group corresponded to the homodimers.

Mutations That Disrupt the Dimerization and Anchoring of RIIbeta Have No Effect on cAMP Binding Activity and the Inhibition of the Catalytic Subunit

A caveat in the interpretation of the preceding experiments is that the replacement of Leu-Leu, Leu alone, or Phe with Ala might elicit the misfolding of the entire polypeptide. Since the partial proteins contained only residues 1-158 of RIIbeta, it was not possible to assess the long-range impact of amino acid substitutions on cAMP binding activity and the ability of RIIbeta to reconstitute the holoenzyme and inhibit the catalytic subunit.

In order to address these points cDNAs encoding selected full-length mutant and wild-type RIIbeta subunits were inserted into baculovirus (see ``Experimental Procedures''). RIIbeta and RIIbeta variants were then expressed as native (non-fusion) proteins in infected Sf9 cells. When Sf9 cytosol containing wild-type human RIIbeta was analyzed by gel filtration on a calibrated column of Sephacryl S-300, a large peak of [^3H]cAMP binding activity emerged at fraction 73 (Fig. 8A). The Stokes radius for the cAMP-binding protein was 4.9 nm, a value that is in excellent agreement with that determined for RIIbeta from bovine brain(35) . No cAMP binding activity was observed in cytosol from Sf9 cells infected with baculovirus lacking the cDNA insert. In contrast, RIIbeta containing Ala-Ala (Fig. 8B), Ala alone (not shown), or Ala (not shown) eluted at fractions 78 or 79 and had Stokes radius of 3.9 nm. Western immunoblot analysis disclosed that intact RIIbeta proteins (approximate M(r) 54,000) were produced in each instance, thereby indicating that the results were not attributable to proteolysis (Fig. 8C). After determining sedimentation coefficients as described above (data not shown), molecular weights of 51,000 to 53,000 were calculated for the RIIbeta variants. The M(r) of wild-type RIIbeta dimer is 97,000. Thus, as expected from the characterization of the NH(2)-terminal partial peptides, the mutant proteins were monomeric.


Figure 8: Determination of the Stokes radius for wild-type RIIbeta and an RIIbeta variant. RIIbeta and RIIbeta containing Ala-Ala in place of Leu-Leu were expressed in the baculovirus/Sf9 cell system (see ``Experimental Procedures'') and characterized by gel filtration on a column of Sephacryl S-300 as described under ``Experimental Procedures.'' Samples containing 1 mg of total cytosol protein (including 15 µg of RIIbeta or mutant RIIbeta) were applied to the column. Aliquots (50 µl) of the fractions were assayed for [^3H]cAMP binding activity. A, elution pattern for wild-type RIIbeta. Only the relevant fractions are shown. B, elution pattern for Ala-Ala RIIbeta. C, samples (15 µl) of the peak fractions were characterized by electrophoresis in a 0.1% SDS-10% polyacrylamide gel and subsequent Western immunoblot analysis with anti-RIIbeta IgGs(5, 13) . Lanes 1 and 2 contained wild-type RIIbeta; lanes 3 and 4 received Ala-Ala RIIbeta; lanes 5 and 6 received a sample of the Ala RIIbeta peak that was isolated in separate gel filtration experiment. The even-numbered lanes were probed with anti-RIIbeta IgGs in the presence of excess (300 ng) RIIbeta. The apparent M(r) of RIIbeta is 54,000. The Western blot was developed by an enhanced chemiluminescence procedure (ECL, Amersham) as described previously(9, 13) . Chemiluminescence signals that were recorded on x-ray film are presented.



Despite the difference in oligomerization state the mutant and wild-type RIIbeta polypeptides were equipotent in binding cAMP (Fig. 9A) and inhibiting the activity of the catalytic subunit of PKAIIbeta (Fig. 9B). Hence, domains proximal and distal to the NH(2) terminus of RIIbeta are correctly folded and function normally.


DISCUSSION

A combination of mutagenesis, recombinant protein expression, binding analysis, and hydrodynamic characterization revealed a group of amino acids in RIIbeta that mediate subunit dimerization and promote the binding of RIIbeta with AKAP75. The crucial residues are located in a dimerization/AKAP binding region that includes amino acids 13-36 at the NH(2) terminus of RIIbeta. Mutations introduced immediately upstream and downstream from the core region had little effect on the properties of partial polypeptides.

Leu, Phe, and the amino acid pairs Val-Leu and Leu-Leu play central roles in establishing a high-affinity binding site in RIIbeta that couples with AKAP75. Each of these amino acids has a bulky side chain that can participate in structure-stabilizing hydrophobic interactions(36, 37) . However, amino acids in this group exert their actions by two different mechanisms.

Replacement of residues at the NH(2) and COOH termini of the core region (Leu, Phe) with Ala markedly diminished or extinguished AKAP75 binding activity (Fig. 3Fig. 4Fig. 5). The loss of binding activity was associated with a change in oligomerization potential. The Ala RIIbeta-N158 and Ala RIIbeta-N158 mutants are monomeric proteins ( Fig. 6and Fig. 7). When the same mutations were introduced into full-length RIIbeta, monomeric proteins were again generated. However, the abilities of mutant RIIbeta monomers and wild-type RIIbeta dimer to bind cAMP and inhibit the catalytic subunit were indistinguishable (Fig. 9). Thus, the reduction in the size of the hydrophobic side chain at either residue 13 or 36 selectively disrupts dimerization, but has no effect on the folding of either proximal or distal segments of the RIIbeta polypeptide.

The deletion of NH(2)-terminal segments (45-90 residues) of R subunits produces monomeric proteins that retain two cAMP binding sites and the capacity to inhibit the catalytic subunit of PKA(14, 15, 17) . These observations on truncated R subunits and the present results on intact RIIbeta that contains a contiguous, but dimerization-defective NH(2)-terminal sequence, preclude subunit oligomerization as a key (positive or negative) factor in the generation of functional domains that regulate kinase activity.

Substitution of Leu with Ala is a conservative mutation. The introduction of Ala should neither alter the net charge of the protein nor affect substantially the propensity of the NH(2)-terminal sequence to fold into an alpha-helical or beta-strand conformation(38) . The hydrophobic character of the side chain is also preserved. Nevertheless, the functional outcome of this single mutation is both striking and illuminating: RIIbeta is unable to dimerize. An apparent consequence of the monomerization of RIIbeta is that the high-affinity binding site for AKAP75 is lost. The importance of the size of the aliphatic side chain at residue 13 was analyzed further by constructing additional RIIbeta-N158 variants. Mutant proteins that contained Ile-Ile, Val-Val (Fig. 5), or Ile alone^2 were dimers and readily formed complexes with AKAP75, although their affinities for the anchor protein were 2-3-fold lower than that exhibited by the wild-type protein. Thus, a bulky aliphatic side chain at residue 13 is sufficient to ensure the stabilization of dimeric RIIbeta and support binding with the anchor protein. Both conservative (Ala) and non-conservative (Glu) substitutions for the contiguous Leu residue failed to abolish either the dimerization or AKAP binding functions of the NH(2) terminus. Evidently, the position of the long-chain aliphatic amino acid in the primary sequence and presumably, its precise orientation in the folded, higher order structure of the NH(2)-terminal domain, play pivotal roles in conferring the properties of RIIbeta oligomerization and indirectly, the intracellular targeting of PKAIIbeta to the cytoskeleton. It seems likely that similar considerations apply to the role Phe. Further mutational analysis is underway to test this proposition.

In an earlier study Scott et al.(14) employed truncation analysis to investigate the interaction of murine RIIalpha with microtubule-associated protein 2, an RIIalpha-selective anchor protein (16, 25) . Deletions of 35, 30, or 14 residues from the NH(2) terminus of RIIalpha disrupted dimerization and abolished microtubule-associated protein 2 binding activity. The properties of the truncated RIIalpha subunits and the characterization of loss-of-function mutations in RIIbeta (Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8) suggest that subunit dimerization may be a pre-requisite for subsequent interactions with PKAII anchoring proteins. Key findings relevant to the model (14) are that ``native'' RIIbeta monomer (produced in eukaryotic cells) binds cAMP and inhibits PKA catalytic activity, yet it cannot be sequestered by AKAP75. Furthermore, the discovery of central roles for Phe and Leu in promoting RIIbeta-RIIbeta interactions provides an initial insight into the structural basis for dimerization.

While this paper was in review Hausken et al.(39) reported that deletion of 10 residues at the NH(2) terminus of RIIalpha generates a monomeric protein which is unable to bind an AKAP. Deletion of residues 1-5 did not compromise dimerization or AKAP binding activity. These data suggest that the sequence PPGLT contains a residues(s) that contributes significantly to the stabilization of RIIalpha (and perhaps, RIIbeta) dimers. Alternatively, the elimination of 10 NH(2)-terminal residues may alter the orientation of the side chain of Leu (which becomes residue 3), thereby diminishing its ability to promote RIIalpha-RIIalpha interaction. These possibilities can be evaluated by site-directed mutagenesis.

Hausken and co-workers (39) also showed that a small fusion protein containing residues 1-30 of RIIalpha dimerized although it lacks Phe. The reason for this difference between RIIbeta and RIIalpha is not known. The limited segment of RIIalpha sequence may not be an exact analog of a large RIIbeta subunit containing a single amino acid substitution. Alternatively, since the NH(2)-terminal segments (residues 1-30) of RIIbeta and RIIalpha are only 67% identical, it is possible that non-conserved residues in RIIalpha facilitate dimerization in an isoform-specific manner.

Certain hydrophobic amino acid residues that are positioned between Leu and Phe subserve an essential, but distinctly different role in the tethering of RIIbeta by AKAP75. Characterization of a prototypic RIIbeta-N158 mutant, in which Val and Leu were replaced with Ala, provided evidence for this conclusion. Ala-Ala RIIbeta-N158 exhibits the hydrodynamic properties of a dimeric protein. However, AKAP75 binds wild-type RIIbeta 25-fold more avidly than the Ala-Ala mutant protein (Fig. 4). Studies on Ala-Ala RIIbeta-N158 yielded similar results. Evidently, the large aliphatic side chains of residues 20, 21, 31, or 33 facilitate the high-affinity tethering of RIIbeta(2) to AKAP75. In contrast, reduction in the size of the hydrophobic groups at these positions has little or no impact on the homodimerization of RIIbeta. Likewise, substitution of Ala for Ile^3 and Ile^5 in RIIalpha results in a dimeric protein with reduced affinity for AKAPs(39) . Thus, the dimerization of RIIbeta, although necessary, is not sufficient to generate an optimal binding site for AKAP75. Aliphatic hydrophobic residues within the binding region may provide a specific hydrophobic surface that interacts with a complementary surface generated by Leu and Ile residues previously implicated in the tethering site on AKAP75(9) .

The properties of Ala-Ala RIIbeta-N158 are anomalous. The expressed protein is monomeric, suggesting that Leu and/or Leu participate in the stabilization of the dimeric configuration. On the other hand, the protein binds AKAP75, albeit with modest affinity ( Fig. 3and Fig. 4). A possible explanation is that contact with the AKAP75 tethering region induces the dimerization of the mutant polypeptide. Alternatively, substitution of Ala at residues 28 and 29 may elicit local changes in structure that partially mimic the arrangement of Val-Leu, Phe, and Leu in the RIIbeta dimer, thereby generating a modest binding capacity. Further mutational analysis and physicochemical studies are needed to address this issue.

The hydrophobic residues that drive the dimerization of RIIbeta and its coupling with AKAP75 are conserved in the NH(2) terminus of human and bovine RIIalpha, with the exception of a Val for Leu replacement at residue 29(40, 41) . This conservation is consistent with the dimeric nature of RIIalpha and RIIbeta subunits and the ability of the two RII isoforms to share binding sites on a variety of AKAPs (14, 15) . Divergent sequences elsewhere in the NH(2) termini of the two proteins must account for the exclusive homodimerization of RII isoforms and their differential binding affinity for certain AKAPs (29, 42) .

Further structural divergence is evident in the NH(2) terminus of mouse RIIalpha(43) , which contains Val-Gly instead of Val-Leu. This raises the possibility that the conserved Val residue plays a central role in mediating the avid binding of RIIbeta with AKAP75 while Leu makes a lesser contribution. Such a situation would parallel the discovery that the branched aliphatic side chain of Leu is crucial for RIIbeta dimerization, whereas, substitution of adjacent Leu with Ala or Glu has little effect on oligomerization and AKAP75 binding activity ( Fig. 5and text of ``Results''). An alternative possibility is that mouse RIIalpha may bind AKAP75 with lower affinity than RIIalpha isoforms from other species. Further mutational analysis and AKAP binding studies are needed to address this issue.

Roles for bulky hydrophobic side chains in the stabilization of protein structures and protein-protein interactions are well-established(36, 37) . The fundamentally important functions subserved by ``leucine zippers'' in the oligomerization of transcription factors illustrate the latter point(44, 45) . Our results can be rationalized in the context of the elegant studies of Matthew's laboratory (31) on the functions of Leu and Phe residues in bacteriophage T4 lysozyme. Biochemical, biophysical, and x-ray crystallographic measurements document that the replacement of selected Leu and Phe residues with Ala can destabilize protein structure as a function of two parameters. One component is the difference in energy required to desolvate Ala relative to the larger hydrophobic moieties. Second, the decrease in hydrophobic surface creates solvent-free cavities of varying size in the folded structure that cannot be fully compensated by the local rearrangement of hydrophobic side chains. The generation of relatively large cavities minimizes favorable van der Waals contacts and substantially weakens the classical hydrophobic effect(31) .

Finally, the binding of partially unfolded proteins by the molecular chaperone BiP, provides a relevant paradigm for protein-protein association driven by long aliphatic side chains(46) . BiP sequesters proteins by coupling with a linear stretch of 7 amino acid residues. Leu, Ile, Met, and Val are highly preferred at several internal positions in such sequences. Moreover, thermodynamic analysis indicates that the binding of proteins to BiP is mediated by same types of interactions that stabilize hydrophobic domains of T4 lysozyme (31) and other proteins.

Overall, our data suggest the speculation that the principal roles of the side chains of Leu and Phe are to interact with and bury otherwise exposed hydrophobic moieties, thereby providing a favorable free energy increment for dimerization. The dimerization of RIIbeta would, in turn, alter the disposition of hydrophobic side chains within the NH(2)-terminal region (e.g. Val-Leu) to create a characteristic exposed binding surface that is sequestered by the concerted actions of Leu, Leu, Ile, and Leu in AKAP75(9) . Ultimately determination of the three-dimensional structure of the relevant domains of RIIbeta, AKAP75, and RIIbetabulletAKAP75 complexes will be required to thoroughly evaluate this scheme and provide detailed molecular mechanisms for dimerization and AKAP binding. However, the structure-function studies described herein elucidate the identities and functions of amino acid residues that mediate the dimerization and cytoskeletal targeting of RIIbeta. Certain crucial amino acids (Val-Leu, Phe-Leu) that are essential for optimal binding of RIIbeta with AKAP75 play no major role in the dimerization reaction. In contrast, the dominant effects of Phe and Leu seem to be due to their indispensable contributions to dimerization. Thus, higher-order structural elements that govern RIIbeta-RIIbeta interactions and AKAP75bulletRIIbeta(2) complex formation may be partially shared and partially distinct.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM22792. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be sent: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2505; Fax: 718-829-8705.

(^1)
The abbreviations used are: PKAIIbeta, the type IIbeta isoform of cyclic AMP-dependent protein kinase; PKA, cyclic AMP-dependent protein kinase; AKAP, protein kinase A anchor protein; R, regulatory subunit of cAMP-dependent protein kinase; RIIbeta-N158, a protein with an amino acid sequence that corresponds to residues 1-158 at the NH(2) terminus of RIIbeta; Mops, 4-morpholinepropanesulfonic acid.

(^2)
Y. Li and C. S. Rubin, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Jonathan H. Freedman for the preparation of Fig. 4and Fig. 6-9 for publication. Ann Marie Alba provided expert secretarial services.


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