Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-dependent Protein Kinase
1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês
10.1074/jbc.273.41.26739
ISSN1083-351X
Autores Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded β-barrel, and a B and C helix (1Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N. Ten Eyck L.F. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of RI was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1–3 micromolar range is composed of residues 94–169, while residues 236–244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5–10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a C-bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B. The two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded β-barrel, and a B and C helix (1Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N. Ten Eyck L.F. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of RI was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1–3 micromolar range is composed of residues 94–169, while residues 236–244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5–10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a C-bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B. cAMP-dependent protein kinase cAMP-dependent protein kinase regulatory subunit cAMP-dependent protein kinase catalytic subunit type Iα regulatory subunit of cyclic AMP-dependent protein kinase type IIα regulatory subunit of cyclic-AMP-dependent protein kinase heat-stable protein kinase inhibitor glutathioneS-transferase peripheral recognition site 1 and 2, respectively nitrilotriacetic acid 4-morpholinepropanesulfonic acid. Of fundamental importance for understanding how signal transduction pathways are regulated is to define in molecular terms how various protein kinases interact with inhibitor proteins. cAMP-dependent protein kinase (PKA),1 probably the best understood protein kinase biochemically, has two known classes of endogenous physiological inhibitors: the regulatory (R) subunits and the heat-stable protein kinase inhibitors (PKIs) (2Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). Both bind the catalytic (C) subunit with high affinity (<1 nm) in a mutually exclusive manner. The R subunits bind in the absence of cAMP forming an inactive tetrameric holoenzyme (R2C2). Cooperative binding of cAMP to the R subunit dissociates the complex, thereby unleashing the catalytically active C subunits. PKI binds to free C in a cAMP-independent manner (3Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar). Both the R subunits and PKIs share a common mechanism for inhibiting C. Each contains an inhibitory consensus site sequence, Arg-Arg-Xaa-Ser/Thr/Ala-Yaa, where Xaa is any amino acid and Yaa is a hydrophobic residue (4Zetterqvist Ö. Z. Ragnarsson U. Engstrom L. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1990: 171-187Google Scholar). This pentapeptide resembles a substrate or inhibitor and binds to the active site cleft of the C subunit (3Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar, 5Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (806) Google Scholar,6Buechler Y.J. Taylor S.S. J. Biol. Chem. 1991; 266: 3491-3497Abstract Full Text PDF PubMed Google Scholar). PKI and R are thus competitive inhibitors. In addition to serving as inhibitors of C, however, PKI and the R subunits share other common features. Both are modular proteins, and both play roles in subcellular localization; neither is solely an inhibitor of C. PKI, for example, in addition to its inhibitor site at the N terminus (3Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar), has near its C terminus a nuclear export signal that is capable of actively shuttling the C·PKI complex out of the nucleus (7Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 1-20Abstract Full Text PDF PubMed Scopus (115) Google Scholar). The R subunits are also modular proteins composed of several distinct, well defined, and stable domains (Fig. 1). At the N terminus is a dimerization/docking domain that not only maintains the R subunits as a stable dimer but also provides a surface that docks to a variety of A kinase anchoring proteins, thereby localizing the enzyme to specific subsites within the cell (8Scott J.D. McCartney S. Mol. Endocrinol. 1994; 8: 5-11Crossref PubMed Scopus (152) Google Scholar). The "hinge" region that follows the dimerization/docking domain contains the consensus site sequence that binds to the active site cleft of C in the absence of cAMP. At the C terminus are two tandem gene-duplicated cAMP binding domains. Although each has a functional cAMP binding site, the two domains serve distinct roles. cAMP binding domain A interacts directly with the C subunit and is essential for high affinity binding to C, whereas cAMP binding domain B is not (9Saraswat L.D. Ringheim G.E. Bubis J. Taylor S.S. J. Biol. Chem. 1988; 263: 18241-18246Abstract Full Text PDF PubMed Google Scholar). Instead, its role is to modulate access of cAMP to domain A and to contribute cooperativity to the activation process. In the holoenzyme, only domain B is accessible (10Øgreid D. Døskeland S.O. FEBS Lett. 1981; 129: 282-286Crossref PubMed Scopus (37) Google Scholar, 11Øgreid D. Døskeland S.O. FEBS Lett. 1981; 129: 287-292Crossref PubMed Scopus (57) Google Scholar). cAMP thus binds first to domain B, which induces a conformational change. The subsequent binding of cAMP to domain A causes dissociation of C and activation of the holoenzyme (47Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1996; 35: 2934-2942Crossref PubMed Scopus (107) Google Scholar). To achieve high affinity binding to C, both PKI and the R subunits utilize a bipartite mechanism. Occupancy of the active site cleft by the small consensus site peptide is not sufficient to convey high affinity binding; additional interaction sites are required. Two additional but different surfaces on the large lobe of the C subunit, peripheral recognition sites 1 and 2 (PRS1 and PRS2), were shown to be essential for high affinity binding of PKI and R, respectively (13Gibson R.M. Ji-Buechler Y. Taylor S.S. J. Biol. Chem. 1997; 272: 16343-16350Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). PRS1 lies N-terminal to the region where the consensus site peptide docks (5Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (806) Google Scholar, 14Glass D.B. Cheng H.-C. Mende-Mueller L. Reed J. Walsh D.A. J. Biol. Chem. 1989; 264: 8802-8810Abstract Full Text PDF PubMed Google Scholar, 15Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1446) Google Scholar), while PRS2, essential for the high affinity binding of the R subunits, lies on a surface that follows the consensus site pocket. For PKI, the recognition sequence is linear; residues 5–24, where residues 18–21 constitute the consensus site, are sufficient to achieve nearly full binding affinity (16Reed J. de Ropp J.S. Trewhella J. Glass D.B. Liddle W.K. Bradbury E.M. Kinzel V. Walsh D.A. Biochem. J. 1989; 264: 371-380Crossref PubMed Scopus (31) Google Scholar). An amphipathic helix lies N-terminal to the consensus site, and this helix docks to the mostly hydrophobic PRS1 surface (3Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Peptides and Protein Phosphorylation. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar, 5Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (806) Google Scholar, 17Wen W. Taylor S. J. Biol. Chem. 1994; 269: 8423-8430Abstract Full Text PDF PubMed Google Scholar). In contrast to PKI, the consensus site plus the immediate flanking sequences are not sufficient to convey high affinity binding for the R subunits. 3L. J. Huang and S. S. Taylor, unpublished results.3L. J. Huang and S. S. Taylor, unpublished results. The binding of R is, therefore, not as simple as PKI and involves regions that are not contiguous to the consensus site. Binding of R to C is also coupled with the release of cAMP. Limited proteolysis combined with genetic engineering of deletion mutants established previously that the region extending from the consensus site through cAMP binding domain A is sufficient for high affinity binding to C (2Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar, 18Ringheim G.E. Taylor S.S. J. Biol. Chem. 1990; 265: 4800-4808Abstract Full Text PDF PubMed Google Scholar, 19Herberg F.W. Dostmann W.R.G. Zorn M. Davis S.J. Taylor S.S. Biochemistry. 1994; 33: 7485-7494Crossref PubMed Scopus (78) Google Scholar). Point mutations also have identified a number of specific residues in addition to and not contiguous with the inhibitor site that are important for R binding to C (13Gibson R.M. Ji-Buechler Y. Taylor S.S. J. Biol. Chem. 1997; 272: 16343-16350Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Orellana S.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4726-4730Crossref PubMed Scopus (114) Google Scholar, 21Orellana S.A. Amieux P.S. Zhao X. McKnight G.S. J. Biol. Chem. 1993; 268: 6843-6846Abstract Full Text PDF PubMed Google Scholar). By using a yeast two-hybrid screen with the C subunit as bait and then engineering additional deletion mutants, we have been able to further dissect cAMP binding domain A into two distinct subsites: one that recognizes C and complements the PRS2 site and the other that binds cAMP. A critical helix is also identified as a switch that bridges the two subsites and modulates interactions of this domain with cAMP, with C, and with cAMP binding domain B. Reagents were obtained from the following sources: ATP, benzamidine, cAMP, EDTA, MOPS, phenylmethylsulfonyl fluoride, Triton X-100, and GST-agarose resin, from Sigma; nickel-NTA resin from Qiagen; urea, 5-bromo-4-chloro-3-indoyl β-d-galactoside and enzymes used for DNA manipulations from Life Technologies, Inc.; and a DNA sequencing kit from U.S. Biochemical Corp. The peptide substrate LRRASLG was synthesized at the peptide and oligonucleotide facility at the University of California, San Diego. The oligonucleotides were synthesized with a 380B DNA synthesizer from Applied Biosystems Inc. A yeast two-hybrid screen was performed according to Vojtek et al. (22Vojtek A.B. Hollenburg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1658) Google Scholar). Plasmids and the yeast strain L40 for the two-hybrid system were obtained from Dr. Stan Hollenburg (Vollum Institute). The LexA-C fusion protein was constructed as the bait and was transcriptionally inactive but able to bind LexA operators. To make LexA-C, the full-length cDNA of C was cloned into the pEG202 vector to obtain the proper restriction sites and then subcloned into the bait plasmid, pBTM116, usingEcoRI and SalI. This bait was used to screen a mouse embryonic library, which was fused to the VP16 activation domain and introduced into the yeast strain L40. Transformants (2 × 105) were screened by first growing on selective medium (Ura−, Trp−, Leu−, His−), and positive colonies were then streaked onto Ura− Trp−Leu−/5-bromo-4-chloro-3-indoyl β-d-galactoside plates for determination of β-galactosidase activity. The prey plasmids of the positive clones were rescued by electroporating competent KC8 cells with crude yeast cell lysates and selecting on Leu− plates. The specificity of these positive clones was then determined by cotransformation of these rescued plasmids with different LexA fusion baits. In this study, LexA-vMos and LexA-laminin were used for specificity analysis. Twenty-two positive clones that interacted specifically with the LexA-C bait were subject to [35S]dideoxy-ATP DNA sequence analysis and then analyzed in the DNA data base (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52503) Google Scholar). Twenty of these clones coded for a protein fragment corresponding to residues 94–235 of RIα, and the other two coded for a protein fragment corresponding to residues 18–169 of RIα. To determine whether the two truncation fragments of RIα isolated from the two-hybrid screen bind C in vitro, plasmids containing these fragments were engineered, and the resulting proteins were purified as GST fusion proteins. To make GST-RI-(94–235) and GST-RI-(18–169), a linker containing a NotI site was first ligated into pGEX-KG vector using NdeI and HindIII to generate pGEX-KG-NotI. The cDNAs of RI-(94–235) and RI-(18–169) were then excised from the rescued prey plasmids of the two-hybrid system and then subcloned into the NotI site of pGEX-KG-NotI. RI-(94–235) was also expressed as a polyhistidine-tagged protein, His6RI-(94–235). To make His6RI-(94–235), cDNA of RI-(94–235) was excised from pGEX-KG-RI-(94–235) and subcloned into pRSETb. A truncation fragment RI-(94–169), corresponding to the overlapping region of the two positive clones was engineered and expressed as a polyhistidine-tagged protein. To make His6RI-(94–169), a NheI site was created at residue 93 and three stop codons were introduced at residue 169 in pRSETb-RI using site-directed mutagenesis to generate the deletion mutant (24Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4894) Google Scholar). The DNA corresponding to the N-terminal 93 residues was then excised by digestion withNheI. His6RI-(94–260) and His6RI-(94–244) were constructed by creating aNheI site at residue 93 and three stop codons at position 261 and 245, respectively, in pRSETb-RI using site-directed mutagenesis. The DNA corresponding to the N-terminal 93 residues was then excised by digestion with NheI. All constructs were confirmed by DNA sequence analysis (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52503) Google Scholar). All fusion proteins were expressed in Escherichia coli BL21(DE3). The BL21(DE3) cell strain was a gift from Bill Studier (Brookhaven National Laboratory, Upton, NY). Recombinant wild type RI was expressed in E. coli 222 and purified by DEAE-52 chromatography as described previously (25Saraswat L.D. Filutowics M. Taylor S. Methods Enzymol. 1988; 159: 325-336Crossref PubMed Scopus (14) Google Scholar). GST-RI-(18–169) and GST-RI-(94–235) were expressed in E. coli BL21(DE3) and purified first on a glutathione-agarose column in 10 mm sodium phosphate buffer, pH 7.4, containing 150 mm NaCl, 5 mmEDTA, 5 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, 0.05% Triton X-100, and 3 mm β-mercaptoethanol. GST-RI-(18–169) was further purified on a gel filtration column using Superdex 200. All polyhistidine-tagged proteins were purified on a nickel-NTA column in 10 mm sodium phosphate buffer as described by Huanget al. (26Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. J. Biol. Chem. 1997; 272: 8057-8064Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Untagged RI-(94–235) was expressed first as a GST fusion protein, and then the GST was cleaved off of the fusion protein by thrombin. Untagged RI-(94–235) was then purified further on a gel filtration Superdex 75 column. The C-subunit was purified by phospocellulose chromatography and then resolved into discrete isoelectric variants on a MonoS HR10/10 column using fast protein liquid chromatography (Amersham Pharmacia Biotech) as described by Herberg et al. (27Herberg F.W. Bell S.M. Taylor S.S. Protein Eng. 1993; 6: 771-777Crossref PubMed Scopus (109) Google Scholar). Isozymes I and II were used for all experiments. To obtain cAMP-free RI subunit, purified recombinant wild type RI was unfolded in 8 murea as described by Buechler et al. (28Ji-Buechler Y. Herberg F.W. Taylor S.S. J. Biol. Chem. 1993; 268: 16495-16503Abstract Full Text PDF PubMed Google Scholar). After removing the cAMP by passing the solution over a prepacked Sephadex G-25 column (NAP10 column), proteins were then refolded by dialyzing overnight in 150 mm KCl, 10 mm MOPS, and 5 mmβ-mercaptoethanol at pH 7. cAMP-free polyhistidine fusion proteins were obtained by purification with nickel-NTA resin under 8m urea denaturing conditions. In summary, cells expressing the fusion proteins were lysed in 10 mm sodium phosphate buffer containing 8 m urea and centrifuged, and the supernatant was then passed through a nickel-NTA column. Polyhistidine fusion R subunits were then eluted from the column with sodium phosphate buffer containing 100 mm imidazole and dialyzed extensively against sodium phosphate buffer, pH 7.4 for refolding. All activity assays were performed using the spectrophotometric method described by Cooket al. (29Cook P.F. Neville M.E. Vrana K.E. Hartl F.T. Roskoski R. Biochemistry. 1982; 21: 5794-5799Crossref PubMed Scopus (346) Google Scholar) in an assay mix containing 1 mm ATP and 10 mm MgCl2. Briefly, 20–50 nmof C was incubated in a 1-ml assay volume containing 1 mmphosphoenolpyruvate, 100 μm NADH, 6 units of lactate dehydrogenase, and 2 units of pyruvate kinase. Kemptide (60–200 μm) was then added to initiate the reaction. The progress of the reaction was monitored continuously by a decrease in absorbance at 340 nm due to the oxidation of NADH in a Hewlett Packard 1587 diode array spectrophotometer. Reaction velocity was constant over 60 s. Values for K m and V max were determined using Michaelis-Menten kinetics. To determine apparent inhibitory constants, C (20–50 nm) was incubated with different concentrations of R mutants for 1 min at room temperature. The reactions were initiated by the addition of Kemptide. The percentage of inhibition for each reaction was plotted as a function of inhibitor concentration, and the apparent inhibition constant, Ki (app), was determined by fitting of the data to the equation,fb=[I]/([I]+ki(app))(Eq. 1) where f b is the fraction of inhibitor bound, [I] is the inhibitor concentration, and K i (app) is the apparent dissociation constant for the inhibitor. Since in these studies, various RI subunits acted as competitive inhibitors of C when Kemptide was used as a substrate, the inhibition constant,K i, can be further calculated from Ki(app) using the Morrison equation,Ki=ki(app)/(1+[S]/Km)(Eq. 2) where [S] is the substrate Kemptide concentration, and K m is the Michaelis constant of Kemptide. In these studies, the K m for Kemptide was determined to be 16 μm. Since His6RI-(94–244) and His6RI-(94–260) inhibited C readily with affinities in the range of the enzyme concentration used in the assay, their inhibitory constants were determined by fitting the inhibition curves to the equation, v/v0=1−(E+I+Ki(app))−((E+I+ki(app))2−4E[I]0.5)(Eq. 3) where v and v 0 are the measured kinetic rates of C in the presence and absence of inhibitor, respectively; E and I are the total enzyme and inhibitor concentrations, respectively. The true K i can then be calculated from the K i (app) using the Morrison equation. In all inhibition studies, each data point represented triplicate measurements. Apparent activation constants (K a) were measured for holoenzymes formed by C with either wild type RI or the various deletion mutants, His6RI-(94–244) or His6RI-(94–235). Holoenzyme for wild type C and RI was formed by two methods. In method 1, C and RI were dialyzed for 24 h at room temperature against 20 mm potassium phosphate, 100 mm KCl, 5 mm β-mercaptoethanol, 5% glycerol, 100 μmATP, and 1 mm MgCl2 at pH 6.5. In method 2, 40 nm C and 35 nm cAMP-free RI were incubated for 30 min in assay mix at room temperature. These two methods were also used to obtain holoenzyme for His6RI-(94–244) except that the dialysis was carried out at 4 °C instead of at room temperature. Holoenzyme for His6RI-(94–235) was obtained by method 2 with 40 nm C and 0.2 μm cAMP-free His6RI-(94–235). In all cases, the reaction was initiated by the addition of Kemptide followed by activation with varying concentrations of cAMP. The activity of the free C subunit was then monitored spectrophotometrically. Each data point was measured in triplicate, and the mean was used for calculation. A yeast two-hybrid screen was used to identify proteins that associate with the C subunit of PKA. Using LexA-C as the bait, 22 out of 2 × 105 transformants interacted specifically with the C subunit bait in screening a mouse embryonic library. All 22 positive clones coded for fragments of the RIα-subunit. Twenty coded for residues 94–235, while the other two coded for residues 18–169. Fig. 1 shows the amino acid sequence of RIα with these two fragments indicated. Both fragments include the inhibitor consensus site (Arg94-Arg-Gly-Ala-Ile98). No other R subunit isoforms were isolated, nor were any PKIs isolated from the screen. To determine whether these fragments interact with C in vitro, both fragments were expressed as GST fusion proteins or polyhistidine fusion proteins. GST-RI-(94–235) and His6RI-(94–235) were expressed in quantities about 10-fold higher than GST-RI-(18–169) and were near homogeneity after affinity purification on either glutathione-agarose or nickel-NTA resin. GST-RI-(18–169) was purified further by gel filtration to near homogeneity. In a spectrophotometric assay, a fixed amount of wild-type C subunit was incubated with increasing amounts of GST-RI-(18–169), GST-RI-(94–235), or His6RI-(94–235) for 1 min at 22 °C in the presence of MgATP. Kemptide was then added to initiate the kinase reaction. The activity of the C subunit was inhibited by both fusion proteins. To further characterize this inhibition, theK m and V max for Kemptide were measured with increasing concentrations of inhibitor His6RI-(94–235). As shown in Fig. 2 A, the fusion protein, His6RI-(94–235), was a competitive inhibitor for C with respect to Kemptide. The calculated K iwas 0.3 μm. Based on the fact that all of the constructs had the consensus sequence, we assumed that the other subsequent constructs would behave also as competitive inhibitors to C. By this criterion, all apparent inhibitory constants (K i (app) values) were calculated by hyperbolic fitting of the binding curves (Fig. 2 B). To demonstrate that tagging the fragment with GST or polyhistidines does not seriously affect the K i values, different forms of RI-(94–235) were expressed and characterized. TheK i values of GST fusion, polyhistidine fusion, and untagged RI-(94–235) were 0.3, 0.2, and 0.13 μm, respectively, indicating that the inhibitory ability was not drastically altered by the presence of the fused protein. These apparent inhibitory constants in the presence of MgATP are summarized in Table I.Table IInhibitory constants for different forms of RI-(18–169) and RI-(94–235) in the presence of MgATPProteinsKiμmGST-RI-(18–169)0.25 ± 0.01GST-RI-(94–2350.3 ± 0.03His6RI-(94–235)0.2 ± 0.01RI-(94–235)0.13 ± 0.03 Open table in a new tab To determine the minimum required sequence for inhibiting C, the overlap piece of the two fragments identified in the two-hybrid screen was constructed as a polyhistidine fusion protein and characterized for its ability to inhibit C. This overlap piece contained residues 94–169 of RI, which includes the consensus site and part of cAMP binding domain A. His6RI-(94–169) inhibited the C subunit with a K i of 2 μm. This inhibitory constant was still 100-fold lower than that of an inhibitor peptide where the Ser residue of Kemptide (LRRASLG) is replaced with Ala (30Whitehouse S. Feramisco J.R. Casnellie J.E. Krebs E.G. Walsh D.A. J. Biol. Chem. 1983; 258: 3693-3701Abstract Full Text PDF PubMed Google Scholar). A peptide corresponding to residues 89–111 of RIwas not able to compete with Kemptide in the assay (data not shown). Therefore, residues 94–169 of RI were defined as the minimum required sequence for RI to inhibit C in the low micromolar (1–3 μm) range. All apparentK i values with respect to the different deletion constructs are summarized in Fig. 3. As described by Herberg et al. (19Herberg F.W. Dostmann W.R.G. Zorn M. Davis S.J. Taylor S.S. Biochemistry. 1994; 33: 7485-7494Crossref PubMed Scopus (78) Google Scholar) and by other proteolysis experiments (9Saraswat L.D. Ringheim G.E. Bubis J. Taylor S.S. J. Biol. Chem. 1988; 263: 18241-18246Abstract Full Text PDF PubMed Google Scholar, 18Ringheim G.E. Taylor S.S. J. Biol. Chem. 1990; 265: 4800-4808Abstract Full Text PDF PubMed Google Scholar), the N-terminal 91 residues do not contribute to high affinity binding of RI to the C subunit; (Δ1–91)RI is as potent an inhibitor as the wild type RI. Both GST-RI-(94–235) and GST-RI-(18–169), however, showed a dramatic decrease in affinity for C, compared with the 0.2 nm K i obtained for cAMP-free full-length RI(19Herberg F.W. Dostmann W.R.G. Zorn M. Davis S.J. Taylor S.S. Biochemistry. 1994; 33: 7485-7494Crossref PubMed Scopus (78) Google Scholar). To map the regions on RI responsible for this 1000-fold decrease in affinity, His6RI-(94–260) was constructed, expressed, and purified on a nickel-NTA affinity column. This mutant, which contains the entire cAMP binding domain A, is monomeric because it lacks the N-terminal dimerization domain. In the spectrophotometric assay, His6RI-(94–260) inhibited C rapidly and efficiently with an inhibitory constant of 5–10 nm. Therefore, by comparison with GST-RI-(94–235), residues 236–260 must contribute significantly to the high affinity binding between RI and C. This segment corresponds to the C helix of cAMP binding domain A. Here we chose not to compare the inhibitory constants of these mutants in their cAMP-free form, since the process of stripping off cAMP may cause some degree of structural damage in these mutants. To more specifically map the region on the C helix responsible for the 2-order of magnitude increase in affinity for binding to C, theK i of His6RI-(94–244), which lacks the latter part of the C helix and the connecting loop, was measured. The K i of this mutant was also 5–10 nm, similar to the K i of His6RI-(94–260). The region between Ser236 and Tyr244 on the C helix is thus sufficient to increase the affinity for C by 2–3 orders of magnitude. TheK a (app) for cAMP for the type I holoenzyme was 144 nm, while the half-maximum activation for holoenzyme formed with His6RI-(94–235) had an apparent K a of 225 nm (Fig. 4). Half-maximal activation of holoenzyme formed with His6RI-(94–244) occurred at 763 nm cAMP, similar to the K a of RI-(93–259) at 1.1 μm (17Wen W. Taylor S. J. Biol. Chem. 1994; 269: 8423-8430Abstract Full Text PDF PubMed Google Scholar). In contrast to wild type RI, activation with both mutants showed no cooperativity, typical of other monomeric forms of R. Since holoenzymes formed with His6RI-(94–235) had aK a 3-fold lower than holoenzymes formed with His6RI-(94–244), residues 236–244 in the C helix of cAMP binding domain A are important for modulating access of cAMP to site A. This could be due to steric interference or to changes in the equilibrium that favor association with C over binding to cAMP. Holoenzyme formed with His6RI-(94–169) cannot be activated by cAMP (data not shown). As summarized in Table II, the modular domains of the regulatory subunit all interact with multiple protein partners, whether it is another domain or a separate molecule. For example, the dimerization/docking domain at the amino terminus not only interacts with the other R protomer in the dimer; the dimeric domain also serves as a docking surface for A kinase anchoring proteins (31Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 32Faux M.C. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 33Faux M.C. Scott J.D. Trends Biochem. Sci. 1996; 21: 312-315Abstract Full Text PDF PubMed Google Scholar). In some cases, such as cAMP binding site A (the cA:A domain), the partners also differ depending on the activation
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