Dissecting the Cooperative Reassociation of the Regulatory and Catalytic Subunits of cAMP-dependent Protein Kinase
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.31998
ISSN1083-351X
AutoresRobin M. Gibson, Susan S. Taylor,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoThe catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active. The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active. Cyclic AMP-dependent protein kinase (cAPK) 1The abbreviations used are: cAPK, cAMP-dependent protein kinase; R, cAMP dependent protein kinase regulatory subunit; RI, type Iα regulatory subunit of cAMP-dependent protein kinase; RII, type IIα regulatory subunit of cAMP-dependent protein kinase; C, cAMP-dependent protein kinase catalytic subunit; β-ME, β-mercaptoethanol; 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide·HCl PKI, heat stable protein kinase inhibitor; PRS, peripheral recognition site; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: cAPK, cAMP-dependent protein kinase; R, cAMP dependent protein kinase regulatory subunit; RI, type Iα regulatory subunit of cAMP-dependent protein kinase; RII, type IIα regulatory subunit of cAMP-dependent protein kinase; C, cAMP-dependent protein kinase catalytic subunit; β-ME, β-mercaptoethanol; 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide·HCl PKI, heat stable protein kinase inhibitor; PRS, peripheral recognition site; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. is a tightly regulated enzyme that plays a multitude of roles in growth regulation, gene transcription, and metabolism. Of equal physiological importance to the specificity of this enzyme for its protein substrates is its ability to be turned on and off in response to cellular signals. There are two known physiological inhibitors of cAPK, the regulatory subunits (RI and RII) and the heat-stable protein kinase inhibitors (PKIs) (1Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (953) Google Scholar). The cooperative binding of cAMP to the inactive tetrameric holoenzyme, (R2C2), causes the complex to dissociate, releasing two active catalytic subunits (C) and a dimeric regulatory subunit saturated with four molecules of cAMP (R2cAMP4). PKIs interact only with the free C subunit and carry, in addition to a cAPK inhibition site, a nuclear export signal that shuttles the complex actively out of the nucleus (2Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (996) Google Scholar).The R subunits and PKIs inhibit C by a common mechanism. Both contain an inhibitory consensus sequence, RRXS/TΨ, that is shared by most substrates and inhibitors of cAPK, where the phosphorylation (P) site is either Ser, Thr, or Ala, X is any amino acid, and Ψ, at the P + 1 site, is a hydrophobic residue. In RI and PKI, the P site is an Ala, whereas in RII, this site is a phosphorylatable Ser (3Titani K. Sasagawa T. Ericsson L.H. Kumar S. Smith S.B. Krebs E.G. Walsh K.A. Biochemistry. 1984; 23: 4193-4199Crossref PubMed Scopus (140) Google Scholar, 4Scott J.D. Fischer E.H. Takio K. Demaille J.G. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5732-5736Crossref PubMed Scopus (64) Google Scholar, 5Takio K. Smith S.B. Krebs E.G. Walsh K.A. Titani K. Biochemistry. 1984; 23: 4200-4206Crossref PubMed Scopus (95) Google Scholar). This consensus site sequence binds competitively to the active site cleft and is similar for both R and PKI. However, this interaction is not sufficient to achieve high affinity binding. Binding to C is bipartite. Both R and PKI require an additional interaction site.As indicated in Fig. 1, the C subunit contains two distinct additional surfaces on the large lobe that are important for recognition of PKI and R. One surface, referred to here as peripheral recognition site 1 (PRS1), is essential for high affinity binding of PKI, and another is required for high affinity binding of R. The interactions that account for the high affinity of C and PKI are well defined in the crystal structure of C containing the 20 amino acid inhibitor peptide, IP20, derived from residues 5–24 of PKI (6Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-h. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1438) Google Scholar, 7Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (803) Google Scholar, 8Cheng H.-C. van Patten S.M. Smith A.J. Walsh D.A. Biochem. J. 1986; 231: 655-661Crossref Scopus (75) Google Scholar). Replacement of the P-11 Phe and the P-6 Arg in PKI (9Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Kemp B.E. CRC Reviews. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar) as well as mutation of Arg-133, located near the PRS1 surface on C (10Wen W. Taylor S.S. J. Biol. Chem. 1994; 269: 8423-8430Abstract Full Text PDF PubMed Google Scholar), all abolished high affinity binding of PKI. The realization that mutation of Arg-133 did not interfere with binding of RI indicated that other surfaces were important. In the absence of a crystal structure, many approaches have been used to map the surface that is important for R binding and at the same time to identify the regions of R that are essential for tight binding to C.The surface on the C subunit that is important for high affinity binding of R is referred to here as peripheral recognition site 2, PRS2 (Fig. 1). This surface is dominated by the essential phosphorylation site, Thr-197, located on the activation loop. The importance of this phosphorylation site for regulation was originally identified in yeast (11Levin L.R. Kuret J. Johnson K.E. Powers S. Cameron S. Michaeli T. Wigler M. Zoller M.J. Science. 1988; 240: 68-70Crossref PubMed Scopus (44) Google Scholar, 12Levin L.R. Zoller M.J. Mol. Cell. Biol. 1990; 10: 1066-1075Crossref PubMed Scopus (50) Google Scholar). The PRS2 surface is quite basic, and several of these basic residues, identified originally by charge to Ala scanning mutagenesis in the yeast C homolog, TPK1, specifically Lys-213 and Lys-217, are important for R/C interaction (13Gibbs C.S. Knighton D.R. Sowadski J.M. Taylor S.S. Zoller M.J. J. Biol. Chem. 1992; 267: 4806-4814Abstract Full Text PDF PubMed Google Scholar). 2Gibson, R. M., Buechler, Y. J., and Taylor, S. S. (1997) Protein Sci. 6, 1825–1834. 2Gibson, R. M., Buechler, Y. J., and Taylor, S. S. (1997) Protein Sci. 6, 1825–1834. H87Q, W196R, T197A, and L198K/S were also identified using an in vivo genetic screen in human JEG-3 cells as mutations that disrupted regulation of C by RI and RII but not PKI (15Orellana S.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4726-4730Crossref PubMed Scopus (113) Google Scholar, 16Orellana S.A. Amieux P.S. Zhao X. McKnight G.S. J. Biol. Chem. 1993; 268: 6843-6846Abstract Full Text PDF PubMed Google Scholar). Mutation of either His-87 or Thr-197, in recombinantly expressed C, subsequently confirmed the importance of these residues for holoenzyme formation (17Cox S. Taylor S.S. Biochemistry. 1995; 34: 16203-16209Crossref PubMed Scopus (30) Google Scholar, 18Cox S. Taylor S.S. J. Biol. Chem. 1994; 269: 22614-22622Abstract Full Text PDF PubMed Google Scholar, 19Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (132) Google Scholar). These amino acids thus define a basic surface with a node at the center comprised of Thr-197, His-87, and Trp-196. Thr-197, as discussed previously, is linked through hydrogen bonds to the active site and is important for assembly of the active conformation of this loop (19Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (132) Google Scholar, 20Yonemoto W. McGlone M.L. Grant B. Taylor S.S. Protein Eng. 1997; (in press)PubMed Google Scholar, 21Johnson L.N. Noble M.E.M. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar). His-87, on the other hand, located at the beginning of the C-helix in many protein kinases in the small lobe, interacts directly with Thr(P)-197 only in the closed conformation of the C subunit (22Zheng J. Knighton D.R. Xuong N.-H. Taylor S.S. Sowadski J.M. Ten Eyck L.F. Protein Sci. 1993; 2: 1559-1573Crossref PubMed Scopus (281) Google Scholar). Trp-196 is fully exposed on the surface.To understand further the importance of the PRS2 surface, and, in particular, Trp-196, this exposed tryptophan was replaced with Arg and Ala in recombinantly expressed murine Cα. The Ala mutation was designed to remove the hydrophobic character of Trp-196 without introducing a positive charge. Mutation of the site corresponding to Trp-196 in TPK1, Tyr-240, to Ala also disrupted R/C interaction in yeast (13Gibbs C.S. Knighton D.R. Sowadski J.M. Taylor S.S. Zoller M.J. J. Biol. Chem. 1992; 267: 4806-4814Abstract Full Text PDF PubMed Google Scholar). The mutant C subunits were characterized for their kinetic properties and their ability to associate with PKI, RI, and RII. In addition, the W196R mutant C subunit was paired with mutant forms of RI, containing deletions as well as defects in their cAMP binding properties. These mutant pairs highlight the conformational flexibility of the R subunits and the important role this plays in the formation of the inhibited holoenzyme complex.DISCUSSIONThe activation loop at the edge of the cleft interface on the large lobe of the catalytic subunit is an important segment for all protein kinases. As seen in Fig. 1, many parts of the molecule communicate with this surface. His-87 from the small lobe, Arg-165 preceding the catalytic loop, and Lys-189 in β-strand 9 all contact the inward facing phosphate on Thr-197. In contrast, the residues that flank Thr-197 contribute directly to substrate/inhibitor binding. Leu-198 is a determinant for the P + 1 site, whereas Trp-196, as shown here, is a critical part of the surface that recognizes the R subunit.In many protein kinases this activation loop is only transiently assembled into its active conformation as a consequence of phosphorylation by a heterologous protein kinase (21Johnson L.N. Noble M.E.M. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar). In cAPK, however, this segment is assembled during the initial processing of the enzyme into a fully active protein phosphorylated on both Thr-197 and Ser-338 (41Steinberg R.A. Cauthron R.D. Symcox M.M. Shuntoh H. Mol. Cell. Biol. 1993; 13: 2332-2341Crossref PubMed Google Scholar). Activation is then controlled through the binding and release of the regulatory subunit. The crystal structures of the C subunit reveal open and closed conformations, and the environment of this node at the edge of the cleft is certainly influenced by this movement with His-87 from the small lobe making contact only in the closed conformation (22Zheng J. Knighton D.R. Xuong N.-H. Taylor S.S. Sowadski J.M. Ten Eyck L.F. Protein Sci. 1993; 2: 1559-1573Crossref PubMed Scopus (281) Google Scholar). However, these are relatively subtle conformational changes. Furthermore, the ledge of the large lobe on which substrates and inhibitors dock does not change as a consequence of substrate or inhibitor binding (42Narayana N. Cox S. Xuong N.-H. Ten Eyck L.F. Taylor S.S. Structure. 1997; 5: 921-935Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). It is therefore a stable docking surface (43Narayana N. Cox S. Taylor S.S. Xuong N.-H. Biochemistry. 1997; 36: 4438-4448Crossref PubMed Scopus (103) Google Scholar).In contrast to C, there is much evidence to suggest that major conformational changes take place in the R subunit upon holoenzyme formation. As illustrated schematically in Fig.7, the R subunit exists in two distinct physiological states. Either it is bound to cAMP (the B-form) or it is in a complex with the catalytic subunit in the holoenzyme, R2C2 (H-form) (44Dostmann W.R.G. FEBS Lett. 1995; 375: 231-234Crossref PubMed Scopus (63) Google Scholar). Considerable kinetic, biophysical, and mutational data indicate that there is tightly coupled communication between the two domains that correlates with the cooperative binding and release of cAMP. We know, for example, that only site B is exposed in the holoenzyme; whereas cAMP binding site A is masked (45Ogreid D. Doskeland S.O. FEBS Lett. 1981; 129: 282-286Crossref PubMed Scopus (37) Google Scholar, 46Ogreid D. Doskeland S.O. FEBS Lett. 1981; 129: 287-292Crossref PubMed Scopus (57) Google Scholar). Binding of cAMP to site B leads to a conformational change that "opens up" site A. It is the binding of cAMP to site A that then leads to the dissociation of C (26Herberg F.W. Taylor S.S. Dostmann W.R.G. Biochemistry. 1995; 35: 2934-2942Crossref Scopus (106) Google Scholar). The evidence presented here indicates that this is also the surface where Trp-196 interacts. cAMP and C, via the Trp-196 surface, both compete for the A domain of the R subunit.Figure 7The H- and B-forms of the regulatory subunit. The two major conformations that the R subunits assume in the presence (B) and absence (H) of cAMP are shown schematically.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The only crystallographic structure of R that has been solved to date is that of the monomeric RI deletion mutant (Δ1–91)RI (47Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N.-H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar). The first 20 amino acids of (Δ1–91)RI, containing the autoinhibitory region, were not resolved in the structure, indicating that this region is disordered. In the full-length free R subunit, this segment is highly sensitive to proteolytic cleavage (48Potter R.L. Stafford P.H. Taylor S.S. Arch. Biochem. Biophys. 1978; 190: 174-180Crossref PubMed Scopus (36) Google Scholar), suggesting that it is probably flexible in the free R subunit. Both cAMP binding sites are saturated in this structure. Proteolytic and deletion mutational analysis of R demonstrated that neither the N terminus nor the B domain is required for high affinity binding to C (28Ringheim G.E. Saraswat L.D. Bubis J. Taylor S.S. J. Biol. Chem. 1988; 263: 18247-18252Abstract Full Text PDF PubMed Google Scholar, 49Weldon S.L. Taylor S.S. J. Biol. Chem. 1985; 260: 4203-4209Abstract Full Text PDF PubMed Google Scholar). Thus, the region that contributes to high affinity binding to C is localized between the inhibitory consensus site and the end of the A domain. Several acidic sites within this region were protected by the C subunit from chemical modification by the water-soluble carbodiimide, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide·HCl, and were shown subsequently to be important for binding to the basic surface of C which surrounds the PRS2 node formed by Trp-196, His-87, and Thr-197 (50Gibson 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). Recently, an electrostatic interaction between Lys-213 on C and Glu-143 on RI was also identified.2As shown in Fig. 8, the structure of the deletion mutant,(Δ1–91)rRI, reveals the orientation of the two cAMP binding domains as well as the molecular features of the cAMP binding sites. The structure also predicts that significant conformational changes would occur when cAMP is released. The extensive hydrogen bonding network surrounding the cAMP, for example, including the short helix between β-strands 6 and 7, will not exist in the absence of the nucleotide. When cAMP is removed, this network will be destroyed. However, when stripped of cAMP in vitro, the R subunit is much less stable, and the surface that docks to C can be readily accessed by both rC(WT) and rC(W196R) (34Le-n D.A. Dostmann W.R.G. Taylor S.S. Biochemistry. 1991; 30: 3035-3040Crossref PubMed Scopus (20) Google Scholar). In the deletion mutant, however, where the B domain is no longer present, rC(W196R) can no longer form a stable complex. The cAMP binding site of the A domain is shielded in the full-length R subunit. Furthermore, the B domain is tightly associated, primarily through hydrophobic interactions, with the C-helix of the A domain. Thus, the B domain through its modulation of the C-helix controls access to cAMP binding site A. This is also consistent with recent deletion analysis of the A domain that shows the C terminus of the C-helix, residues 245–260 in domain A, is not essential for either cAMP binding or binding to C. When the B domain is absent, it is likely that the C-helix of domain A collapses onto the β-barrel, similar to the conformation it assumes in both the B domain and in the catabolite gene activation protein (CAP) (47Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N.-H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar, 51Weber I.T. Steitz T.A. Bubis J. Taylor S.S. Biochemistry. 1987; 26: 343-351Crossref PubMed Scopus (116) Google Scholar). The best evidence for this comes from affinity labeling with 8-N3-cAMP. In the wild-type R subunit, Trp-260 is labeled by 8-N3-cAMP bound to site A (52Bubis J. Taylor S.S. Biochemistry. 1987; 26: 3478-3486Crossref PubMed Scopus (25) Google Scholar, 53Bubis J. Saraswat L.D. Taylor S.S. Biochemistry. 1988; 27: 1570-1576Crossref PubMed Scopus (21) Google Scholar). In the deletion mutant, rRI(Δ260–379), however, Tyr-244 is labeled instead (14Bubis J. Taylor S.S. Biochemistry. 1987; 26: 5997-6004Crossref PubMed Scopus (16) Google Scholar). As seen in Fig. 8, labeling of Tyr-244, which is far from the cAMP binding pocket in full-length RI, requires this movement. In this conformation, where the normal signaling from the B domain is gone and Tyr-244 is close to cAMP, rC(W196R) cannot form an inhibited complex because the site that binds to C is shielded.Figure 8Crystallographic model of the type Iα regulatory subunit of cAPK. A, hydrogen bond interactions within the cAMP binding domain A of the type I regulatory subunit. Potential hydrogen bonds are indicated by dashed lines(distances <3.3 Å). Arg-209 is shown interacting with Asp-170 and the backbone carbonyl of Asn-171 as well as the oxygen on the exocyclic phosphate of cAMP. The backbone amide of Gly-199 and the side chain of Glu-200 form hydrogen bonds with the 3′-OH of the ribose ring. Glu-200 is also within hydrogen bonding distance of Trp-260 and Arg-241. Arg-241 resides on the C-helix of the A domain where it also interacts with Asp-267 on the A-helix of the B domain. B, model of the regulatory subunit (Δ1–91)RI. The N terminus begins at Arg-113. One cAMP molecule occupies each of the cAMP binding domains, A(white) and B (dark).Shaded residues indicate acidic sites (D140, E143, E255, and D258) protected from chemical modification in the holoenzyme complex. Y244, R209, and R333 are also indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In rRI(R333K) the B domain is still present; however, because of its reduced affinity for cAMP, the site is empty. This mutant R subunit when stripped of cAMP can form a complex with rC(W196R) but the complex is incompletely formed. Based on 1) the altered kinetic behavior of rC(W196R) in the presence of stripped rRI(R333K), 2) the subnanomolar affinity of this complex measured by surface plasmon resonance, and 3) the fact that this active complex could be isolated by gel filtration, the partial inhibition of rC(W196R) in the presence of the various mutant forms of RI summarized in Table IVis most likely due to the formation of a complex that has undergone an initial docking event but cannot undergo the subsequent conformational switch that leads to complete inhibition, a step that correlates with occupancy of the active site cleft by the inhibitor segment (Fig.9). Whether the initial docking in wild-type C is through the inhibitor site at the cleft interface or through the PRS2 surface, in the final inhibited complex, both sites are filled and there must be a conformational change in R to enable both sites to be occupied simultaneously. Neither the initial docking nor this conformational switch can take place for rC(W196R) when cAMP is present. This is true for wild-type RI as well as for the mutant RI subunit. rC(W196R) can, however, form a stable complex with rRI(R333K), where cAMP binding site B is defective, once cAMP has been removed from site A. With this mutant, docking occurs via the PRS2 surface in the absence of cAMP; however, the switch mechanism that allows the inhibitor site to occupy the active site cleft is defective. The R subunit cannot change conformation so that the inhibitor recognition site is occupied. Thus, a stable complex is formed, whereby the active site cleft is empty and capable of turning over substrates, albeit with less catalytic efficiency. The electrostatic interaction between Glu-143 and Lys-213 as well as the inability of the B deletion mutants to bind to rC(W196R) are consistent with this interpretation. The K d values for wild-type C and RI as well as for the two mutants, rC(W196R) and rRI(R333K), also indicate that the interaction with the PRS2 site provides most of the affinity for the interaction between RI and C.Table IVInhibition of mutant RI-subunits with rC(W196R)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Figure 9Model for the formation of partially inhibited holoenzyme complex. In the formation of wild-type holoenzyme, an initial docking event is followed by a conformational change that triggers the release of cAMP. This process requires both the inhibitor recognition site (IRS) and the PRS2 surface on C. When rC(W196R) was paired with cAMP-free rRI(R333K), a stable complex formed, but it was unable to undergo the final conformational switch (inset). This complex was partially active.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To understand fully the interactions between the regulatory and catalytic subunits of cAPK at a molecular level, a high resolution crystallographic model of the tetrameric holoenzyme complex is required. Crystallographic models of mutant holoenzyme complexes such as that formed between rRI(R333K) and rC(W196R) will test the model shown in Fig. 9, which may represent an intermediate in the holoenzyme-forming pathway. Cyclic AMP-dependent protein kinase (cAPK) 1The abbreviations used are: cAPK, cAMP-dependent protein kinase; R, cAMP dependent protein kinase regulatory subunit; RI, type Iα regulatory subunit of cAMP-dependent protein kinase; RII, type IIα regulatory subunit of cAMP-dependent protein kinase; C, cAMP-dependent protein kinase catalytic subunit; β-ME, β-mercaptoethanol; 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide·HCl PKI, heat stable protein kinase inhibitor; PRS, peripheral recognition site; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: cAPK, cAMP-dependent protein kinase; R, cAMP dependent protein kinase regulatory subunit; RI, type Iα regulatory subunit of cAMP-dependent protein kinase; RII, type IIα regulatory subunit of cAMP-dependent protein kinase; C, cAMP-dependent protein kinase catalytic subunit; β-ME, β-mercaptoethanol; 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide·HCl PKI, heat stable protein kinase inhibitor; PRS, peripheral recognition site; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. is a tightly regulated enzyme that plays a multitude of roles in growth regulation, gene transcription, and metabolism. Of equal physiological importance to the specificity of this enzyme for its protein substrates is its ability to be turned on and off in response to cellular signals. There are two known physiological inhibitors of cAPK, the regulatory subunits (RI and RII) and the heat-stable protein kinase inhibitors (PKIs) (1Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (953) Google Scholar). The cooperative binding of cAMP to the inactive tetrameric holoenzyme, (R2C2), causes the complex to dissociate, releasing two active catalytic subunits (C) and a dimeric regulatory subunit saturated with four molecules of cAMP (R2cAMP4). PKIs interact only with the free C subunit and carry, in addition to a cAPK inhibition site, a nuclear export signal that shuttles the complex actively out of the nucleus (2Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (996) Google Scholar). The R subunits and PKIs inhibit C by a common mechanism. Both contain an inhibitory consensus sequence, RRXS/TΨ, that is shared by most substrates and inhibitors of cAPK, where the phosphorylation (P) site is either Ser, Thr, or Ala, X is any amino acid, and Ψ, at the P + 1 site, is a hydrophobic residue. In RI and PKI, the P site is an Ala, whereas in RII, this site is a phosphorylatable Ser (3Titani K. Sasagawa T. Ericsson L.H. Kumar S. Smith S.B. Krebs E.G. Walsh K.A. Biochemistry. 1984; 23: 4193-4199Crossref PubMed Scopus (140) Google Scholar, 4Scott J.D. Fischer E.H. Takio K. Demaille J.G. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5732-5736Crossref PubMed Scopus (64) Google Scholar, 5Takio K. Smith S.B. Krebs E.G. Walsh K.A. Titani K. Biochemistry. 1984; 23: 4200-4206Crossref PubMed Scopus (95) Google Scholar). This consensus site sequence binds competitively to the active site cleft and is similar for both R and PKI. However, this interaction is not sufficient to achieve high affinity binding. Binding to C is bipartite. Both R and PKI require an additional interaction site. As indicated in Fig. 1, the C subunit contains two distinct additional surfaces on the large lobe that are important for recognition of PKI and R. One surface, referred to here as peripheral recognition site 1 (PRS1), is essential for high affinity binding of PKI, and another is required for high affinity binding of R. The interactions that account for the high affinity of C and PKI are well defined in the crystal structure of C containing the 20 amino acid inhibitor peptide, IP20, derived from residues 5–24 of PKI (6Knighton D.R. Zheng J. Ten Eyck L.F. Ashford V.A. Xuong N.-h. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1438) Google Scholar, 7Knighton D.R. Zheng J. Ten Eyck L.F. Xuong N.-H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (803) Google Scholar, 8Cheng H.-C. van Patten S.M. Smith A.J. Walsh D.A. Biochem. J. 1986; 231: 655-661Crossref Scopus (75) Google Scholar). Replacement of the P-11 Phe and the P-6 Arg in PKI (9Walsh D.A. Angelos K.L. Van Patten S.M. Glass D.B. Garetto L.P. Kemp B.E. CRC Reviews. CRC Press, Inc., Boca Raton, FL1990: 43-84Google Scholar) as well as mutation of Arg-133, located near the PRS1 surface on C (10Wen W. Taylor S.S. J. Biol. Chem. 1994; 269: 8423-8430Abstract Full Text PDF PubMed Google Scholar), all abolished high affinity binding of
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