Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1
1997; Springer Nature; Volume: 16; Issue: 8 Linguagem: Inglês
10.1093/emboj/16.8.1876
ISSN1460-2075
Autores Tópico(s)Muscle Physiology and Disorders
ResumoArticle15 April 1997free access Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1 Marie-Pierre Egloff Marie-Pierre Egloff Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Deborah F. Johnson Deborah F. Johnson MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Greg Moorhead Greg Moorhead MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Patricia T. W. Cohen Patricia T. W. Cohen MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Philip Cohen Philip Cohen MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author David Barford Corresponding Author David Barford Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Marie-Pierre Egloff Marie-Pierre Egloff Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Deborah F. Johnson Deborah F. Johnson MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Greg Moorhead Greg Moorhead MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Patricia T. W. Cohen Patricia T. W. Cohen MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author Philip Cohen Philip Cohen MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK Search for more papers by this author David Barford Corresponding Author David Barford Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Author Information Marie-Pierre Egloff1, Deborah F. Johnson2, Greg Moorhead2, Patricia T. W. Cohen2, Philip Cohen2 and David Barford 1 1Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford, OX1 3QU UK 2MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN UK The EMBO Journal (1997)16:1876-1887https://doi.org/10.1093/emboj/16.8.1876 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (GM) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 Å resolution, of PP1c in complex with a 13 residue peptide (GM[63–75]) of GM. The residues in GM[63–75] that interact with PP1c are those in the Arg/Lys–Val/Ile–Xaa–Phe motif that is present in almost every other identified mammalian PP1-binding subunit. Disrupting this motif in the GM[63–75] peptide and the M110[1–38] peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown. Introduction The reversible phosphorylation of proteins regulates most aspects of cell life. About a third of all mammalian proteins are now thought to contain covalently bound phosphate and, since protein kinases and phosphatases probably account for ∼2–3% of all human gene products (Hunter, 1995), many of these enzymes must typically phosphorylate/dephosphorylate numerous proteins in vivo. However, it is becoming increasingly clear that some protein kinases and phosphatases do not find their physiological substrates by simple diffusion within cells and that they frequently are directed to particular loci in the vicinity of their substrates by interaction with targeting subunits. In this way, the actions of protein kinases and phosphatases with inherently broad specificities are restricted and their properties tailored to the needs of a particular subcellular location, organelle or process (reviewed in Hubbard and Cohen, 1993; Faux and Scott, 1996). The paradigm for the targeting subunit concept is protein phosphatase-1 (PP1), one of the major serine/threonine-specific protein phosphatases of eukaryotic cells (Stralfors et al., 1985). This enzyme is involved in controlling diverse cellular functions including glycogen metabolism, muscle contraction, the exit from mitosis and the splicing of RNA (Cohen, 1989; Mermoud et al., 1992; Shenolikar, 1994; Wera and Hemmings, 1995). These different processes appear to be regulated by distinct PP1 holoenzymes in which the same catalytic subunit (PP1c) is complexed to different targeting or regulatory subunits. The latter class of subunits act to confer in vivo substrate specificity not only by directing PP1c to the subcellular loci of its substrates, but also by enhancing or suppressing its activity towards different substrates. In addition, the regulatory subunits allow the activity of PP1 to be modulated by reversible protein phosphorylation and second messengers in response to extracellular stimuli. Several mammalian PP1c targeting subunits have been isolated and characterized, including the GM subunit that targets PP1c to both the glycogen particles and sarcoplasmic reticulum of striated muscle (Tang et al., 1991), the GL subunit that targets PP1c to liver glycogen (Doherty et al., 1995; Moorhead et al., 1995), the M110 subunits responsible for the association of PP1c with the myofibrils of skeletal muscle (Alessi et al., 1992; Moorhead et al., 1994) and smooth muscle (Alessi et al., 1992; Chen et al., 1994), the p53-binding protein p53BP2 (Helps et al., 1995) and the nuclear protein NIPP-1 (Jagiello et al., 1995; Van Eynde et al., 1995). PP1c is also reported to interact with other mammalian proteins such as the retinoblastoma gene product (Durfee et al., 1993), an RNA-splicing factor (Hirano et al., 1996), ribosomal protein L5 (Hirano et al., 1995) and RIPP-1 (Beullens et al., 1996), a 110 kDa nuclear protein yet to be identified (Jagiello et al., 1995) and small cytosolic proteins, inhibitor-1, DARPP-32 and inhibitor-2 (reviewed in Cohen, 1989, 1992; Hubbard and Cohen, 1993). Moreover, a number of distinct PP1 regulatory subunits have been identified in yeast (reviewed by Stark, 1996). It seems likely that many further PP1 targeting subunits remain to be identified, and the exploitation of powerful new techniques such as microcystin–Sepharose affinity chromatography (Moorhead et al., 1994) and the yeast two-hybrid system (Helps et al., 1995) are accelerating the rate at which new PP1 targeting subunits are being discovered. Each form of PP1c that has been isolated contains just one PP1c-binding subunit, implying that the interaction of different targeting subunits with PP1c is mutually exclusive and that the binding site(s) for different targeting subunits is (are) identical or overlapping. This would suggest that most, if not all, targeting subunits have a common PP1c-binding motif. Surprisingly, elucidation of the amino acid sequences of a number of targeting subunits initially failed to reveal significant sequence similarities common to all these proteins. However, comparison of GM and GL identified three short highly conserved regions, one being residues 63–86 of GM (Doherty et al., 1995). Peptides comprising residues 63–93, 63–80 and 63–75 of GM were therefore synthesized and found to bind to PP1c (Johnson et al., 1996). We then sought to identify the region of the M110 subunit that binds to PP1c by deletion analysis and peptide synthesis. These studies led to the finding that the N-terminal 38 residues (M110[1–38]) mimic the intact M110 subunit in enhancing the rate at which PP1c dephosphorylated the 20 kDa myosin light chain (MLC20) subunit of smooth muscle myosin (Johnson et al., 1996). The finding that GM[63–93] disrupted the interaction between PP1c and the M110 subunit and prevented M110 from enhancing the MLC20 phosphatase activity of PP1c implies that the binding of M110 and GM to PP1c is mutually exclusive. To understand the basis for the recognition by PP1c of regulatory subunits, and peptides derived from these subunits, we have co-crystallized a complex of PP1c with the GM[63–75] peptide and determined the structure at 3.0 Å resolution. These experiments have demonstrated that residues 64–69 of the peptide are bound in an extended conformation to a hydrophobic channel within the C-terminal region of PP1c. The residues in GM[63–75] that interact with PP1c lie in an Arg/Lys–Val/Ile–Xaa–Phe motif common to M110[1–38] and almost all known mammalian PP1-binding proteins. Substituting Val or Phe by Ala in the GM[63–75] peptide, and deleting the VXF motif from the M110[1–38] peptide, abolished the ability of both peptides to interact with PP1c. Moreover, a peptide from p53BP2 that contains the RVXF motif also bound to PP1c. These findings identify a recognition site on PP1c for a critical structural motif involved in the interaction with its targeting subunits. Results and discussion Structure determination Crystallographic data to 3.0 Å were measured at the ESRF beam-line BL4 at Grenoble and at PX9.6, Daresbury (Table I). The relatively high merging R-factors and low I/σI values of the crystallographic data result from the weak diffraction observed from the PP1–GM[63–75] peptide complex crystals. This is attributable to both the small crystal size (∼25 μm×25 μm×5 μm) and long c-axis of the unit cell. In addition, the high X-ray photon dose required to obtain usable diffraction images resulted in X-ray radiation damage to the crystals, despite being maintained at a temperature of 100 K during the course of the experiment. The structure was solved by the molecular replacement method using as a search model the 2.5 Å refined coordinates of PP1c (Egloff et al., 1995). Phases obtained from a single cycle of simulated annealing refinement of the protein coordinates alone using X–PLOR (Brünger, 1992), and improved by 2-fold non-crystallographic symmetry averaging and solvent flattening, were used to calculate an electron density map. This map revealed clear density corresponding to residues Val66′, Ser67′ and Phe68′ of the GM peptide (where the prime denotes residues of the peptide) and provided a starting point for further refinement of the PP1–GM[63–75] peptide complex. The final model of the complex was refined at 3.0 Å resolution with a crystallographic R-factor of 0.22 and R-free of 0.31 (Figure 1). The two molecules of PP1c within the asymmetric unit are similar with a root-mean-square deviation (r.m.s.d.) between main-chain atoms of 0.6 Å. Residues 6–299 and 8–297 from molecules 1 and 2, respectively, are visible in the electron density map. Similar to the structures of native PP1γ1 (Egloff et al., 1995) and PP1α in complex with microcystin LR (Goldberg et al., 1995), residues C-terminal to 299 are disordered. Figure 1.Stereo views of electron density maps. (A) Electron density corresponding to residues Gly63–Ala69 of the GM[63–75] peptide. The map was calculated using 3Fo−2Fc coefficients and phases calculated from the final refined model. (B) Electron density map of the GM[63–75] peptide and corresponding region of PP1c. Displayed using TURBO-FRODO (Roussel and Cambillau, 1992). Download figure Download PowerPoint Table 1. Crystallographic data and refinement statistics Crystallographic data Space group P41212 Unit cell parameters (Å) a = b = 62.50; c = 361.30 Molecules per asymmetric unit (N) 2 Crystals used during data collection (N) 4 Temperature (K) 100 Total measured reflections (N) 290 671 Unique reflections (N) 15 509 Mean I/σ(I) 7.5 Completeness (%) 87 R-mergea (%) 14.7 Refinement statistics Reflections used for refinement (N) 13 078 Resolution range (Å) 8.0–3.0 Rcrystb (work) 0.223 Rcryst (free) 0.308 Protein and peptide atoms (N) 5861 Water molecules (N) 14 R.m.s.d. from ideal bond lengths (Å) 0.012 R.m.s.d. from ideal angles (°) 1.863 a R-merge: ΣhΣi|I(h)–Ii(h)|ΣhΣiIi(h) where Ii(h) and I(h) are the ith and mean measurements of the intensity of reflection h. b Rcryst: Σh|Fo−Fc|ΣhFo where Fo and Fc are the observed and calculated structure factor amplitudes of reflection h. Overall structure of PP1 The conformation of PP1c in the PP1–GM[63–75] peptide complex is virtually identical to that of native PP1c in complex with tungstate (Egloff et al., 1995) with an r.m.s.d. between equivalent main-chain atoms of 1.0 Å. PP1c is folded into a single elliptical domain consisting of a central β-sandwich of two mixed β-sheets surrounded on one side by seven α-helices and on the other by a sub-domain consisting of three α-helices and a three-stranded mixed β-sheet (Figure 2A). The interface of the three β–sheets at the top of the β-sandwich creates a shallow catalytic site channel. Three loops connecting β-strands with α-helices within a β-α-β-α-β motif in sheet 1 (strand order β4-β3-β2-β13-β14) together with loops emanating from the opposite β-sheet (sheet 2; strand order β1-β5-β6-β10-β12-β11) provide the catalytic site residues. The catalytic site of PP1 contains a binuclear metal site consisting of Mn2+ and Fe2+ (Egloff et al., 1995) and, in the PP1–GM[63–75] peptide complex, oxygen atoms of a sulfate ion of crystallization coordinate both metal ions, similar to what is seen in the PP1–tungstate (Egloff et al., 1995) and PP2B–phosphate complexes (Griffith et al., 1995). Figure 2.Structure of PP1–GM[63–75] peptide complex. (A) A ribbons diagram of PP1c to indicate the position of the peptide-binding channel at the interface of the two β-sheets of the β-sandwich. The GM peptide atoms are represented as ball-and-stick. The position of one of the metal ions at the catalytic site is indicated as a green sphere and the Cα of Cys273, whose side chain forms a covalent bond with the Mdha side chain of microcystin LR is shown as a yellow sphere (MOLSCRIPT, Kraulis, 1991). (B) Two perpendicular views of the surface of PP1c to show the hydrophobic peptide-binding channel. Residues 63′–69′ (GRRVSFA) of the GM[63–75] peptide are shown as sticks. The position of the sulfate ion bound at the catalytic site is indicated in one view and the N- and C-termini of PP1 are labelled. Drawn with TURBO-FRODO (Roussel and Cambillau, 1992). Pink: hydrophobic; purple: hydrophilic; cyan: acidic; blue: basic residues. (C) Stereo view of the residues 64′–69′ of the GM[63–75] peptide at the recognition site of PP1 to indicate polar interactions between peptide and protein and the formation of the β-sheet between Ser67′–Ala69′ and β14 of PP1. Drawn with TURBO-FRODO (Roussel and Cambillau, 1992). (D) Solvent-accessible surface and surface electrostatic potential of the PP1–GM[63–75] peptide complex calculated with PP1c coordinates alone and showing the peptide as a stick representation in the vicinity of the peptide-binding site. The protein surface is coloured according to electrostatic potential from red (most negative) to blue (most positive). The figure shows pronounced negative electrostatic potential in the region surrounding the N-terminus of the peptide-binding site that results from seven conserved acidic residues. Residues of PP1c in the vicinity of the peptide-binding site are labelled. The figure was created with GRASP (Nicholls and Honig, 1991). (E) Details of the structure of the peptide-binding site to show hydrophobic interactions between PP1c and Val66′, Phe68′ and Ala69′ of the GM[63–75] peptide (MOLSCRIPT, Kraulis, 1991). Download figure Download PowerPoint PP1c–GM[63–75] peptide interactions Six residues of the GM[63–75] peptide (Arg64′–Ala69′) are clearly visible in the electron density map of the complex of molecule 2; the remaining residues are not visible and are assumed to be disordered (Figure 1). Density is not visible for Arg64′ of the peptide bound to molecule 1, otherwise equivalent residues of the peptide are similar within the two complexes. The six residues (RRVSFA) of the GM[63–75] peptide in complex 2 adopt an extended conformation and bind to a hydrophobic channel on the protein surface with dimensions 25 Å×10 Å that is formed at the interface of the two β-sheets of the β-sandwich opposite to the catalytic site channel and is therefore remote from the catalytic site (Figure 2A and B). This site differs from the position of the regulatory B-subunit binding site of the PP2B catalytic subunit (Griffith et al., 1995; Kissinger et al., 1995). The location of a regulatory subunit-binding site at a region distinct from the catalytic site of PP1c is also consistent with the discovery that PP1c attached to microcystin–Sepharose affinity columns maintains an intact regulatory subunit-binding site (Moorhead et al., 1994). The residues that form this channel occur on three regions of PP1c, namely: (i) the N-terminus of β5 and the β5/β6 loop of sheet 2; (ii) the three edge β-strands of sheet 2: β10, β12 and β11; and (iii) β13, the β13/β14 loop and β14 of the edge of sheet 1 (Figure 2A). The total solvent-accessible surface area buried on formation of the complex is 980 Å2. Three residues of the peptide (Ser67′–Ala69′) form a β-strand which is incorporated into β-sheet 1 of PP1c as a sixth β-strand parallel to the N-terminus of the edge β-strand, β14 (residues Leu289–Leu296) (Figure 2C). Main-chain atoms of Ser67′ and Ala69′ form H-bonds to the main-chain atoms of residues of β14. In addition, the main-chain nitrogen of Val66′ forms a H-bond with the side chain of Asp242. Other polar interactions include the guanidinium group of Arg64′ with the main-chain carbonyl of Glu287 and a salt bridge to the side chain of Asp166. Both Asp166 and Asp242 are invariant in mammalian PP1 isoforms. A water molecule bridges the main-chain carbonyl of Arg65′ and side-chain hydroxyl of Ser67′ with the main-chain carbonyl of Thr288 of PP1c (Figure 2C). A notable feature of the peptide-binding site is the presence of a negatively charged region created by seven acidic residues (with one Lys residue) surrounding the hydrophobic channel at the N-terminus of the peptide in the vicinity of Arg64′ and Arg65′ that includes Asp166 and Asp242 (Figure 2D). This would suggest a favourable electrostatic environment for the side chains of Arg64′ and Arg65′. The predominant interactions between the peptide and PP1c involve hydrophobic contacts between the side chains of Val66′ and Phe68′ and solvent-exposed, invariant, hydrophobic residues of PP1c that form the hydrophobic channel (Figure 2C and E). In particular, the binding site for the side chain of Val 66′ is formed from the side chains of Ile169, Leu243, Leu289 and Cys291, whereas that for the side chain of Phe68′ is formed from the side chains of Phe257, Cys291 and Phe293. Details of peptide–PP1c contacts are given in Table II. The structure of the GM[63–75] peptide-binding site is likely to be conserved in other forms of PP1 from diverse species. Each hydrophobic residue of PP1c that interacts with the Val66′ and Phe68′ residues of the GM[63–75] peptide is invariant, and the acidic residues that surround the N-terminus of the peptide-binding site are highly conserved amongst all isoforms of PP1 from species as diverse as yeast, Drosophila, mammals and higher plants (Barton et al., 1994). However, since these residues are not conserved within the PP2A and PP2B sequences, these proteins will not recognize PP1 regulatory subunits. Table 2. PP1–peptide interactions Peptide atom Protein atom Water molecule Distance (Å) Polar interactions Molecule 1 Arg65′ O 7W 3.2 Val66′ N Asp242 OD2a 3.0 Ser67′ N Leu289 O 3.3 Ser67′ OG 7W 2.7 Ser67′ O Cys291 Nb 3.2 Ala69′ N Cys291 Ob 2.8 Molecule 2 Arg64′ NH1 Glu287 Oa 2.6 Arg65′ O 7W 2.8 Val66′ N Asp242 OD2a 3.2 Ser67′ N Leu289 Ob 3.1 Ser67′ OG 7W 2.6 Ser67′ O Cys291 Nb 3.0 Ala69′ N Cys291 Ob 3.3 Peptide residues Protein residues Hydrophobic interactions Val66′ Ile169b, Leu243b, Asp242a, Leu289b, Cys291b Phe68′ Phe257b, Cys291b, Phe293b Ala69′ Met290a a Highly conserved residues (Barton et al., 1994). b Invariant residues in all known PP1 sequences. The mode of interaction between PP1c and the GM[63–75] peptide is similar to that observed in complexes of phosphotyrosine-binding (PTB) domains (Zhou et al., 1995) and PDZ domains (Doyle et al., 1996) with their cognate peptide ligands. In these complexes, short peptides of 4–6 residues engage the protein by forming anti-parallel hydrogen bonding interactions with edge β-strands that occur within a β-barrel. The peptide binding sites occur within hydrophobic channels created at the interface of secondary structural elements, namely a β-sheet and an α-helix. For PP1c the two secondary structural elements are two β-sheets. Formation of H-bonds between edge β-strands is observed at protein interfaces within a number of protein–protein complexes. For example, the streptococcal protein-G domain interaction with the CH domain of IgG (Derrick and Wigley, 1992); the Ras-binding domain of Raf kinase with Rap1A (Nassar et al., 1995) and the interaction of p27Kip1 with Cdk2 within a ternary p27Kip1–cyclin A–Cdk2 complex (Russo et al., 1996). Presence of an (R/K)(V/I)XF motif in other PP1c regulatory proteins Over a dozen regulatory subunits of PP1c are now known which appear to bind to PP1c in a mutually exclusive manner that suggests an overlapping binding site or sites. Although sequence comparisons initially revealed little overall similarity between different PP1 targeting subunits, we found that M110 and p53BP2 could be aligned in the region of residues 774–900 of p53BP2 (Naumovski and Cleary, 1996), that binds to PP1c (Helps et al., 1995). Comparison of p53BP2[774–900] and M110[13–137] aligned the two ankyrin repeats in p53BP2 with the second and third ankyrin repeats of M110 and identified a conserved motif (R/K)VKF (residues 35–38 of M110 and residues 798–801 of p53BP2) preceding the ankyrin repeats. This sequence is similar to the RVSF motif found in GM[63–75] and the homologous region of GL. The motif is also the last four residues of the peptide M110[1–38] which was shown previously to bind to PP1c (Johnson et al., 1996; Figure 3A). Moreover, a 32 residue peptide from p53BP2 (residues 780–811), which contains this motif, disrupted the interaction of the M110 subunit with PP1c, as shown by a decrease in the rate of dephosphorylation of the MLC20 subunit of smooth muscle myosin and by an increase in the rate of dephosphorylation of glycogen phosphorylase (Figure 4A). This peptide also disrupted the interaction of the GL subunit with PP1c, as shown by an increase in the rate of dephosphorylation of glycogen phosphorylase (Figure 4B). This result indicates that the RVKF sequence in p53BP2 is important in the interaction with PP1c. Inspection of the sequences of other mammalian PP1-binding proteins also revealed an (R/K)(V/I)XF motif (Figure 3A), which was present in fragments of NIPP-1 (Beullens et al., 1992; Van Eynde et al., 1995) and an RNA splicing factor (Hirano et al., 1996), known to interact with PP1c. Figure 3.Sequence alignment of PP1 regulatory subunits in the vicinity of the (R/K)(V/I)XF motif. (A) Mammalian PP1-binding subunits. GM (Tang et al., 1991); GL (Doherty et al., 1995); GL-related protein (Doherty et al., 1996); p53BP2 (Helps et al., 1995); NIPP-1 (Van Eynde et al., 1995); splicing factor PSF (Hirano et al., 1996); M110 subunit (Chen et al., 1994); inhibitor-1 (Aitken et al., 1982); DARPP-32 (Williams et al., 1986). (B) PP1-binding proteins in S.cerevisiae. GAC1 (Francois et al., 1992); PIG2 (P.J.Roach, personal communication); GIP1, GIP2, YIL045W (Tu et al., 1996); REG1, REG2 (Tu and Carlson, 1995; Frederick and Tatchell, 1996); SCD5 (Nelson et al., 1996; Tu et al., 1996). The region similar to the RRVSFA sequence of GM which interacts with PP1c is boxed. Download figure Download PowerPoint Figure 4.Disruption of the interactions between PP1c and the GL and M110 subunits by a synthetic peptide from p53BP2. (A) PP1M from chicken gizzard smooth muscle (Alessi et al., 1992) was diluted and incubated for 15 min at 30°C with the peptide GKRTNLRKTGSERIAHGMRVK- FNPLALLLDSC, corresponding to the sequence in p53BP2 that contains the RVXF motif. Reactions were started with either 32P-labelled MLC20 or glycogen phosphorylase, and the MLC20 phosphatase (○) and phosphorylase phosphatase (PhP, •) activities were determined. The results are expressed as a percentage of the activity determined in control incubations where the p53BP2 peptide was omitted (100%). Similar results were obtained in three separate experiments. (B) Same as (A), except that the peptide was incubated with diluted hepatic glycogen particles containing PP1–GL before measuring the PhP activity. Similar results were obtained in three separate experiments. Download figure Download PowerPoint In further support of the notion of a common PP1c recognition motif present within PP1-binding proteins, previous studies had revealed that the sequence KIQF (similar to the R/KVXF motif) at the N-terminus of protein inhibitor 1 and its homologue DARPP-32 (Figure 3A) is necessary for mediating the inhibition of PP1c by these proteins. Loss of Ile10 of the KIQF motif of inhibitor 1 disrupts the inhibitory effects on PP1c by phosphoinhibitor-1 (Aitken and Cohen, 1982; Endo et al., 1996) and the binding of either dephosphoinhibitor-1 or phosphoinhibitor-1 to PP1c (Endo et al., 1996). A similar result was found on disrupting the equivalent residue (Ile9) of DARPP-32 (Hemmings et al., 1990; Desdouits et al., 1995). These results were interpreted to indicate that inhibitor-1 and DARPP-32 bind to PP1 through two low affinity binding sites, one that encompasses the sequence KIQF and another which includes the phosphorylated Thr residue (35 in I-1, 34 in DARPP-32) and which presumably binds at the catalytic site. Analysis of the PP1–GM[63–75] peptide complex structure suggests that an isoleucine residue could be accommodated readily within the peptide-binding site in place of Val66′ such that the additional methyl group on Ile compared with Val would contribute to favourable van der Waals interactions between the peptide and Leu243 and Cys291 of PP1. More bulky hydrophobic residues such as Leu, Met and Phe cannot be accommodated, however. It is interesting to note that, as well as the (R/K)(V/I)XF motif shared by PP1 regulatory subunits, the four residues N-terminal to this motif contain an abundance of basic residues. These residues may provide further favourable interactions with the negative electrostatic surface potential at the N-terminus of the GM[63–75] peptide-binding site of PP1c (Figure 2D). Mutagenesis of the (R/K)(V/I)XF motif The structural studies presented here suggest a dominant role for Val66′ and Phe68′ in stabilizing the interaction between GM[63–75] and PP1c, and this notion is reinforced further by the finding that other PP1 regulatory subunit sequences contain an (R/K)(V/I)XF motif yet share little overall sequence similarity. To test the hypothesis that Val66′ and Phe68′ are required for the interaction of GM[63–75] with PP1c and also that the KVKF sequence present within the M110[M1–F38) peptide is important in mediating its interaction with PP1c, we synthesized variations of the GM and M110 peptides where the R/KVXF motif was disrupted. The two variants of the GM peptide were Val66′ and Phe68′ to Ala substitutions. In order to disrupt the (R/K)(V/I)XF present within the M110 peptide, a peptide corresponding to residues Met1–Lys35 was synthesized which no longer contains the sequence VKF of the VXF motif, which is present at residues 36–38. The results for the M110[1–38] and M110[1–35] peptides (Figures 5 and 6A) are unequivocal. Whereas M110[1–38] stimulates the myosin light chain phosphatase activity of PP1c with a half-maximal effect at 10 nM reaching maximal (3-fold) activation at a peptide concentration of 1 μM as reported previously (Johnson et al., 1996), the M110[1–35] peptide was at least 104-fold less effective at activating PP1c (Figure 5). Unlike M110[1–38], the M110[1–35] peptide was also unable to activate the phosphorylase phosphatase activity of liver PP1–GL (Figure 6A). This latter result suggests two conclusions. First, that although M110[1–38] is able to bind to PP1c and disrupt the interactions between PP1c and the GL subunit, hence reversing the inhibitory effects of GL on the ability of PP1c to dephosphorylate phosphorylase, loss of the VKF seq
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