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J. Biol. Chem., Vol. 278, Issue 21, 18817-18823, May 23, 2003
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From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received for publication, January 8, 2003
, and in revised form, March 24, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Mammalian genomes contain three genes that encode isoforms of PP1 (1, 3). These isoforms (35–38 kDa) are about 90% identical, and the differences are mainly concentrated in the extremities. Although some PP1 interactors such as the neurabins (5, 6) interact with PP1 in an isoform-specific manner, most interactors do not discriminate between PP1 isoforms, implying that the major interactor binding sites reside in the catalytic core, i.e. the central three-quarters of the protein. The surface of the catalytic core is too small to harbor specific binding sites for each of the 65 known mammalian interactors. The available evidence rather suggests that PP1 interactors compete for a limited number of common or over-lapping binding sites (discussed in Ref. 1). The binding to these sites is mainly mediated by short (4–6 residues) degenerate motifs, and this accounts for the lack of structural similarity between PP1 interactors. Combined with observations that most PP1 interactors have multiple PP1 binding sites, this inspired us to suggest that PP1 is subject to a combinatorial control (1). According to this model, the activity and substrate specificity of PP1 is (partially) determined by the interactors that occupy different combinations of the available binding sites. Even with a limited number of interactor binding sites, the interactors could thus "combine" with PP1 in many different ways. It is not yet understood exactly how protein interactors of PP1 affect the activity and substrate specificity of the enzyme, but it can be envisaged that binding of the interactors induces conformational changes or has steric consequences that affect the accessibility of the catalytic site. An additional control by the interactors is usually conferred by targeting domains and binding sites for specific substrates.
Only a few interactor binding sites of PP1 have been mapped in detail. One of these is the catalytic site that binds some of the interactors (inhibitor-1, PHI, and Mypt1) in their phosphorylated form as pseudosubstrate inhibitors (7, 8, 9). Another interactor binding site is represented by the 
4/5/6 triangle of PP1, which mediates binding to Sds22 (10). The 
12-13 loop of PP1, which overhangs the catalytic site, is important for inhibition by both toxins and protein inhibitors (11). By far the best-characterized interactor binding site of PP1 is the hydrophobic "RVXF" binding groove, which is remote from the catalytic site and centered by the edge of one of the two -sheets (12). Most interactors of PP1 contain an RVXF-variant that binds to this hydrophobic channel (1, 3). Surprisingly, binding of the RVXF motif in itself has little effect on the conformation and activity of PP1 (1, 12); yet, considerable evidence suggests that the RVXF motif is required for interactor-specific effects on PP1. Thus, synthetic RVXF-containing peptides competitively disrupt the PP1-interactor binding or affect the enzymatic properties of various PP1 holoenzymes (12, 13, 14, 15). Likewise, mutation of the RVXF motif is often sufficient to prevent the high-affinity binding of an interactor to PP1. One interpretation for these findings is that the binding of the RVXF motif to PP1 increases the local concentration of the interactor and thereby promotes the binding of secondary binding sites to PP1, which determines the activity and/or substrate specificity of the enzyme (1). In one report on the binding of the myosin targeting protein Mypt1 to PP1 as studied by surface plasmon resonance spectroscopy, it was concluded that the interaction of the RVXF motif is a prerequisite for the (cooperative) binding of other motifs (16).
One aim of the present study was to further define the consensus RVXF sequence using both alignments of established RVXF sequences and site-directed mutagenesis. This analysis revealed that the RVXF motif is rather degenerate and that "X" can be any residue except Pro. The second aim was to explore the function of the RVXF motif by swapping studies using variant RVXF motifs and unrelated PP1 interaction motifs. Our data suggest that the RVXF motif has only a "proximity" effect and thereby promotes the binding of secondary interaction sites to PP1.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-32P]ATP by purified phosphorylase kinase (15). Glutathione-Sepharose and blue Sepharose were obtained from Amersham Biosciences. Ni2+-pentadentatechelator-Sepharose was purchased from Affiland. Okadaic acid was obtained from Calbiochem. All peptides were synthesized by the N-(9-fluorenyl-)methoxycarbonyl method on a Milligen 9050. Preparation of Recombinant Proteins—Polyhistidine-tagged NIPP1-(143–224) was purified as described by Beullens et al. (15). Recombinant human inhibitor-1 was expressed as a fusion protein with glutathione S-transferase (GST) in BL21(DE3)pLys cells transformed with a pGEX-2T-inhibitor-1 plasmid provided by Dr. S. Shenolikar (Duke University). The fusion protein was purified on glutathione-Sepharose as described (7). The purified protein was cleaved with thrombin, and free inhibitor-1 was purified as a heat-resistant fragment after centrifugation. Inhibitor-1 was phosphorylated with the catalytic subunit of protein kinase A (PKA) (7). Inhibitor-2 was purified by chromatography on blue Sepharose of heat-treated lysates of BL21(DE3)pLys cells that had been transformed with the pET8d-inhibitor-2 plasmid, donated by Dr. A. DePaoli-Roach (Indiana University) (19). The pET15b plasmid encoding polyhistidine-tagged rabbit glycogen synthase kinase-3 (gift of Dr. P. Roach, Indiana University) was transformed into BL21(DE3)pLys cells, and the expressed fusion protein was purified on Ni2+-Sepharose.
Cell Lysates—COS-1 cells were washed three times in phosphate-buffered saline, resuspended in lysis buffer containing 20 mM Tris at pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, and 5 µg/ml leupeptin and lysed by three passages through 18- and 22-gauge needles. After 10 min of incubation on ice, the suspension was centrifuged (10 min at 10,000 x g), and the supernatant was used as the cell lysate.
Assays—NIPP1-(143–224), inhibitor-1, inhibitor-2, and their mutants were assayed as inhibitors of the phosphorylase phosphatase activity of the catalytic subunit of PP1 as described (20). The substrate was added after preincubation of the reaction mixture for 10 min at 30 °C. Phosphorylase phosphatase assays in cell lysates were performed identically, but the assays were made specific for PP1 by the addition of 10 nM okadaic acid. The inhibitor-2-mediated inactivation of PP-1 was monitored by the time-dependent decrease in trypsin-revealed phosphorylase phosphatase activity. For that purpose, the catalytic subunit was incubated at 25 °C in the presence of 20 nM inhibitor-2 in a buffer containing 20 mM imidazole at pH 7.5, 0.5 mM dithiothreitol, 5 mM -mercaptoethanol, and 1 mg/ml bovine serum albumin. The phosphorylase phosphatase activity was then measured after prior incubation of aliquots with trypsin (0.2 mg/ml) for 5 min at 30 °C and the addition of trypsin inhibitor (0.4 mg/ml). After the inactivation by inhibitor-2, PP1 was reactivated by the addition of glycogen synthase kinase-3 (GSK-3), MgCl2 (2 mM), and ATP (0.2 mM) for the indicated times at 30 °C, and the phosphorylase phosphatase activity was again determined after a trypsin treatment.
Mutagenesis—pET16b-NIPP1-(143–224) (21), pGEX-2T-inhibitor-1 and pET8d-inhibitor-2 served as templates for site-directed mutagenesis using the QuikChange protocol (Stratagene). All mutations were confirmed by DNA sequencing.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-strand conformation when bound to PP1 (12), and proline is well known to prevent -strand formation. In the muscle-type glycogen targeting subunit GM/RGl, the X in the RVXF sequence is a serine, and phosphorylation of this residue has been shown to prevent the interaction of this sequence with PP1 (12, 30, 31). We found that the T202E mutation did not have any effect on the IC50 value of NIPP1-(143–224), whereas the T202D mutation resulted in a 5-fold higher IC50 (Fig. 1). These data suggest that the effects of phosphorylation of the RVXF motif can be partially mimicked by an Asp at position X. Surprisingly, the effects of phosphorylation were not mimicked by a Glu at this position, suggesting than the additional hydrophobic CH2-group in the side chain compensates for the lower affinity induced by the acidic carboxyl group. A Phe at position X was also associated with a somewhat higher IC50 (Fig. 1).
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Our data seem at variance with a study by Liu et al. (32) who reported that the substitution of either Ala or Val for Ser in the RVSF sequence of the GM/RGl subunit abolished the binding of PP1. The presence of an Ala or Val at this position in an otherwise identical RVXF sequence clearly did not affect the inhibitory potency of NIPP1-(143–224) (Fig. 1). Moreover, it should be pointed out that in some regulators of PP1, for example the murine glycogen-targeting subunit R5/PTG (3) and the apoptotic regulator Bcl2 (Table I), the penultimate position of the RVXF motif is Val or Ala. Because the inhibitory potency of NIPP1-(143–224) is totally dependent on a functional RVXF motif (Figs. 2A and 4), the potent inhibition by the T202A or T202V mutants cannot be accounted for by secondary PP1 interaction sites. It is possible, however, that the corresponding mutations in the RVXF motif of the GM/RGl subunit turn this sequence into a binding site for a remote regulatory element and, thereby, make the RVXF motif inaccessible for binding to PP1.
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Swapping of RVXF Sequences between Inhibitor-1 and NIPP1—The RVXF motif is essential for inhibition of PP1 by inhibitor-1 and NIPP1, but inhibitor-1 is a less potent inhibitor than is NIPP1 (7, 15). To study whether this difference in potency is related to their RVXF sequence, we replaced the RVTF sequence in the central domain of NIPP1 with the RVXF sequence of human inhibitor-1 (KIQF). In Fig. 2A it is shown that this mutation turned NIPP1-(143–224) into a 3.5-fold less potent inhibitor, suggesting that the KIQF sequence indeed represents an RVXF motif that binds to PP1 with a relatively low affinity. In accordance with previous data (21), NIPP1-(143–224) was no longer inhibitory when the RVXF motif was destroyed by substitution of Ala for Val and Phe. The data in Fig. 2A were corroborated by competition studies with synthetic RVXF-containing decapeptides (Fig. 2B). Indeed, the addition of 50 µM of NIPP1-(197–206), which comprises the RVTF sequence and three flanking residues at each side, increased the IC50 of NIPP1-(143–224) 
16-fold. On the other hand, NIPP1-(197–206), with the RVXF motif from inhibitor-1 (KIQF), was a very poor competitor, and the V201A/F203A mutant was not a competitor at all.
Because the KIQF sequence emerged as a rather poor RVXF motif, we wondered whether the inhibitory potency of inhibitor-1 could be increased by replacement of the KIQF sequence by the NIPP1-derived RVTF sequence. In Fig. 3 it is shown that this mutation indeed turned phospho-inhibitor-1 into a more potent inhibitor of the PP1 catalytic subunit as well as the PP1 holoenzymes that are present in cell lysates. In accordance with previous reports (33, 34), the inhibition of the holoenzymes required a higher concentration of inhibitor-1. Non-phosphorylated inhibitor-1 with the RVXF sequence from NIPP1 was not inhibitory at all (not shown). This demonstrated that the inhibition of PP1 by the mutated inhibitor-1 was still entirely phosphorylation-dependent and that the increased inhibitory potency could not be accounted for by the creation of an additional inhibitory binding site. Thus, it seems likely that the mutated inhibitor-1 inhibited PP1 by the same mechanism as did the wild-type inhibitor, i.e. by the binding of phospho-Thr34 as a pseudosubstrate to PP1 (7). The increased inhibitory potency of the mutated inhibitor-1 can then be explained by the higher binding affinity of the NIPP1-derived RVXF motif for PP1, which is expected to increase the rate of association and/or decrease the rate of dissociation of phospho-Thr34.
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The RVXF Motif Can Be Functionally Replaced by Other PP1-binding Motifs—The above data are in accordance with the view that RVXF motifs serve to anchor R-subunits to PP1 and promote thereby the interaction of secondary binding sites with PP1 (see Introduction). A corollary of this view is that the R-subunits could still be functional if their RVXF motif was replaced by another PP1 anchoring motif. To test this experimentally, we have made use of two established PP1 binding sequences of inhibitor-2, which were originally described as the "IKGI" (35, 36) and "KLHY" sequences (37). Alignments showed that part of these sequences belong to phylogenetically conserved pentapeptide motifs that conform to the consensus sequences K-[GS]-I-L-K and R-[KR]-X-H-Y (Table II). Based on the human sequences and the nature of the conserved residues, we will further refer to these sequences as the KGILK and the RKXHY motifs, respectively.
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Following replacement of the RVTF sequence plus the preceding Ser by the KGILK and RKLHY sequences from human inhibitor-2, NIPP1-(143–224) was still inhibitory with IC50 values of 143 and 7 nM, respectively (Fig. 4). Because the replacement of the RVTF sequence by the RATA sequence completely abolished inhibition by NIPP1-(143–224), these data suggested that the KGILK and RKXHY sequences could at least in part functionally replace the RVXF motif. There are at least two obvious and not mutually exclusive explanations for why the NIPP1-(143–224) mutants with the KGILK or RKLHY sequences were less potent inhibitors than the wild-type protein. First, it is possible that the KGILK and RKLHY sequences bound to PP1 with less affinity than did the RVTF sequence. This view is supported by observations that the addition of the competitor NIPP1-(197–206) (1 mM) increased the IC50 for NIPP1-(143–224) some 200-fold, whereas the addition of the same concentration of the corresponding decapeptides of NIPP1-(143–224) with the RKLHY or KGILK sequences only increased the IC50 of the mutated NIPP1-(143–224) by 5- and 3-fold, respectively (not shown). Secondly, it is conceivable that the binding of the KGILK or RKLHY sequences to a site of PP1 that differs from the RVXF binding groove hampers the optimal interaction of the secondary binding sites of NIPP1-(143–224) with PP1.
Swapping of the RKXHY and KGILK Motifs of Inhibitor-2 for an RVXF Motif—Our observation that the function of the RVXF motif can be (partially) taken over by the KGILK or RKXHY motifs (Fig. 4) can only be used as new evidence of the anchoring role of the RVXF motif if these motifs do not all interact identically with PP1. Although convincing evidence has been presented showing that the KGILK motif binds to a site that differs from the RVXF binding groove (36), some modeling data and experiments with synthetic peptides suggested that the KLHY sequence of human inhibitor-2 also interacts with the RVXF-binding channel of PP1 (37). However, three independent lines of evidence argue against the latter view. First, although the KLHY sequence of human inhibitor-2 still resembles to some extent an RVXF motif, the consensus sequences for both motifs are entirely different (Tables I and II). More specifically, the Leu in position two, which was considered to be equivalent to the Val in the RVXF motif (37), is not conserved. Actually, in some species an Ala is present at this position (Table II), and this is known to be incompatible with a functional RVXF motif (12). Second, RVXF-containing peptides were shown to antagonize inhibition by inhibitor-2 (15, 37), whereas the deletion or mutation of the RKXHY motif did not have major effects on the inhibitory potency of inhibitor-2 (35, 37). This strongly indicates that the competition by these peptides cannot be accounted for by the displacement of the RKXHY motif and, thus, that the RVXF and RKXHY motifs have different binding sites on PP1. Third, we found that the KLHY sequence of human inhibitor-2 could not be functionally replaced by the NIPP1-derived RVTF sequence. Indeed, although the wild type and mutated inhibitor-2 were equally potent inhibitors of the catalytic subunit of PP1 (Fig. 5A), the mutated inhibitor-2 was a much poorer inhibitor of PP1 holoenzymes in cell lysates (Fig. 5B). However, the lesser inhibition of PP1 holoenzymes by mutated inhibitor-2 was not affected by the simultaneous addition of 50 µM synthetic peptide that comprises residues 135–158 of inhibitor-2 and includes the KLHY sequence (not shown). One way to account for these data is that the KLHY sequence plays a role in the disruption of PP1 holoenzymes that accompanies their inhibition by inhibitor-2 (38), a function that can apparently not be mimicked by the RVTF sequence. We also found that the characteristic time-dependent and trypsin-resistant inactivation of PP1 by inhibitor-2 (39) was faster with the mutated inhibitor (Fig. 6A) and that the MgATP-dependent reactivation of the inactive complex by glycogen-synthase kinase-3 occurred more slowly and to a lesser extent with inhibitor-2-(RVTF). (Fig. 6B). In conclusion, the KLHY 
RVTF mutant of inhibitor-2 was a poorer inhibitor of PP1 holoenzymes but promoted the conversion of the catalytic subunit into an inactive conformation.
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Although the RKXHY motif of inhibitor-2 does not appear to be essential for inhibition of the catalytic subunit of PP1 (Fig. 5A, and Ref. 37), the inhibitory potency of inhibitor-2 was severely decreased by mutation of the KGILK motif into EGGLK (Fig. 7) in accordance with data from Huang et al. (35). However, when the last four residues of the KGILK motif were replaced by RVTF, the IC50 for inhibition of PP1 only increased from 1 to 2 nM (Fig. 7). Moreover, the replacement did not significantly affect the inhibition of PP1 holoenzymes (not shown). Thus, the KGILK motif can be functionally replaced by an RVXF motif, indicating that the KGILK motif of inhibitor-2 has a similar function as the RVXF motif in most other PP1 interactors, although it does not bind to the RVXF binding groove (36).
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Conclusion—The consensus RVXF motif that we have delineated here as [RK]-X0–1-[VI]-{P}-[FW] occurs in about a third of all eukaryotic proteins, and only a small fraction of these are PP1 interactors. Therefore, the presence of an RVXF consensus sequence in itself is not sufficient to classify a protein as a putative PP1 interactor. Additional information on the functionality of an RVXF consensus sequence could come from the use of competing RVXF-containing peptides or from RVXF mutants. It is currently not entirely clear why the majority of the RVXF-consensus sequences do not mediate binding to PP1. However, crystal structure studies of p53BP2, which represents the C-terminal part of ASPP2, have revealed that the RVXF motif in the unbound interactor is not very structured (40). On the other hand, a peptide comprising the RVXF motif of the muscle-type G-subunit was shown to bind to the RVXF binding groove as a -strand (12). Thus, RVXF consensus sequences may only function as PP1 interaction sites when they are present in a flexible and exposed loop that can be modeled into a -strand.
Although our alignment and swapping studies strongly suggest that the RVXF motif contains four or five residues, this does, of course, not rule out the possibility that flanking residues can affect the affinity of this motif for PP1. By panning a random peptide display library, Zhao and Lee (41) identified a number of peptides that bind to PP1. The consensus PP1-binding peptide sequence that they delineated, i.e. [RK]-[RK]-X0–2-V-[RH]-[FW]-X-[DE], is surprisingly similar to the consensus RVXF motif that we have proposed. The main difference is that the consensus sequence proposed by Zhao and Lee contains an additional N-terminal basic residue and a C-terminal acidic residue. Interestingly, the established RVXF motifs are also often preceded by one or more basic residues and followed by one or more C-terminal acidic residues (Table I), which is in accordance with the view that these flanking residues affect the binding affinity of the RVXF motif.
Our swapping experiments suggest that both the ubiquitous RVXF motif and the inhibitor-2-specific KGILK motif have no other function than to mediate binding to PP1. By increasing the local concentration of the interactor, this initial binding promotes the interaction with PP1 of secondary lower affinity binding sites, which have interactor-specific effects on the activity and substrate specificity of PP1. In agreement with this view, PP1 interactors with a mutated RVXF motif still have the same effects on PP1, but these effects are only seen at much higher concentrations. For example, both NIPP1 and inhibitor-1 are still inhibitory after mutation of their RVXF motif, and their inhibitory potency is still controlled by phosphorylation, but the inhibitory concentrations are much higher. Our view of RVXF motifs as "anchors" for the binding to PP1 is consistent with the function deduced for the RVXF motif based on surface plasmon resonance spectroscopy (16) and peptide competition studies (12, 13, 14, 15). Although the KGILK sequence may act as an anchor for the binding of inhibitor-2 to PP1, the RKXHY motif does not appear to have this function, because its mutation or deletion had no major effect on the inhibition of the catalytic subunit of PP1 by inhibitor-2 (35, 37), and because the RKXHY motif could not be functionally replaced by the RVXF motif (Figs. 5B and 6).
We have used here a combination of bioinformatics tools and mutagenesis studies to delineate the consensus sequence and function of three PP1-binding motifs. The same approach could also be used to explore other PP1-binding motifs such as the recently described FXXRXR motif, which also appears to be shared by various PP1 interactors (42).
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FOOTNOTES |
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Postdoctoral fellow of the National Fund for Scientific Research-Flanders.
To whom correspondence should be addressed: Afdeling Biochemie, Campus Gasthuisberg, Katholieke Universiteit Leuven, Herestr. 49, B-3000 Leuven, Belgium. Tel.: 32-16-345701; Fax: 32-16-345995; E-mail: Mathieu.Bollen{at}med.kuleuven.ac.be.
1 The abbreviations used are: PP1, protein phosphatase 1; PP2, protein phosphatase 2; NIPP1, nuclear inhibitor of PP1.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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