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Volume 272, Number 45, Issue of November 7, 1997 pp. 28368-28372
(Received for publication, July 24, 1997, and in revised form, September 5, 1997)
From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT 
An unusually large number of regulatory or targeting proteins that bind to the catalytic subunit of protein phosphatase-1 have been recently reported. This can be explained by their possession of a common protein motif that interacts with a binding site on protein phosphatase-1. The existence of such a motif was established by the panning of a random peptide library in which peptide sequences are displayed on the Escherichia coli bacterial flagellin protein for bacteria that bound to protein phosphatase-1. There were 79 isolates containing 46 unique sequences with the conserved motif VXF or VXW, where X was most frequently His or Arg. In addition, this sequence was commonly preceded by 2-5 basic residues and followed by 1 acidic residue. This study demonstrates that binding to protein phosphatase-1 can be conferred to a protein by the presentation of a peptide motif on a surface loop. This binding motif is found in a number of protein phosphatase-1-binding proteins.
INTRODUCTION
Protein phosphatase-1, originally studied in the context of
glycogen metabolism as the enzyme that converts phosphorylase a to phosphorylase b (1), has been implicated in
the regulation of a number of important cellular processes (for
reviews, see Refs. 2 and 3). Biochemical studies have revealed that it consists of a catalytic subunit (4-7) of 37 kDa
(PP1)1 that forms a number of
heterodimeric enzymes with different subunits, which include a
glycogen-binding subunit, a myosin-binding subunit (8), and inhibitor-2
(9). These subunits function to target the catalytic subunit to the
subcellular or molecular proximity of its substrates and may serve to
provide regulation of activity as well as modulation of substrate
specificity (8). In addition, PP1 is regulated by several inhibitory
proteins; these include inhibitor-1 (10), DARPP-32 (10), inhibitor-2
and NIPP (a nuclear inhibitory protein) (11). The use of the yeast
two-hybrid system has led to the discovery of a surprisingly large
number of PP1-binding proteins. Mammalian PP1-binding proteins include
the retinoblastoma gene product (12), HSP78 (13), p53BP2 (14), splicing
factor PSF (15), ribosomal protein L5 (16), herpesvirus
134.5 protein (17), and HOX11 (18). In yeast, over a
dozen genes that encode PP1-binding proteins have been identified,
based largely on the use of a two-hybrid screen. These include genes
that are variously required for control of glycogen metabolism, glucose
repression, meiosis and/or sporulation, and mitotic cell cycle
regulation (19-21). Thus, PP1 is unusual in that this single enzyme is
involved in the regulation of a number of diverse cellular
processes.
Few, if any, of the PP1 proteins share any major sequence identity, although examination of different glycogen-binding subunits has revealed the presence of two small regions of sequence similarity that is shared between several glycogen-binding proteins (20, 22-25). The unusually large number of PP1-binding proteins that have been described suggests either that PP1 contains a motif that is recognized by a common binding structure on this diverse group of proteins or that the latter contain a protein motif that is recognized by a single binding site on PP1. To test the latter possibility, we have used a panning technique to isolate PP1-binding peptides using a random peptide display library and found that there is a consensus binding motif for PP1.
EXPERIMENTAL PROCEDURES
Recombinant PP1 was expressed in
Escherichia coli (26) and purified as described previously
(27). The preparation was further purified by gel filtration on a
Sephacryl S-200 column (1.5 × 60 cm).
The FliTrx random peptide library was obtained from Invitrogen (San Diego, CA) and is based on the system described by Lu et al. (28). Growth of the cultures and general panning methods were essentially as described in the manufacturer's protocol or as by Lu et al. (28). E. coli strain GI826 harboring the pFliTrx plasmids was grown in IMC medium (6 g/liter Na2HPO4, 3 g/liter KH2PO4, 0.5 g/liter NaCl, 1 g/liter NH4Cl, pH 7.4, 0.2% casamino acids, 0.5% glucose, and 1 mM MgCl2) containing 100 µg/ml ampicillin. Expression of the thioredoxin-flagellin fusion proteins containing the peptide inserts was induced by growth in IMC medium containing 100 µg/ml ampicillin and 100 µg/ml tryptophan at 25 °C for 6 h.
Immobilization of PP1 on Culture Plates
PP1 was
immobilized on 60-mm plastic Petri dishes by adsorption from a solution
of PP1 (1 ml, 100 µg of PP1/plate) with gentle agitation for 1 h. Adsorption was confirmed by testing for activity of the bound enzyme
toward phosphorylase a. The plates were washed with 10 ml of
sterile water and agitated for 1 h with 10 ml of blocking solution
(1% nonfat dry milk, 150 mM NaCl, 1% -methylmannoside, and 100 µg/ml ampicillin in IMC medium).
The following
were added to 50 ml of the induced E. coli culture after
6 h of growth: 0.5 g of nonfat dry milk, 1.5 ml of 5 M NaCl, and 2.5 ml of 20% -methylmannoside. The culture
(10 ml) was used in a "panning" technique by addition to the plate containing immobilized PP1. The plate was rotated gently for 1 min at
50 rpm and allowed to incubate for 60 min at room temperature. The
bacterial culture was decanted, and the plate was washed by gentle
agitation for 5 min with 10 ml of IMC medium containing 100 µg/ml
ampicillin and 1% -methylmannoside. After an additional four
washes, the bound bacteria were detached by simple mechanical shearing
(vortexing the plates for 30 s). The detached bacteria were
decanted from the plate and grown as described above. The culture was
then induced by growth with tryptophan-containing medium, and the cycle
of panning was repeated. Six rounds of panning were performed, after
which the cultures were plated out, and individual colonies were
selected for further study. The pFliTrx plasmids were then isolated and
sequenced using the primer 5
-ATTCACCTGACTGACGAC to obtain the sequence
of the inserted peptides.
PP1 (0.25 mg/ml) was dialyzed overnight in 50 mM sodium borate and 1 mM dithiothreitol, pH 8.5, and concentrated on a Centricon-10 filter. One ml of PP1 solution (1 mg/ml) was reacted with 25 µl of digoxigenin carboxymethyl-N-hydroxysuccinimide ester (2 mg/ml in dimethyl sulfoxide; Boehringer Mannheim) for 2 h at room temperature and then passed through a Sephadex G-25 column equilibrated in phosphate-buffered saline. The conjugated enzyme was then diluted 1000-fold in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mg/ml bovine serum albumin and stored at 4 °C. Polyclonal sheep anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim) were used for detection of PP1-binding proteins after SDS-PAGE and transfer to nitrocellulose (29, 30).
Assay of PP1 ActivityPP1 activity was assayed using 32P-labeled rabbit muscle phosphorylase a as the substrate (26). PP1 was diluted in 50 mM imidazole chloride, pH 7.4, 1 mM EDTA, and 0.2 mg/ml bovine serum albumin immediately before assay. The synthetic peptides RPKRKRKNSRVTFSEDDE, SKFERVDKRPNRSERTDK, CGPVRHKRVRFEADVGPC, CGPSTRHVHWDDREAGPC, and CGPRVSRHVHWADLEGPC were synthesized by GENEMED Synthesis Inc. (South San Francisco, CA). The peptides were dissolved in water and diluted in 50 mM imidazole chloride, pH 7.4.
RESULTS
The FliTrx library displays random dodecapeptides flanked by the
sequences CPG and GPC at their N and C termini, respectively, and has a
diversity of 1.77 × 108 individual peptides. The
peptides are inserted into the active-site loop of thioredoxin, which
is itself fused into the major flagellin protein of E. coli.
The peptides are thus displayed on the flagella. Six rounds of panning
on Petri dishes to which PP1 was adsorbed were performed (see
"Experimental Procedures"), after which the plasmids from
individual E. coli isolates were isolated and analyzed. To
confirm that these isolates were indeed binding to PP1, they were
examined for the expression of thioredoxin-flagellin fusion proteins
that would bind to PP1. This was done by SDS-PAGE of the E. coli proteins and transfer to nitrocellulose, after which PP1
binding was determined using digoxigenin-conjugated PP1. A typical set
of results are shown in Fig. 1. Of 13 isolates examined, it can be seen that there are 10 positives for a
protein of 68 kDa. This is consistent with the expected size of the
fusion protein since E. coli thioredoxin has a molecular
mass of ~12 kDa (31) and E. coli flagellin has a molecular
mass of ~60 kDa (32).
[View Larger Version of this Image (75K GIF file)]
DNA sequence analysis of the encoded peptides from a total of 104 isolates was performed. Seventy-nine of these isolates harbored peptide
sequences that could be aligned as shown in Table
I. These represent 46 unique sequences
that share the common motif VX(F/W). Twenty-three of the
sequences fell in the VXF family (Table I, series A-C),
with the most frequent of the motifs being VRF (series A1-A16).
Nineteen of the sequences fell in the VXW family (series D
F), of which 13 were VHW. Four other peptides (series G) were not as
well conserved. In the VXF series, X was also
found as His, Ser, Thr, and Lys, whereas in the VXW series, X was also found as Arg, Thr, Ser, Met, and Ala. Both the
VXF and VXW series were generally preceded by
2-5 basic residues and most commonly by 2 basic residues immediately
preceding the conserved valine. The residue immediately N-terminal to
the conserved valine was frequently Arg or Lys, although Gln and His
were also well represented. On the C-terminal side of the
VX(F/W) motif, it was also evident that an acidic residue
was present in 1 of the next 2 residues. Thus, the consensus motif that
emerges is
(R/K)(R/K)X0-2V(R/H)(F/W)X(D/E). The
frequency of amino acids at various positions is shown diagrammatically in Fig. 2. These findings demonstrate the
existence of a peptide binding motif for PP1.
|
[View Larger Version of this Image (33K GIF file)]
We tested several synthetic peptides for their ability to inhibit PP1
since a number of the proteins that contain the binding motif are
inhibitors of PP1. The peptide sequence derived from the NIPP sequence
(11), which contains the motif RVTFSE, and a scrambled sequence of the
same amino acids were tested for the ability to inhibit phosphorylase
phosphatase activity. The NIPP peptide was inhibitory, with an
IC50 of ~3 µM (Fig.
3). That this inhibition is specific is
supported by the failure of the scrambled sequence to inhibit PP1
activity at concentrations up to 10 µM. Above this value,
the scrambled NIPP peptide was inhibitory, with an IC50 of
100 µM. We also tested a peptide (Table I, A1) that harbors the VRF motif. This peptide was inhibitory, with an
IC50 of ~10 µM. This is consistent with the
finding that a N-terminal peptide (residues 349-430) of the splicing
factor PSF, which harbors an RVRF motif, is inhibitory to PP1 (15). Two
peptides (Table I, D1 and D2) containing the most frequently
encountered VHW motif were not inhibitory at low concentrations and
behaved in a similar way to the scrambled NIPP peptide in that
inhibition was observed at high concentrations. Because of the basic
nature of these peptides, it is possible that this inhibition is
similar to the inhibition of phosphorylase phosphatase activity by
polyamines, which has previously been shown to be likely a
substrate-directed effect (33).
),
the scrambled NIPP peptide (
), A1 (
), D1 (
), and D2
(
).
[View Larger Version of this Image (24K GIF file)]
DISCUSSION
In this study, we have used a random peptide display library in
which peptides are inserted into a surface loop at the active site of
thioredoxin (31), which itself is fused into a nonessential domain of
the bacterial flagellin protein (28). Our results clearly demonstrate
that a short peptide sequence containing the general motif
VXF or VXW preceded by 1 or more basic residues is sufficient to generate a capacity for the fusion proteins to bind to
PP1. These results strongly support the thesis that the binding of
proteins to PP1 can be specified by presentation of this binding motif.
During the course of this work, the crystal structure of PP1
in a
complex with a peptide corresponding to the region on the muscle
glycogen-binding subunit, GM, that had previously been
implicated in binding to PP1 was determined (34). The crystal structure
reveals that the sequence RRVSFA of GM binds in an extended
conformation to a hydrophobic groove, adjacent to a region of acidic
residues that accommodates the N-terminal basic residues. This site is
located on a side of PP1 that is nearly opposite to the active site and
is thus ideally situated as a site for the binding of targeting
subunits. Taken together with our findings that binding to this region
can be specified by a relatively short consensus peptide sequence,
there is now a strong structural basis for the concept that PP1
contains a site that recognizes a novel peptide motif that can confer
PP1-binding ability to a given protein.
An examination of the coding regions of a number of yeast genes as well
as the sequences of mammalian PP1-binding proteins for the presence of
the binding motif is shown in Table II.
Table II includes sequences encoded by the yeast genes
GAC1, REG1, REG2, SCD5, GIP1, SHP1, and GIP2 and
several yeast open reading frames, most of which were identified by a
two-hybrid screen (20, 21), and the mammalian proteins p53bp2, NIPP,
splicing factor PSF, the M110 subunit and several variants
of the glycogen-binding subunits, and the herpes simplex virus protein
134.5 (12, 14-18, 22-24). With one exception, these
all exhibit the VXF motif, and only one occurrence of the
VXW sequence was found. Both inhibitor-1 and DARPP-32
contain sequences at their N termini (KIQF) that are similar to the
RVXF motif and that are required for inhibition of PP1 (35,
36). Interestingly, the results of the random peptide screen did not
yield any substitution of Val for Ile. It should also be noted that
there are several sequences that fit the consensus in GIP2, SCD5, and
YFL023W (Table II), but that are inconsistent with the results of the
two-hybrid screen in that the fusions are ones in which these sequences
are deleted. Clearly, identification of any of these sequences as a
PP1-binding motif needs to be experimentally confirmed by mutagenesis
studies.
|
Our data indicate that binding of the peptides containing the NIPP sequence and a peptide containing a VRF motif was inhibitory in the micromolar range. This is much weaker than the inhibition observed with several inhibitory proteins of PP1, which have IC50 values in the nanomolar range (2, 3, 10). On the other hand, two other peptides containing the VHW motif were not inhibitory. The differential effects on the inhibition of phosphorylase phosphatase activity observed with the peptides are not surprising, given that the binding of the GM subunit has been shown to partially inhibit phosphorylase phosphatase activity, but to increase activity toward glycogen synthase (15, 22, 34). It remains a possibility that binding of variants of the PP1-binding motif could produce differential effects on either activity or substrate specificity. In this regard, it is of interest to note that both inhibitor-1 and DARPP-32 contain sequences at their N termini (KIQF) that are similar to the RVXF motif and that are required for inhibition of PP1 (35, 36). Irrespective of whether occupancy of the VXF binding site can affect either activity or substrate specificity, there is support for a model in which a high affinity interaction with PP1 involves at least two binding sites of lower affinity, as has been suggested for inhibitor-1 and DARPP-32 (34-36).
The analysis of putative PP1-binding proteins for motifs that could represent a binding sequence is obviously facilitated by the information provided by the availability of the family of peptides that are shown in Table I. The structural information that only a small peptide motif is involved in the interaction with the PP1 binding site (34) and the demonstration that presentation of peptides conforming to a consensus motif on a surface loop is sufficient to endow PP1 binding to the thioredoxin-flagellin fusion protein have other implications for the identification of PP1-binding proteins. The motif K(V/I)XF occurs in 10% of known sequences, so that the occurrence of the motif is very common, and the issue of whether such a sequence functions to bind PP1 may depend on its structural context (34). Thus, caution is needed in the sole use of the yeast two-hybrid system for the identification of PP1-binding proteins, particularly since this commonly involves the ability of partial protein sequences to interact with PP1. Expression of a fusion protein with a partial sequence or even fusion of a full-length sequence may lead changes in the structural context of sequences that have the potential for PP1 binding, but that are cryptic in the native protein, such that these are now surface-accessible and functionally active in PP1 binding. The identification of putative PP1-binding proteins that are of physiological relevance by the two-hybrid system needs to be confirmed experimentally by the biochemical demonstration of binding of the native protein with PP1. For example, it was found that the PSF protein inhibited PP1 with an IC50 that is ~30-fold higher than for a partial fragment, and it was concluded that PSF, at least in an unmodified form, was unlikely to function to inhibit PP1 in vivo (15). The same caveat holds for the use of digoxigenin-conjugated PP1 for the detection of PP1-binding proteins following SDS-PAGE. Nevertheless, it is evident that the number of mammalian PP1-binding proteins is likely to be quite large, as demonstrated by the use of immobilized microcystin affinity chromatography (37).
Recent findings have now provided a basis for understanding the complex enzymology and regulation of PP1, which have been an enigma for half a century (1-3). First, the primary structure of the catalytic subunit of PP1 is one of the most highly conserved in evolution (38). Second, PP1 exhibits a broad specificity that is seemingly inconsistent with its multiple regulatory functions. Third, targeting of PP1 to the molecular proximity of its substrates appears to be essential for the expression of its function. This is a critical concept, first shown for the effect of mutations of PP1 in yeast that prevent its association with the yeast glycogen-binding subunit (39, 40) and also by the deletion of yeast genes for the targeting subunits (20). These considerations suggest that the evolutionary route for PP1 took a different direction from that of the protein kinases. Rather than the evolution of multiple catalytic subunits with different functions (41), the route taken was the acquisition of multiple regulatory subunits that individually define both specificity and function. This process would have been facilitated through the agency of the PP1-binding motif that is the focus of this study. A new perspective of PP1 and its regulatory proteins emerges, in which PP1 heterodimers can be viewed as a family of protein phosphatases of far more diversity than previously could be considered from the standpoint of the existence of PP1 as a single catalytic entity. This evolutionary strategy provides a novel paradigm for the development of an enzyme family that encompasses diverse cellular functions. It is also noteworthy that a parallel in the use of targeting proteins exists for the protein kinases such in the AKAP family of proteins (42, 43).
Finally, a knowledge of the binding site of PP1 and of its specificity for variations of the binding motif provides the foundation for design of peptide inhibitors that could target this site, with the possibility of selective interference with PP1 functions. The idea that PP1 may be a useful target for future therapeutic purposes has recently been enhanced by the observations that targeting of PP1 by a herpesvirus protein is part of the viral mechanism for overcoming host defenses (17) and by the observation that HOX11, which is oncogenic in human T-cell leukemia, interacts with PP1 and PP2A (6).
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4057; Fax: 914-594-4058.
REFERENCES
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