(Received for publication, September 27, 1996)
From the Muscle Biology Group, University of Arizona, Tucson, Arizona 85721
Myosin phosphatase from smooth muscle consists of a catalytic subunit (PP1c) and two non-catalytic subunits, M130 and M20. Interactions among PP1c, M20, and various mutants of M130 were investigated. Using the yeast two-hybrid system, PP1c was shown to bind to the NH2-terminal sequence of M130, 1-511. Other interactions were detected, i.e. PP1c to PP1c, M20 to the COOH-terminal fragment of M130, and dimerization of the COOH-terminal fragment of M130. Mutants of M130 were constructed to localize the PP1c and light chain binding regions. Results from the two-hybrid system indicated two binding sites for PP1c on M130: one site in the NH2-terminal 38 residues and a weaker site(s) in the ankyrin repeats region. Inhibition of PP1c activity with phosphorylase a by the M130 mutants also was consistent with the assignment of these two sites. Overlay assays showed binding of phosphorylated light chain to the ankyrin repeats, probably in the COOH-terminal repeats. Activation of PP1c with phosphorylated light chain required binding sites for PP1c and substrate, plus an additional sequence COOH-terminal to the ankyrin repeats. Thus, activation of phosphatase and binding of PP1c and substrate are properties of the NH2-terminal one-third of M130.
Phosphorylation of the two regulatory light chains of myosin is an important mechanism in controlling the contractile activity of smooth muscle (1). The extent of phosphorylation depends on the balance of activities of two enzymes: the Ca2+/calmodulin-dependent myosin light chain kinase and a myosin phosphatase (MP).1 Under some conditions, for example stimulation with certain agonists, the extent of phosphorylation at suboptimal Ca2+ concentrations can increase, and this has been traced to an inhibition of MP activity (2). The pathway from receptor occupancy to phosphatase inhibition is not established but is thought to involve trimeric (2) and monomeric (2-6) G proteins. In addition it has been suggested that MP inhibition is caused by dissociation of the subunits by arachidonic acid (2, 7) or by phosphorylation of the large MP subunit (8, 9).
A classification of protein phosphatases which is used widely was based
on substrate preferences and inhibition by inhibitors 1 and 2 (10). Two
classes were identified, 1 (PP1) and 2 (PP2) with the latter divided
into three subgroups, A, B, and C. Several years ago it was suggested
that the smooth muscle MP was a type 1 enzyme (11). Subsequently three
laboratories have reported that MP from gizzard (12, 13) and pig
bladder (14) consists of three subunits: a catalytic subunit of about
38 kDa; a large subunit of 110-130 kDa (based on motilities on
SDS-PAGE), termed M130; and a subunit of about 20 kDa, M20. The
cDNAs for each subunit have been sequenced. The PP1c from gizzard
is the isoform (13), also referred to as the
isoform (12). In
gizzard, two isoforms of the large subunit exist, M130 and M133 (13);
these are similar to, but not identical with, the large subunit from
rat aorta, termed M110 (15). Another isoform of this subunit was
detected from a rat kidney cDNA library and represented the
NH2-terminal 72.5-kDa fragment (16). The derived sequence
of M20 from gizzard (15) indicates several leucine zipper sequences at
the COOH-terminal end. This structure is similar to the COOH terminus
of rat aorta M110 (15) but different from the two gizzard isoforms (13) which lack leucine zippers.
A possible regulation mechanism for phosphatases, and kinases, involves targeting (12) or anchoring subunits (17). These bind to the catalytic subunit and to the substrate or other specific target. In addition to localizing the phosphatases they may also modify the activity of the catalytic subunit. Thus the two non-catalytic subunits of MP could act as targeting subunits (12). In support of this idea it is known that the holoenzyme has enhanced activity toward myosin, compared with PP1c (12, 14), and also that the holoenzyme binds to myosin (13). The sites for interactions among subunits are not defined, but it has been shown that the NH2-terminal portion of M130 probably contains the PP1c site(s) and myosin binding site(s) (16, 18). An interesting feature of the M130 molecule is that it contains several ankyrin repeat sequences (13, 15) close to the NH2 terminus which could provide a platform for multiple interactions.
To investigate interactions of the subunits of MP the yeast two-hybrid system was employed. This powerful technique to study protein-protein interactions was developed by Fields and Song (19) and subsequently has been used for many systems, including screening of a gizzard cDNA library for PP1c-binding proteins (20, 21). In this present application various subunits, or fragments thereof, were inserted as bait or prey with the objectives of obtaining an outline of the subunit interactions of the holoenzyme. Where applicable the interactions were also monitored by enzyme assays or by overlay techniques.
Oligonucleotides were synthesized at the Macromolecular Structure Facility at the University of Arizona and by National Biosciences Inc. Plymouth, MN. The antibody to the hexahistidine tag (MRGSHis) was from Qiagen. Enzymes and media for bacterial and yeast cultures and radionucleotides were as listed previously (20).
Bacteria and Yeast StrainsEscherichia coli
DH5 was used as the transformation recipient for plasmid
constructions in the two-hybrid system. Saccharomyces cerevisiae strain Y190 (20) and Y187 (Clontech) were used for the
two-hybrid assay, as a recipient of bait and prey plasmid, respectively. E. coli JM109 and M15[pREP4] were used as
transformation recipient and as expression host for the pQE expression
system (Qiagen), respectively.
Plasmid pAS1
was used to construct the bait plasmid encoding the GAL4 DNA binding
domain hybrid protein (20). Plasmid pACT2 (Clontech) or pGAD424 (20)
was used to construct the prey plasmid encoding the GAL4 activation
domain hybrid protein. All cDNA inserts were obtained by PCR
amplification with Taq DNA polymerase (Boehringer Mannheim).
A cDNA of the full-length coding region of the catalytic subunit of
type 1 protein phosphatase (PP1) was obtained as described (20). The
cDNAs for some of the plasmids were obtained by PCR amplification
directly from a chicken gizzard Uni-Zap cDNA library (13). These
included the cDNAs for two NH2-terminal fragments of
M130 containing residues 1-511 and 1-633 (referred to as
M1301-511 and M1301-633, respectively); a
COOH-terminal fragment of M130 (M130512-963 or
M130514-963); and the coding region of M20, minus the
COOH-terminal 28 residues (M201-158). In some plasmid
constructions the insert was transferred from a preexisting plasmid to
another, using PCR amplification of the insert DNA. All primers were
designed to contain restriction sites for ligation. In some cases a
stop codon was inserted in the primer. The bait plasmids thus obtained
were pAS1-PP1
, pAS1-PP1
1-108, pAS1-M20,
pAS1-M1301-511, and pAS1-M130512-963. The
prey plasmids were pACT2-PP1
, pACT2-M1301-511,
pACT2-M1301-633, pACT2M130514-963,
pGAD424-PP1
, pGAD424-M20, and pGAD424-M1301-511. The
DNA sequences of all plasmids were determined.
Various truncation mutants of M130 were constructed for use in the two-hybrid system and for expression as hexahistidine-tagged proteins (18). All cDNAs for the truncation mutants were obtained by PCR amplification with pACT2-M1301-633 as the template DNA. The sense and antisense primers were designed to contain BamHI and SalI sites for ligation to the vector plasmid, respectively. Pfu DNA polymerase (Stratagene) was used for the PCR. The BamHI and SalI-digested PCR products were ligated to the BamHI- and XhoI-digested pACT2 for the prey constructs in the two-hybrid system and to BamHI- and SalI-digested pQE32 for expression as hexahistidine-tagged proteins. Truncation mutants obtained were M1301-38, M1301-69, M1301-104, M1301-137, M1301-171, M1301-296, M1301-674, M13040-511, M130168-511, and M130304-511.
The Two-hybrid Assay Using Yeast MatingThe principle of
the two-hybrid assay for protein-protein interaction was described
elsewhere (22). In the standard two-hybrid assay, haploid yeast cells
are cotransformed with two plasmids, bait and prey. This standard assay
was used to examine interactions between the phosphatase subunits with
pAS1 and pGAD424 as bait and prey vectors, respectively. However, the
sensitivity of this assay was low. To increase sensitivity the
two-hybrid assay was carried out using a different prey vector, pACT2,
and also by mating two types of yeast strains expressing either the
bait or prey hybrid proteins. The advantages of this approach were
first, that pACT2 gives higher levels of expression than pGAD424 (see Clontech literature); and second, that the mated diploid cells (Y190
and Y187) contain two copies of the lacZ reporter gene, and
this should lead to a higher level of -galactosidase expression compared with a single copy. The bait and prey plasmids were introduced into S. cerevisiae strain Y190 (MATa) and Y187
(MAT
), respectively, using the lithium acetate method
(23). The two strains were mixed and incubated overnight in YPD medium.
The mated cells were selected by plating on SC-Try-Leu agar, and this
plate was used for lacZ reporter assay. The expression of
all bait and prey protein was confirmed by Western blot analysis of the
yeast homogenate with anti-hemagglutinin antibody (20), anti-PP1
(20), or anti-M130 (8).
The filter lift assay for
-galactosidase was as described (20). To quantitate
-galactosidase activity all colonies were collected after the filter
lift assay and cultured in SC-Try-Leu broth. Activities were determined
using chlorophenol red-
-D-galactopyranoside as substrate
and expressed as units defined by Bartel and Fields (22).
The GAL1-HIS3 reporter gene utilized in Y190 had residual HIS3 expression sufficient to allow growth without exogenous histidine, even in the absence of GAL4 (24). The stringency of the His selection can be modulated by varying the concentrations of 3-aminotriazole. Therefore, the level of HIS3 reporter gene expression was semi-quantitatively evaluated by examining growth of yeast colonies on SC-Try-Leu-His containing various concentrations of 3-aminotriazole (0, 5, 10, 20, 30, and 50 mM). The mated yeast cells were streaked on SC-Try-Leu-His + 3-aminotriazole and allowed to grow for 5-10 days. The extent of growth for each concentration of 3-aminotriazole was then evaluated.
Expression and Purification of Hexahistidine-tagged Truncation MutantsE. coli cells M15[pREP4], containing the pQE
expression plasmid, were cultured in LB broth supplemented with 100 µg/mg ampicillin and 25 µg/ml kanamycin at 37 °C overnight. A
100-fold dilution of the overnight culture was grown at 37 °C until
the absorbance at 600 nm reached between 0.7 and 0.9. Expression then
was induced by addition of
isopropyl--D-thiogalactopyanoside to 0.4 mM.
Cells were allowed to grow for 3-4 h and collected by centrifugation at 15,000 × g for 15 min at 4 °C. After washing
with 40 mM Tris-HCl (pH 8.0), 1 mM EDTA and
centrifugation, the cell pellets were stored at
80 °C. After
thawing at 4 °C, cells were homogenized in 6 M guanidine
HCl, 50 mM sodium phosphate, 10 mM Tris-HCl, 100 mM NaCl (pH 8.0; buffer A). The homogenate was
clarified by centrifugation at 70,000 × g for 20 min
at 4 °C, and the supernatant was mixed for 20 min with metal
affinity resin (Talon, Clontech) equilibrated in buffer A. The resin
was loaded into a column and washed extensively with 6 M
guanidine HCl, 50 mM sodium phosphate, 100 mM
NaCl (pH 7.0). The bound protein was eluted by 100 mM
imidazole, 6 M guanidine HCl, 20 mM Tris-HCl
(pH 8.0), 100 mM NaCl. The eluate was dialyzed against 30 mM Tris-HCl (pH 7.5), 30 mM NaCl, 0.5 mM dithiothreitol. The dialysate was clarified by
centrifugation at 12,000 × g for 10 min at 4 °C,
and diisopropyl fluorophosphate and leupeptin were added to 0.5 mM and 10 µg/ml, respectively. The yield of soluble
protein varied with the mutant. Some examples, given in mg of
protein/liter of culture, were 0.5 for M1301-38 and 12 for
M1301-171. In general, the purity was estimated by
SDS-PAGE to be 70-95%.
32P-Labeled myosin light chain (11) or phosphorylase a (25) was used as a substrate at 5 µM. The assay mixtures contained approximately 1 nM PP1c prepared from turkey gizzard and various concentrations of mutants in 30 mM Tris-HCl (pH 7.5), 30 mM NaCl, 0.5 mM dithiothreitol, and 0.4 mg/ml bovine serum albumin. Caffeine, 3 mM, was added for assays with phosphorylase a. After a 3-min incubation at 30 °C reactions were started by the addition of substrate and terminated by the addition of trichloroacetic acid and bovine serum albumin to final concentrations of 7% and 4 mg/ml, respectively. Aliquots were cooled to 0 °C and the precipitated protein sedimented at 15,000 × g for 2 min. Radioactivity (i.e. released 32P) of the supernatant was determined by Cerenkov counting.
Light Chain OverlayLC20 was isolated from gizzard myosin
(26) by the procedure of Hathaway and Haeberle (27). Approximately 7 mg
of freeze-dried LC20 was dissolved in 0.1 M KCl, 30 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, and 10 mM dithiothreitol and after 2 h at 4 °C dialyzed against this solvent minus dithiothreitol under N2.
N-[6-(Biotinamide)hexyl]-3-(2
-pyridyldithio)propionamide, Biotin-HPDP (Pierce) was coupled to LC20 according to the
manufacturer's protocol. The biotinylated LC20 (2.5 ml at 2.8 mg/ml)
was dialyzed versus 0.1 M KCl, 30 mM
Tris-HCl (pH 7.5) and thiophosphorylated by incubation at 25 °C for
30 min with 1 mM ATP
S, 50 µg/ml myosin light chain
kinase (28), 5 µg/ml calmodulin (29), 0.05 mM CaCl2, 1 mM MgCl2, 30 mM Tris-HCl (pH 7.5), and 50 mM KCl. The ATP
S was removed by dialysis versus 0.15 M
NaCl, 20 mM Tris-HCl (pH 7.5). After SDS-PAGE the various
mutants were transferred to nitrocellulose membranes by standard
procedures (30). Prior to overlay the membranes were blocked in 5%
non-fat dried milk and 0.9 mg/ml dephosphorylated LC20. The overlay was
carried out with 5 µM thiophosphorylated biotinylated
LC20, and binding was detected with the streptavidin-horse radish
peroxidase conjugate (Chemicon) and 4-chloro-1-naphthol (Sigma).
The type 1 catalytic subunit was isolated
from frozen turkey gizzard (31). This is predominantly a 38-kDa
species. Nucleotide sequences were determined with a Sequenase version
2.0 DNA sequencing kit (U. S. Biochemical Corp.) and
-35S-dATP (Du Pont NEN). Other methods as given
previously (20).
To assess the binding
among the three subunits of the holoenzyme, various bait and prey
plasmids were constructed and inserted into the yeast two-hybrid system
to detect interactions. The colorimetric procedure monitoring
-galactosidase was used. The bait plasmids utilized cDNAs from
PP1
, the M20 subunit minus the leucine zipper sequences, an
NH2-terminal part of M130 from residues 1 through 511 (M1301-511), and the COOH-terminal part of M130
(M130512-963). The prey plasmids included PP1
and
M1301-511 plus an NH2-terminal fragment of
PP1
(PP1
1-108), a larger NH2-terminal
fragment of M130 (M1301-633), and a COOH-terminal portion
of M130 (M130514-963). Results from the two-hybrid assays
are shown in Table I. With PP1
as bait, the
interacting bait-prey pairs were PP1
-PP1
, PP1
M1301-511, and PP1
-M1301-633.
The interaction between the catalytic subunit and the
NH2-terminal fragment of M130 was bidirectional and was
shown when M1301-511 was used as bait and PP1
as prey
(Table I). A PP1
mutant containing the NH2-terminal
third of PP1
(PP1
1-108) did not show this
interaction (Table I). Other interactions also were detected and were:
binding of M20 (as bait) and the COOH-terminal fragment of M130
(M130512-963); interaction among the COOH-terminal
fragments of M130; and a weaker interaction between PP1
and the
COOH-terminal fragment of M130.
|
The key point from the results shown in Table I was that PP1c bound to
the NH2-terminal fragment of M130. However, the smallest NH2-terminal fragment used was still relatively large,
i.e. 511 residues, and it was important to define more
precisely the PP1c binding site(s). To achieve this a variety of
mutants was constructed as shown in Fig. 1. These
include progressive COOH-terminal deletions from Asn-511 and
NH2-terminal deletions with Asn-511 fixed as the COOH
terminus. These mutants were used in the two-hybrid system as prey
proteins with PP1 as bait and were also expressed as hexahistidine-tagged fusion proteins.
The results of the two-hybrid assays using both the filter lift assays
and -galactosidase measurements are shown in Table II. All of the mutants that contained sequences
initiated at Met-1 bound PP1
. The shortest NH2-terminal
segment was Met-1 to Phe-38, and this also bound to PP1
. This
sequence precedes the ankyrin repeats that for the gizzard M130/133
isoforms begin at Asp-39 (13). Although the interaction between
M1301-38 and PP1
appeared weaker than for longer
segments of M130 (Table II), it represented a critical interaction
since those mutants lacking the NH2-terminal 39 residues
did not bind PP1
. This is shown in Table II for the mutant
M13040-511. Other mutants lacking longer
NH2-terminal sequences also did not bind PP1
(Table
II).
|
The 3-aminotriazole resistance (an assay of the HIS3
reporter gene) of these bait-prey pairs also was estimated. In general, the same pattern was observed as shown in Table II. The strongest interactions, i.e. PP1-M1301-633 and
PP1
-M1301-69, showed vigorous growth at 30 mM 3-aminotriazole and positive but reduced growth at 50 mM. The weaker interactions had a reduced tolerance of
3-aminotriazole (data not shown).
It was
shown previously that the MP holoenzyme is more active with
phosphorylated myosin or P-LC20 than the isolated catalytic subunit
(12, 14, 18) and that this activation is carried by an
NH2-terminal fragment, i.e.
M1331-674 (18). It is assumed that activation of
phosphatase activity requires the presence of at least two sites on
M130, a binding site for PP1 and one for the substrate, P-LC20. Thus
the M130 mutants, expressed as hexahistidine fusion proteins, were
assayed for their effect on PP1c activity using 32P-LC20 as
substrate (Fig. 2). Three of the mutants containing the
NH2-terminal region of M130 activated PP1c activity. These were M1301-633, M1301-511, and
M1301-374. However, the smallest mutant was less
effective, and activation required higher concentrations. Further
truncation of the NH2-terminal segment caused loss of
activation, as shown for M1301-296. The M130 fragment in
which the first 39 residues were deleted, M13040-511, also
did not activate phosphatase activity (Fig. 2).
One possibility is that the interaction of PP1c and M130 induced a
conformational change in PP1c which resulted in activation of
phosphatase activity. It would be predicted that this effect is
independent of substrate. To test if the activation of PP1c is
dependent only on interaction with M130 and is not specific with
respect to substrate, the effect of various mutants on phosphatase activity was assayed using phosphorylase a as substrate. The
effect of three representative M130 mutants is shown in Fig.
3. Each mutant inhibited phosphatase activity, but the
potency of inhibition varied markedly. The most effective inhibitor was
M1301-511, whereas M13040-511 was
considerably less potent. To categorize this effect each of the mutants
was assayed for its effect on the PP1c- phosphorylase a
system, and IC50 values (i.e. the concentration
of mutant required for 50% inhibition) were calculated. These are
given in Table III. The relative value for each mutant
compared with M1301-633 also is given. These values can be
divided into three groups. For the mutants including the sequence from
1-633 down to 1-296 (top four mutants of Table III) the
IC50 values were in the subnanomolar range. On further
truncation at the COOH-terminal end of M130, i.e. those
mutants including 1-171 to 1-38, the IC50 values were approximately 10-fold higher. Deletion of the NH2-terminal
sequences, notably the first 39 residues, caused a further increase in
IC50 values (bottom three mutants of Table III).
|
To obtain an independent assessment of
P-LC20 binding an overlay procedure was used. Each of the M130 mutants
was screened for binding to thiophosphorylated, biotinylated LC20.
In Fig. 4 are shown the SDS-PAGE patterns of the mutants
and the light chain overlays. For the larger mutants many of the lower
molecular mass bands were proteolysis products since they retained the
hexahistidine tag (as shown by the MRGSHis antibody). For
the smaller mutants (M1301-171 and below) proteolysis
apparently was not a problem. Binding of light chain was detected for
the NH2-terminal fragments containing a complete ankyrin
repeat region M1301-296 and larger and also for mutants
M13040-511 and M130168-511. The latter two
mutants did not activate PP1c. The M130168-511 mutant
contains the COOH-terminal half of the ankyrin repeats, and this
suggests that this part of the molecule is involved in light chain
interaction.
From the initial two-hybrid screen a rough plan of the holoenzyme
can be obtained. The NH2-terminal third of M130 is
involved in the interaction with PP1c, and M20 binds to the
COOH-terminal part of M130. The function of M20 is not known. The
results of the two-hybrid assays indicate that it does not bind to
PP1. Earlier results showed that the complex of PP1c and an
NH2-terminal fragment of M130, i.e. the 58-kDa
component, was similar to the trimeric MP holoenzyme in terms of myosin
dephosphorylation and binding to myosin (33). This complex did not
include M20. Thus a role for M20 in activation of PP1c or in binding to
myosin is unlikely. It has not been determined if M20 is required for
regulation of MP activity. In addition, it is possible that M20 does
not influence phosphatase activity but may serve an auxiliary function such as targeting MP to other proteins and indirectly modifying activity or determining cellular localization.
Various interactions with PP1c (PP1) were indicated. The initial
two-hybrid screen showed that PP1
could self-associate. Dimer
formation of PP1c has been demonstrated (34), and it was also proposed
that myosin light chain phosphatases isolated from gizzard consisted of
a tetramer of catalytic subunits (35, 36). Thus, the association of
PP1c subunits is consistent with earlier results, but the physiological
significance of dimer or tetramer formation is not known. It is
unlikely that a PP1c dimer or tetramer would have a high affinity for
myosin since PP1c in solution does not bind to dephosphorylated or
phosphorylated myosin (18).
Interaction of PP1c and M130 forms an important component of the
function of the MP holoenzyme. The two-hybrid assays for various M130
mutants as prey and PP1 as bait indicated that PP1
binds to the
NH2-terminal part of M130. The surprising observation was
that PP1
bound to the NH2-terminal segment of 38 residues. This precedes the ankyrin repeats that begin at Asp-39. The
binding of PP1
to this segment was weaker than for the longer
NH2-terminal sequences but was an important component of
overall binding since the mutants lacking this sequence,
e.g. M13040-511, did not give a positive
signal. Recently, Endo et al. (37) have shown that an
NH2-terminal peptide of inhibitor-1, KIQF, was required for
full inhibition by phosphorylated inhibitor-1. One possibility,
suggested by these authors, was that the tetrapeptide represented part
of a PP1c binding site distinct from the catalytic site occupied by the
phosphorylated Thr-35 of inhibitor-1. A similar sequence, KVKF, is
found only in one position in M130/133, namely at residues 35-38 (13).
This NH2-terminal sequence also is present in rat aorta
M110 (15) and rat kidney M110 (16). Thus it is possible that this
sequence forms at least part of the PP1c binding site present in the
sequence 1-38. While this manuscript was in preparation, Johnson
et al. (38) also noted the importance of the
NH2-terminal sequence of M130 in binding PP1c. However,
they reported that the sequence 1-38 activated PP1c (38) and
facilitated relaxation in skinned fibers (39), although at relatively
high concentrations. This was not observed in our studies.
The inhibition data from the PP1c-phosphorylase a assays can also be used to assess PP1c binding to the M130 mutants. If it is assumed that there is no specific interaction between M130 and phosphorylase a, then inhibition of phosphatase activity by M130 and its mutants would reflect competitive binding of PP1c to M130 and phosphorylase a. In addition, it is required that the binding sites(s) on PP1c for M130 and phosphorylase a is similar, or the sites overlap. This has recently been shown (38). The most effective inhibitors, therefore, would possess a higher affinity for PP1c. These are represented (see Table III) by those mutants possessing the NH2-terminal 38 residues plus a longer NH2-terminal segment, possibly the ankyrin repeats. Truncation of these mutants at the COOH-terminal end reduces the inhibitory potency. Removal of the four COOH-terminal ankyrin repeats (in M1301-171) produces a less effective inhibitor. The predicted PP1c binding site for this second group of mutants is the NH2-terminal sequence 1-38. The loss of this NH2-terminal sequence generates the third group of inhibitory mutants. Here the inhibitory potency is low, indicating a reduced affinity of binding, and it is difficult to assign the location of the additional PP1c binding site. The very high IC50 for M13040-511 compared with the other two mutants in this group cannot be explained but was a reproducible observation.
The results from the light chain overlays (using the biotinylated thiophosphorylated LC20 as a probe) indicate that the ankyrin repeats are required for light chain binding. The NH2-terminal segment of 39 residues is not involved. Further, it is suggested that the COOH-terminal half of the ankyrin repeats is important, since binding was detected for M1301-296 but not M1301-171. The fifth ankyrin repeat shows a considerably lower homology than the other repeat sequences (13) and in fact was not considered as an ankyrin repeat in the rat M110 (15). Thus it is suggested that repeats 6, 7, and 8 may play a more crucial role.
The smallest NH2-terminal fragment that could activate PP1c
(using 32P-LC20 as substrate) was M1301-374.
This mutant contains in addition to the ankyrin repeats another 78 residues, and in this sequence the notable feature is an acidic cluster, residues 326-372 (13). It is not known if this sequence contains an additional binding site for PP1 or P-LC20 or if it is
necessary for correct folding or orientation of the ankyrin repeats.
In summary, a tentative plan of the M130 molecule can be assembled from the above data, with the emphasis on the NH2-terminal portion. For PP1c at least two sites are indicated: a relatively strong site in the NH2-terminal 38 residue sequence and a second weaker site in the ankyrin repeats, possibly in the COOH-terminal half of the repeats. If such is the case then the NH2-terminal portion of M130 may wrap around PP1c. The binding of P-LC20 is indicated in the ankyrin repeats, and again the COOH-terminal repeats are suggested to be involved. Activation of PP1c by M130 is assumed to require binding to both PP1c and substrate, P-LC20. Thus, theoretically the sequence 1-296 should be sufficient for activation. However, the situation is more complex, and additional COOH-terminal sequence (297-374) was required for activation. Whether this sequence contains additional sites for interaction or is needed to stabilize the NH2-terminal segment is not known. Another possibility is suggested by earlier results (9), namely, that if inhibition of PP1c by phosphorylated M130 results from binding of Thr-654 to the active site of PP1c then the M130 molecule must fold to accommodate this interaction.
We are grateful for the expert technical assistance of M. Hirano and C. Dudas and to Dr. T. Butler (Jefferson Medical College) for many helpful discussions and assistance with the manuscript.