(Received for publication, June 5, 1995; and in revised form, August 8, 1995)
From the
The Mg-dependent serine/threonine protein
phosphatases, also known as type 2C phosphatases (PP2C), belong to a
gene family distinct from the other serine/threonine phosphatases and
tyrosine phosphatases. Here we report the purification to apparent
homogeneity of a novel Mg
-dependent,
Ca
-inhibitable serine/threonine protein phosphatase
from bovine brain. It is a type 2C enzyme in view of its Mg
requirement, resistance to okadaic acid and calyculin A,
inability to use phosphorylase a as substrate, and a segment
of amino acid sequence typical of all PP2C type phosphatases known to
date. However, it differs from the other PP2C enzymes, particularly the
mammalian PP2C
and -
isoforms, in that its molecular weight,
76,000, is considerably larger and that it is inhibited by
Ca
, NaF, and polycations, but not by orthovanadate.
The Ca
inhibition may not be related to its cellular
regulation because of K
values in the
20-90 µM range, but this property permits
distinction of this enzyme from the other phosphatases. Although the
precise physiological role of this phosphatase is not yet known, its
ability to dephosphorylate a wide variety of phosphoproteins and its
broad distribution, as shown by a survey of mouse tissues for its
activity, suggest that it may serve an important cellular function.
Protein phosphorylation-dephosphorylation is a universal
mechanism by which numerous cellular events are regulated. Of the
enzymes catalyzing the reversible reactions, it has become apparent
that there may exist as many as 1,000 phosphatases which, like the
kinases, are just as elaborately and rigorously
controlled(1, 2, 3, 4, 5) .
The serine/threonine-specific phosphatases have been classified into
four main types according to their in vitro specificity for
selected substrates and sensitivity to activators and
inhibitors(6) . Sequence analyses revealed that they can be
sorted into two major gene families. The first one includes type 1
(PP1), ()type 2A (PP2A), and type 2B (PP2B) phosphatases,
which share 37-59% sequence identity (7) in their
catalytic domains and are inhibited by okadaic acid(8) . The
second family, the Mg
-dependent phosphatases, also
designated type 2C (PP2C), share little sequence similarity with the
first family and are insensitive to okadaic acid.
The
Mg-dependent phosphatases have been identified in
animals(9, 10, 11, 12, 13, 14, 15) ,
plants(30) , and yeast(31) , and several have been
purified to
homogeneity(10, 11, 12, 32, 35) .
cDNA sequences of PP2C
and -
from mammalian sources showed
>90%
identity(16, 17, 18, 19, 34) ,
whereas those from yeast were 35% identical (13, 20, 33) and showed 21-24% identity
with the mammalian ones. The mammalian PP2C
and -
are
monomeric cytosolic proteins with molecular weights in the
42,000-45,000 range, and yeast PP2Cs are 31,500-51,400
monomeric enzymes. PP2Cs of larger sizes, however, have been reported.
A rabbit myosin light chain phosphatase has a molecular weight of
70,000(32) , and a bovine pyruvate dehydrogenase phosphatase is
a dimer with a 50,000 catalytic subunit complexed to a 97,000
subunit(35) . The physiological roles of PP2Cs are
unclear(1, 2) , although they have been implicated in
the regulation of fatty acid and cholesterol biosynthesis (21) and heat shock response(13, 20) .
Recently, a M
= 200,000 PP2C-like
phosphatase from HeLa cells has been reported (15) which
dephosphorylates the C-terminal region of RNA polymerase II.
Conceivably, PP2Cs can exist in different molecular sizes and serve
diverse biological functions.
We report here the purification to
homogeneity and characterization of a novel
Mg-dependent, Ca
-inhibitable
protein phosphatase (MCPP) from bovine brain. Although the
Ca
inhibition (K
= 20-90 µM) may not be related to
its cellular control, this unique property permits the differentiation
of this PP2C-type phosphatase from other phosphatases during its
isolation. While MCPP possesses many characteristics common to other
PP2Cs, it is a monomer with a larger molecular weight of 76,000 and
responds dissimilarly to several substrates and inhibitors.
P-Syntide-2 and -Kemptide were prepared
by the same procedure described above except that, after
2 h of
incubation, the reaction mixtures were spotted on P-81 phosphocellulose
filters, washed with 75 mM phosphoric acid, packed into a
column, and then washed extensively with water and eluted with 5 N HCl. Incorporation of
P was measured according to
Roskoski(24) .
[P]Phosphorylase b kinase was prepared under conditions minimizing
autophosphorylation by incubating 6 µM phosphorylase b kinase with 500 microunits of cAMP-dependent protein kinase
catalytic subunit for 4 min at 30 °C in a 1-ml solution containing
1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl
, 0.2 mM [
-
P]ATP in 50 mM Hepes, pH
7.0. The phosphate incorporation (
and
) was 2.0 mol/mol of
protein (
).
[P[Phosphorylase a was prepared
according to the method described by King et al.(25) .
P-Labeled histone H3 and -MBP were phosphorylated by
protein kinase C according to the method of Masmoudi et
al.(26) .
P-Labeled MBP was phosphorylated
by mitogen-activated protein kinase by incubating 2 mg of MBP with 5
mM MgCl
, 0.2 mM ATP (100 µCi), 30
µg of mitogen-activated protein kinase in 50 mM Tris
buffer, pH 7.2, at 30 °C for 5 h. 0.3 mol of
P/mol of
MBP were incorporated.
P-Labeled
-casein was
phosphorylated by casein kinase 2 by incubating 339 µM
-casein with 350 µM ATP (250 µCi), 10 mM MgCl
, 0.1 M KCl, 10 µg of casein kinase
2, in 50 mM Hepes, pH 7.4, at 30 °C for 4 h. 0.5 mol of
P/mol of protein was incorporated.
P-Labeled Raytide, MBP, and casein were phosphorylated
by pp60
tyrosine kinase in the presence of 70 µM ATP (100 µCi), 11 mM MgCl
, 70 µg/ml
bovine serum albumin, 33 µM EDTA in 50 mM Hepes,
pH 7.5, at 30 °C for 14 h. Incorporation of
P was
0.1-0.2 mol/mol protein.
Figure 1:
Elution profiles
of MCPP. The columns were eluted as described under Experimental
Procedures. , absorbance at 280 nm; -
- -, NaCl gradient;
, assay of MCPP by the
±Ca
method;
, MCPP and other PP2Cs
detected by inhibiting PP1 and PP2A with 1 µM okadaic
acid. A, DEAE-cellulose column. Each fraction = 12.5
ml. B, Affi-Gel Blue column. MCPP came out in the wash. Each
fraction = 9 ml. C, high resolution Sephacryl S-300
column. Each fraction = 2.8 ml. D, Mono Q columns from
FPLC. Each fraction = 0.5 ml.
Figure 2: Gel electrophoresis patterns of purified MCPP (fraction 34) from the Mono Q column step. Left panel, 8-16% SDS-PAGE: left lane, molecular weight markers; right lane, MCPP. Right panel, 4-20% nondenaturing PAGE: left lane, MCPP; right lane, molecular weight markers.
Figure 3: Identification of MCPP activity band. Fraction 34 from the Mono Q step was run on 4-20% nondenaturing PAGE in six lanes. One lane was stained with Coomassie Blue to mark the protein band. The other five were cut into strips, and the protein from each section was eluted and assayed for MCPP activity.
Figure 4: Comparison of amino acid sequences of a 24-residue segment deduced from the cDNA of MCPP with segments from 12 other PP2Cs. Residues identical with those of MCPP are underlined. Conservative substitutions (relative to MCPP) are in capital letters. The number given at the end of each sequence marks the position of the last residue. All alignments are obtained from the BLAST Server.
We have purified to homogeneity a new
Mg-dependent protein phosphatase from bovine brain.
It is named Ca
-inhibitable protein phosphatase to
differentiate it from other mammalian Mg
-dependent
phosphatases which have been called PP2C
, PP2C
, or named
after their substrates such as phosphofructokinase
phosphatase(14) , myosin light chain phosphatase (32) ,
and pyruvate dehydrogenase phosphatase(35) . Although PP2C-type
phosphatases have been identified in various sources, only a handful
have been purified to homogeneity, and MCPP is the first 76-kDa enzyme
obtained in pure form.
The catalytic and structural characteristics
of MCPP indicate that it can be classified as a type 2C phosphatase. 1)
It requires Mg (or Mn
) for
activity. 2) It catalyzes the dephosphorylation of
phosphoseryl/threonyl residues of proteins and peptides phosphorylated
by cAMP-dependent protein kinase and protein kinase C (Table 2).
3) It is insensitive to inhibitors like okadaic acid and calyculin A,
heparin, and PP1 inhibitors 1 and 2. 4) It does not attack
phosphorylase a. 5) A segment of its amino acid sequence (24
residues) is homologous to a region found in 12 other PP2C-type enzymes (Fig. 4). In this segment, MCPP is 54.58% identical with and 96%
similar to 6 mammalian PP2C
and -
isoforms; 46-54%
identical with and 88-92% similar to 4 yeast, isolated or
putative, PP2Cs. It should be of interest to note that a CDLLW motif
similar to the CDGIW or CDGLW sequence of PP2Cs is found in practically
every amino acid sequence of PP1, PP2A, and PP2B(7) .
MCPP,
however, possesses properties that differ from PP2C and -
and
other PP2Cs. 1) Its molecular weight, 76,000, is considerably larger
than the M
= 42,000-51,000 mammalian
and yeast enzymes, but comparable to the M
= 70,000 myosin light chain phosphatase (32) . 2)
It is inhibited by polycations and F
ion which do not
inhibit PP2C
and -
, although a Mg
-dependent
phosphatase from turkey gizzard smooth muscle also is inhibited by
F
(10) . 3) PP2Cs are inhibited by millimolar
concentrations of orthovanadate, but MCPP is not. 4) PP2Cs
preferentially dephosphorylate
subunit of phosphorylase b kinase, whereas MCPP has very low activity with this substrate and
shows no preference for either
or
subunit. The M
= 70,000 myosin light chain phosphatase
also exhibited little activity with phosphorylase b kinase as
substrate(32) . 5) MCPP is inhibited by Ca
with K
in the 20-90 µM range,
depending on the substrate used. The effect of Ca
,
however, is on the enzyme since the inhibitory effect was observed with
every substrate tested so far. The shift in K
suggests that Ca
is a noncompetitive inhibitor
which affects both V
and K
for the substrates. Since the K
values are
much higher than the intracellular Ca
level of
0.1-1 µM, it appears that the Ca
inhibition may not be physiologically important. However,
regulation by Ca
may not be excluded because, like
the synergistic effect of glycogen on the Ca
inhibition of glycogen-synthetase phosphatase(37) , the
presence of another component may be involved in amplifying the
Ca
signal in vivo. In this regard, it is
interesting to note that bovine pyruvate dehydrogenase phosphatase is
activated by Ca
with K
in the
24-62 µM range in the absence of EGTA
buffer(36) .
The role of MCPP in the cell, like the other
PP2Cs, is not clear at this time. The rather wide distribution of MCPP,
as shown in the survey presented in Table 4, suggests that MCPP
or its isoforms constitute a new subclass of PP2C and likely serve
important cellular functions. It should be of interest to note that a
SET protein encoded by a set gene (38) copurified with
MCPP until the Mono Q chromatography step. In a case of acute
undifferentiated leukemia, the set gene was fused to a can gene as a result of chromosomal translocation(38) .
Phosphorylation of the SET protein by protein kinase C was blocked by
the presence of trace amounts of MCPP, indicating that SET is an
excellent substrate for the phosphatase. ()Since the set gene is expressed in all tissues of the mouse, particularly during
embryogenesis, SET may play a key role in the cell and MCPP may
regulate the function of SET. The fact that MCPP activity is highest
with MBP (10 times better than any other substrates tested so far) may
also imply a special function for this phosphatase in the brain.