From the
DARPP-32 ( dopamine- and
c AMP- regulated phospho protein,
M
The activity of the catalytic subunit of protein phosphatase-1
(PP-1c)
In striatonigral neurons, DARPP-32 is
phosphorylated under basal conditions on serine by casein kinase II,
whereas inhibitor-1 is not a substrate for this kinase
(9, 10) . However, phosphorylation by casein kinase II
accounts only in part for the phosphorylation of DARPP-32 on serine in
the striatum. The other major site of in vivo phosphorylation
of rat DARPP-32 is located in a very acidic peptide on thermolytic maps
(10) . Since the rat DARPP-32 sequence contains a stretch of 18
acidic amino acids followed by a serine at position 137, which provides
a casein kinase I consensus site, we have suggested previously that
DARPP-32 might be a substrate for casein kinase I
(10) .
Casein kinase I is a highly conserved protein kinase, identified in
yeast, plants, and mammals
(11, 12, 13) . It is
a ubiquitous enzyme which can be found in the nucleus and the cytosol
and bound to the cytoskeleton or to the membrane
(13) . In
vitro, membrane-bound casein kinase I can be inhibited by
exogenous phosphatidylinositol 4,5-bisphosphate
(14) , but this
effect, which is the only form of regulation observed so far, may not
be physiological
(15) . Thus, very little is known about the
modulation of casein kinase I activity in vivo. Moreover, very
few substrates have been shown to be phosphorylated by casein kinase I
in intact cells, and substrates of this kinase remain to be identified
in neurons, where casein kinase I has been shown to be tightly bound to
neurofilaments
(16, 17) .
Here, we show that DARPP-32
is an excellent substrate for casein kinase I and that one of the two
serines phosphorylated in vitro is also phosphorylated in
intact cells. In addition, phosphorylation of this serine induces an
unusual shift in migration of the protein on SDS-PAGE, allowing the
easy assessment of casein kinase I activity in intact cells.
The effects of phosphorylation of DARPP-32 by casein kinase I on
PP-1c inhibition were investigated. Phosphorylation of DARPP-32 by
casein kinase I did not convert the protein into an inhibitor of PP-1c
and the inhibitory activity of phospho-Thr-34-DARPP-32 was not altered
by its phosphorylation by casein kinase I (not shown).
We have identified casein kinase I as a novel DARPP-32 kinase
in vitro and, most probably, in vivo. Compared to
DARPP-32, inhibitor-1 was a poor substrate for this kinase, and the
sites phosphorylated on DARPP-32 are not conserved on inhibitor-1. The
apparent K
Phosphorylation of Ser-137 induces a
more rapid migration of DARPP-32 in SDS-PAGE. This is a very unusual
feature, reported also for cdc2 phosphorylated by MO15
(42) and
connexin 45.6 phosphorylated by an unidentified kinase
(43) ,
since phosphorylation usually retards the migration of proteins in
SDS-PAGE by reducing the binding of SDS and thus the total charge of
the protein. Two mechanisms could account for the increased
electrophoretic mobility of DARPP-32 phosphorylated on Ser-137 in
SDS-PAGE: 1) a charge effect: by taking place in a region which is
likely not to bind SDS (the stretch of 18 acidic amino acids between
position 119 and 136) phosphorylation would increase the net charge of
the protein during electrophoresis and 2) a conformational change which
would persist in the presence of SDS. In the presence of urea the
migration of DARPP-32 was not changed by phosphorylation by casein
kinase I arguing against a charge effect. Moreover, the migration in
SDS-PAGE of the amino-terminal fragment 72-205 of DARPP-32, was
not affected by phosphorylation by casein kinase I, indicating that the
anomalous migration of DARPP-32 upon phosphorylation of Ser-137 was a
characteristic of the entire molecule. DARPP-32 has a very elongated
shape and time-resolved fluorescence studies have indicated that the
amino-terminal part is less flexible than the carboxyl-terminal moiety,
suggesting an intramolecular organization of the protein
(18) .
Hence, phosphorylation of Ser-137 could change the conformation of the
protein in a way which persists in the presence of SDS.
DARPP-32
from rat striatonigral neurons migrates as a doublet. Our results
provide strong evidence that the lower band corresponds to DARPP-32
phosphorylated on Ser-137 and that casein kinase I and II are the main
DARPP-32 kinases active under basal conditions in intact striatonigral
neurons. The stoichiometry of phosphorylation of Ser-137 in vivo appears higher in the substantia nigra than in the striatum. Since
very little is known about the regulation of casein kinase I in intact
cells, it will be important to determine whether this difference in
phosphorylation of Ser-137 is the consequence of a higher activity of
casein kinase I in nerve terminals than in neuronal cell bodies and
dendrites. Casein kinase I is considered usually to be involved in
basic functions in cells
(13) , including germination
(44) , regulation of cell morphology
(45) , and DNA
strand-break repair
(46) in yeast, and segregation of
chromosomes during mitosis in Chinese hamster ovary cells
(47) .
DARPP-32 appears to be an excellent neuronal substrate for casein
kinase I. Phosphorylation of DARPP-32 by casein kinase I did not alter
its ability to inhibit PP-1c which depends only on phosphorylation of
Thr-34. However, we have found recently that phosphorylation of Ser-137
by casein kinase I decreases dephosphorylation of Thr-34 by calcineurin
(48) . Thus, casein kinase I could play a indirect role in
reinforcing PP-1c inhibition by DARPP-32.
Phosphopeptides obtained
by tryptic cleavage of casein kinase I-phosphorylated DARPP-32 were
sequenced as described under ``Experimental Procedures.'' The
peptide numbers refer to the peaks in Fig. 5 A. The sequence of
the peptides, the corresponding position in the rat DARPP-32 sequence
of the phosphorylated residue, and the relative contribution (%) of the
peptide to the total eluted radioactivity are indicated. Sec,
phosphorylated serine modified to S-ethylcysteine; residues in
parentheses were not identified with certainty; X corresponds
to unidentifiable residue, and u.s. to unidentifiable sequence.
Ser-137 and Ser-189 in rat DARPP-32 sequence and
corresponding residues in bovine and human sequences are underlined.
Ser-137 is located after a stretch of 18 (rat and human) or 16 (bovine)
acidic amino acids, and is in a conserved region, whereas Ser-189 is
absent from bovine protein and is located in a more variable region.
Identical amino acids are indicated in boldface type. Sequences are
from Refs. 2, 49, and 50. - indicates gaps made for the alignment
of the sequence of bovine DARPP-32.
Phosphorylation sites have been grouped according to the
presence or the absence of consensus sequences described for casein
kinase I (28, 38). The first group of substrates (classical) contains
an acidic amino acid at position -3 (indicated in boldface type)
or a stretch of acidic amino acids located at the amino terminal side
of the phosphorylated serine. The second group is composed of two
examples of hierarchal phosphorylation. The serine at position
-3, which is indicated in boldface type, has to be phosphorylated
to permit the phosphorylation of the indicated serine. The third group
is composed of ``non-consensus'' sequences (atypical). These
serines are phosphorylated by casein kinase I, but are not surrounded
by conserved acidic amino acids. The serines phosphorylated by casein
kinase I are underlined. All sites indicated in this table, with the
exception of Ser-189 in DARPP-32, have been shown to be phosphorylated
in intact cells.
We thank Dr. S. Halpain for providing us the
immunoprecipitated DARPP-32 from prelabeled striatal slices used in
Fig. 6B, and Hsien-Bin Huang for assistance in
purification of PP-1c. Protein sequence analysis was provided by The
Rockefeller University Protein Sequencing Facility, which is supported
in part by NIH shared instrumentation grants and by funds provided by
the U.S. Army and Navy for purchase of equipment. We also thank Prof.
Jacques Glowinski for his constant support and encouragement.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 32,000) is a potent inhibitor of
protein phosphatase-1 when it is phosphorylated on Thr-34 by
cAMP-dependent protein kinase. DARPP-32 is highly enriched in some
specific cell populations such as striatonigral neurons and choroid
plexus epithelial cells. Here we show that recombinant rat DARPP-32 is
phosphorylated by casein kinase I on seryl residues to a stoichiometry
of
2 mol of phosphate/mol of protein. DARPP-32 is one of the best
known substrates for casein kinase I ( K
= 3.4 ± 0.3 µ
M), whereas the
homologous phosphatase-1 inhibitor, inhibitor-1, is not.
Phosphorylation of DARPP-32 by casein kinase I does not alter its
ability to inhibit protein phosphatase-1. Residues phosphorylated by
casein kinase I were identified as Ser-137 and Ser-189 by site-directed
mutagenesis and by protein sequencing. Ser-137 and the preceding
stretch of 16-18 acidic residues are conserved in DARPP-32 among
all species examined, whereas Ser-189 is not. Phosphorylation of
Ser-137 induces an unusual increase in DARPP-32 electrophoretic
mobility in polyacrylamide gels in the presence of SDS. In
striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the
stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than
in the striatum (soma and dendrites). These results indicate that
casein kinase I is highly active in striatonigral neurons in which it
may play important roles, including in protein phosphatase-1 modulation
via phosphorylation of DARPP-32.
(
)
is under the control of several
inhibitory proteins that are regulated by phosphorylation (reviewed in
Ref. 1). Among these, DARPP-32 ( dopamine- and
c AMP- regulated phospho protein,
M
= 32,000 on SDS-PAGE) and inhibitor-1
become potent inhibitors of PP-1c when they are phosphorylated on
threonine by cAMP-dependent protein kinase. The two proteins display a
high degree of sequence homology in their amino-terminal part, but
differ in their carboxyl terminus
(2) . DARPP-32 is highly
enriched in specific cell populations (reviewed in Ref. 3), whereas
inhibitor-1 has a more widespread distribution
(4) . During
development, DARPP-32 is expressed only at a differentiated stage of
these cell populations, whereas inhibitor-1 is maximally expressed in
the embryo and shortly after birth
(4, 5, 6) . A
striking feature of DARPP-32 expression is its correlation with that of
the dopamine D1 receptor
(7) , and it has been shown that its
state of phosphorylation is regulated by dopamine in striatonigral
neurons
(8) .
Materials
Recombinant rat DARPP-32 was produced
in Escherichia coli and purified as previously described
(18) . Inhibitor-1 from rabbit skeletal muscle
(19) ,
cAMP-dependent protein kinase from rabbit skeletal muscle or calf heart
(20) , and casein kinase I
(21) and casein kinase II
(10) from calf thymus were purified as described. PP-1c was
purified from rabbit skeletal muscle
(22) . Phosphorylase a was
prepared as previously described
(22) . Rats were purchased from
Charles River France, [P]ATP from DuPont
NEN, acrylamide from National Diagnostic, thin layer chromatography
cellulose sheets from Eastman Kodak Co., thermolysin and
dithiothreithol from Calbiochem, CKI-7 from Seikagaku America Inc.,
nitrocellulose membranes (0.2-µm pore size) from Schleicher &
Schuell, and autoradiographic films from Kodak and Amersham Corp.
Site-directed Mutagenesis
Site-directed
mutagenesis of the single-stranded form of an M13 phage containing the
sense full-length rat DARPP-32 cDNA sequence was carried out with a
commercially available kit (Amersham) based on the method of Eckstein
and colleagues
(23) . Briefly, a 33-nucleotide long antisense
oligonucleotide (GACGAAGAAGAAGACGCCCAGGCGGAGGTCCTG) was used to prime
cDNA synthesis. It matched the sequence of rat DARPP-32 cDNA with the
exception of two base replacements, underlined in the sequence,
corresponding to a single amino acid substitution from serine (AGC) to
alanine (GCC). The second strand of DNA was synthesized with the Klenow
fragment of DNA polymerase, in the presence of dCTPS, and ligated
with T4 DNA ligase. Double-stranded DNA was purified by filtration on
nitrocellulose and incubated with NciI, a restriction enzyme
that recognizes a unique site (CCCGG) in the heteroduplex, but cannot
cut the phosphorothioate-containing strand. The nicked heteroduplex was
digested by exonuclease III and repolymerized by polymerase I. TG1
bacteria were transformed with the resulting double-stranded DNA.
Successful mutation was confirmed by DNA sequencing.
Phosphorylation Reactions
The buffer used for
phosphorylation by cAMP-dependent protein kinase contained 50 m
M HEPES, pH 7.4, 10 m
M magnesium acetate, and 1 m
M EGTA. The buffers used for phosphorylation by casein kinases I and
II contained 50 m
M Tris-HCl, pH 7.1, 10 m
M magnesium
acetate, and 100 or 150 m
M KCl, respectively. All
phosphorylation reactions were carried out at 30 °C using an ATP
concentration of 100 µ
M, except for the stoichiometric
phosphorylation of DARPP-32, where it was 150 µ
M.
Phosphoproteins were analyzed by SDS-PAGE (13 or 15% acrylamide, w/v)
(10) . Samples were loaded on gels in ``stop
solution'' (3% SDS (w/v), 62 m
M Tris-HCl, pH 6.8, 5%
glycerol (v/v), 0.3
M -mercaptoethanol, and traces of
pyronine Y and bromphenol blue). Study of the time course of
phosphorylation of recombinant rat DARPP-32, bovine DARPP-32 and
inhibitor-1 were carried out in a final volume of 400 µl containing
40 µg of substrate and 10 ng of casein kinase I. Aliquots were
removed at various times, mixed with stop solution, boiled for 2 min,
and analyzed by SDS-PAGE. Experiments to determine the kinetic
constants of the phosphorylation of DARPP-32 by casein kinase I were as
described elsewhere
(10) . In initial rate conditions,
phosphorylation was linear with time and corresponded to incorporation
of phosphate into less than 10% of the substrate.
Phosphopeptide Sequencing
Three hundred µg of
DARPP-32 or S137A-DARPP-32 were phosphorylated with 150 µ
M [P]ATP (specific activity, 950
cpm/pmol) and 4 µg of casein kinase I in a final volume of 1 ml,
for 90 min, at 30 °C. The reaction was stopped by the addition of
500 µl of acetic acid. ATP was removed by anion exchange
chromatography on a Dowex AG1-X8 column (Bio-Rad) equilibrated in 30%
acetic acid (v/v)
(10) . The eluted proteins were lyophilized
and resuspended in 150 µl of 1
M ammonium carbonate. After
adding 50 µl of 8
M urea and 50 µl of 100 m
M dithiothreitol, samples were kept at 50 °C for 15 min, then
750 µl of water and 30 µg of trypsin were added. Reactions were
carried out at 37 °C for 36 h. The reaction mixtures were kept at
-20 °C until they were injected on reverse phase HPLC. HPLC
was performed on a Vydac C
column (25 cm
2.1 mm)
which had been equilibrated at a flow rate of 1 ml/min in 5 m
M tetrabutyl ammonium phosphate (TBAP), pH 5.4. Peptides were eluted
with linear gradients of increasing concentrations of acetonitrile in
the presence of 5 m
M TBAP: 0-35% acetonitrile
(5-10 min), 35-70% acetonitrile (10-50 min).
Radioactivity of the eluate was monitored on line. Fractions were
collected, every 30 s, in polypropylene microcentrifuge tubes, and
dried before Cerenkov radiation was measured. Peak fractions, with the
exception of peak 10, were resuspended in 400 µl of water and
subjected to a second purification on the same column equilibrated at a
flow rate of 1 ml/min in 0.1% trifluoroacetic acid. The peptides were
eluted with increasing concentrations of acetonitrile in the presence
of 0.075% trifluoroacetic acid: 0-14% acetonitrile (1-3
min), 14-49% acetonitrile (3-20 min), 49-70%
acetonitrile (20-25 min). The large peak 10 was resuspended in
400 µl of 25 m
M ammonium carbonate. Two hundred µl of
this sample were incubated with 10 µg of thermolysin, overnight, at
37 °C. Phosphopeptides were then purified by reversed phase HPLC in
TBAP buffer as described above. Before sequencing of the peptides,
phosphoserine was chemically converted to S-ethylcysteine
(24) . Dried samples were resuspended, under nitrogen, in 50
µl of a mixture containing 60 µl of 10
M ethanethiol,
200 µl of water, 200 µl of dimethylsulfoxide, 80 µl of
ethanol, and 65 µl of 5
M NaOH. After incubation for 1 h
at 50 °C, samples were cooled to room temperature and 10 µl of
acetic acid were added.
Studies with Rat Brain
For studying DARPP-32
mobility shift, rapidly dissected substantia nigra was incubated at 30
°C, in artificial cerebrospinal fluid
(25) , for 0, 10, 20,
or 30 min and frozen in liquid nitrogen. For estimation of the
stoichiometry of phosphorylation by casein kinase I in vivo,
striata and substantia nigra of four rats were rapidly dissected and
directly frozen in liquid nitrogen. Tissues were homogenized in boiling
1% SDS in water (w/v) by sonication and subjected to SDS-PAGE. DARPP-32
in striatum and substantia nigra was analyzed by immunoblotting with a
mixture of two monoclonal antibodies (C24-5a and C24-6a), as previously
described
(26) , except that a horseradish peroxidase-coupled
donkey-anti-mouse IgG secondary antibody was used and that
immunoreactivity was detected with an enhanced chemiluminescence method
(Amersham). The relative amounts of immunoreactive bands were
quantified by computer-assisted densitometry measurement of the films.
To analyze the phosphorylation of DARPP-32 in striatonigral neurons,
prelabeling of striatal slices and immunoprecipitation of DARPP-32 were
carried out as described elsewhere
(10) .
Miscellaneous Methods
Two-dimensional thin layer
phosphopeptide mapping and phosphoamino acid analysis of DARPP-32 and
S137A-DARPP-32 were carried out as previously described
(27) .
2-Nitro-5-thiocyanobenzoic acid (NTCB) cleavage of phosphoproteins was
carried out as described previously
(10) , except that NTCB was
dissolved by sonication. PP-1c inhibition was studied as described,
using phosphorylase a as substrate
(10) .
Casein Kinase I Phosphorylates
DARPP-32
Recombinant DARPP-32 was phosphorylated efficiently by
casein kinase I, whereas the rate of phosphorylation of inhibitor-1 was
8-fold lower (Fig. 1 A). Recombinant rat DARPP-32 was
phosphorylated by casein kinase I with an apparent
K= 3.4 ± 0.3 µ
M (mean ± S.D., n = 3)
(Fig. 1 B). The V
of
phosphorylation ranged from 30 to 100 pmol/min/µg depending on
enzyme batches, values which were approximately 10 times lower than
that observed for casein. The stoichiometry of phosphorylation of
recombinant DARPP-32 was approximately 2 mol of phosphate per mol of
protein (Fig. 2 A), suggesting the existence of at least
two sites of phosphorylation. When phosphorylated by casein kinase I,
DARPP-32 migrated more rapidly on SDS-PAGE (Fig. 2 B).
DARPP-32, phosphorylated to a low stoichiometry ( e.g.
0.2
mol of phosphate/mol of DARPP-32, Fig. 3 A), appeared as
a doublet of phosphoproteins on autoradiograms, suggesting that
phosphorylation of one site, but not the other, could modify the
migration of the protein. This shift in migration on SDS-PAGE was not
observed in the presence of 4.6
M urea (not shown) or when
DARPP-32 was phosphorylated by cAMP-dependent protein kinase or casein
kinase II (Fig. 3 A).
Figure 1:
Phosphorylation of recombinant rat
DARPP-32 () and rabbit inhibitor-1 (+) by casein kinase I.
A, proteins were incubated with casein kinase I and
[
P]ATP in initial rate conditions for
various amounts of time, as indicated under ``Experimental
Procedures.'' B, Lineweaver-Burk plot of the
incorporation of phosphate into DARPP-32 incubated at various
concentrations in the presence of casein kinase I. Phosphorylated
proteins were separated from radioactive ATP by SDS-PAGE and analyzed
by autoradiography. For quantification, bands corresponding to the
phosphorylated proteins were excised and their Cerenkov radiation
measured.
Figure 2:
Phosphorylation of recombinant DARPP-32 by
casein kinase I shifts its electrophoretic mobility. A,
stoichiometry of phosphorylation of DARPP-32 incubated with casein
kinase I, in moles of phosphate incorporated per mol of DARPP-32. One
mg of recombinant DARPP-32 was incubated with 10 µg of casein
kinase I and 150 µ
M [P]ATP, at
30 °C for 4 h. After 2 h of incubation, 5 µg of kinase were
added to the reaction mixture ( arrow). At the indicated times,
aliquots of the reaction mixture were removed and the incorporated
radioactivity measured as described in the legend to Fig. 1.
B, Coomassie Blue staining of the aliquots was analyzed by
SDS-PAGE (15% (w/v) acrylamide, and 2.5 µg of DARPP-32 were loaded
on each lane).
Figure 3:
Phosphorylation of DARPP-32 and
S137A-DARPP-32. A, DARPP-32 was phosphorylated, in initial
rate conditions, by cAMP-dependent protein kinase ( lane 1),
casein kinase II ( lane 2), or casein kinase I ( lane
3) using [P]ATP. B, DARPP-32
( lanes 1 and 2) and S137A-DARPP-32 ( lanes 3 and 4) were incubated in the absence ( lanes 1 and 3) or the presence ( lanes 2 and 4)
of casein kinase I in conditions leading to a stoichiometry of
phosphorylation of
2 mol of phosphate/mol of DARPP-32 (see Fig. 2).
C, DARPP-32 phosphorylated by casein kinase I ( lane
1), cAMP-dependent protein kinase ( lane 3), or casein
kinase II ( lane 4) and S137A-DARPP-32 phosphorylated by casein
kinase I ( lane 2) were subjected to cysteine-specific cleavage
by NTCB, as described under ``Experimental Procedures.'' The
position of amino-terminal ( 1-71) and carboxyl-terminal
( 72-205) fragments of the proteins are indicated.
cAMP-dependent protein kinase phosphorylates DARPP-32 on Thr-34
(1-71 fragment), and casein kinase II phosphorylates DARPP-32 on
Ser-45 and Ser-102 (1-71 and 72-205 fragments,
respectively). D, phosphoamino acid analysis of the lower
( lane 1) and upper ( lane 2) bands of the
phospho-doublet of DARPP-32 and of S137A-DARPP-32 ( lane 3)
partially phosphorylated by casein kinase I under initial rate
conditions, as in A. The positions of unlabeled phosphoamino
acid standards, revealed by ninhydrin staining, and origin are
indicated. P-Ser, O-phospho-
DL-serine;
P-Thr, O-phospho-
DL-threonine;
P-Tyr, O-phospho-
DL-tyrosine.
Phosphoproteins ( A, B, and C) were separated
by SDS-PAGE (13% ( A) or 15% ( B and C)
acrylamide, w/v) and analyzed by Coomassie Blue staining or
autoradiography as indicated. Phosphoamino acids were separated by high
voltage thin layer electrophoresis and detected by
autoradiography.
In several cases, casein kinase I
phosphorylates serines located on the carboxyl-terminal side of a
phosphoamino acid, usually phospho-Ser at position -3, a process
called hierarchal phosphorylation
(28, 29) .
Phosphorylation of DARPP-32 by casein kinase I was not enhanced by
prior phosphorylation by cAMP-dependent protein kinase or casein kinase
II (not shown). Furthermore, recombinant DARPP-32 purified from E.
coli, in an unphosphorylated form (not shown), was nevertheless an
excellent substrate for casein kinase I. Thus it appears that casein
kinase I is not dependent on hierarchal phosphorylation of DARPP-32.
Localization of Casein Kinase I Phosphorylation Sites on
DARPP-32 by Site-directed Mutagenesis and Peptide Mapping, and Effects
on Electrophoretic Mobility
In DARPP-32, the best consensus
sequence for casein kinase I precedes Ser-137. S137A-DARPP-32 (in which
Ser-137 was mutated to alanine) incubated with casein kinase I
incorporated less phosphate than DARPP-32, and its migration on
SDS-PAGE was not altered by phosphorylation (Fig. 3 B).
When DARPP-32 was phosphorylated by casein kinase I to a low
stoichiometry, it migrated as a doublet (Fig. 3 A), and
the thermolytic maps of the lower band contained a major acidic peptide
absent from the maps obtained from the upper band (Fig. 4, A and B). This acidic peptide was also absent from the maps
of S137A-DARPP-32 phosphorylated by casein kinase I, demonstrating that
it contained phospho-Ser-137 (Fig. 4 C). On the other
hand, two basic peptides corresponding to additional phosphorylation
site(s), were the major phosphopeptides in thermolytic maps of the
upper band and S137A-DARPP-32 and minor ones in maps of the lower band.
Taken together, these results demonstrate that phosphorylation of
Ser-137 is responsible for the more rapid migration of DARPP-32 on
SDS-PAGE.
Figure 4:
Phosphopeptide mapping of DARPP-32 and
S137A-DARPP-32 phosphorylated by casein kinase I. DARPP-32 and
S137A-DARPP-32 were phosphorylated, in initial rate conditions (as in
Fig. 3 A), by casein kinase I. Gel pieces corresponding to the
upper ( A) and lower ( B) bands of DARPP-32 and to
S137A-DARPP-32 ( C) were excised and incubated in the presence
of thermolysin (10). Phosphopeptides were separated by thin layer
electrophoresis and chromatography and detected by autoradiography. The
specific phosphopeptide of the lower band of DARPP-32, corresponding to
phospho-Ser-137 (see text), is indicated by an arrowhead. The
positions of the anode (+) and the cathode (-) are
indicated.
Since phosphorylation of S137A-DARPP-32 by casein kinase I
was still significant, attempts to localize the other phosphorylated
residue(s) were made. Phospho-DARPP-32 and phospho-S137A-DARPP-32 were
chemically cleaved at the unique Cys-72 with NTCB into two peptides
corresponding to residues 1-71 and 72-205, respectively.
DARPP-32 and S137A-DARPP-32 were phosphorylated by casein kinase I
almost exclusively on the 72-205 fragment
(Fig. 3 C). Interestingly, the 72-205 fragment from
DARPP-32 phosphorylated either by casein kinase I or by casein kinase
II had the same electrophoretic mobility (Fig. 3 C).
Phosphoamino acid analysis of DARPP-32 and S137A-DARPP-32
phosphorylated by casein kinase I revealed that the proteins were
phosphorylated only on serine (Fig. 3 D).
Identification of Casein Kinase I Phosphorylation Sites
on DARPP-32 by Phosphopeptide Sequencing
To demonstrate directly
that Ser-137 was phosphorylated and to identify the other
phosphorylated serine(s), we sequenced tryptic phosphopeptides obtained
from DARPP-32 phosphorylated by casein kinase I. Tryptic peptides were
resolved on reverse phase HPLC into about 10 phosphopeptides (Fig.
5 A). Seven of these phosphopeptides were sequenced
successfully (). The two major peaks (peptides 5 and 10)
corresponded to phospho-Ser-189 and phospho-Ser-137, respectively.
Ser-137 is located at the carboxyl-terminal end of a stretch of 18
acidic amino acids which is conserved among species ().
Surprisingly, the peptide containing phospho-Ser-137, which is very
acidic and hydrophilic, was strongly retained on the Ccolumn and eluted as a broad asymmetrical peak
(Fig. 5 A). Moreover, the use of a mobile phase
containing TBAP, at a pH
5.4, was required to purify this peptide
since it was not eluted by 70% acetonitrile in standard acidic buffer
containing 1% trifluoroacetic acid (not shown). The peptide containing
phospho-Ser-189 was the major phosphopeptide from the tryptic digest of
S137A-DARPP-32 (Fig. 5 B). Ser-189 is not conserved among
species () and is not located in a typical casein kinase I
consensus site (I). However, its phosphorylation was
inhibited by CKI-7, a specific inhibitor of casein kinase I
(30, 31) , with the same efficiency as that of Ser-137
(not shown), indicating that phosphorylation of this residue is
attributable to bona fide casein kinase I and not to another
protein kinase contaminating the enzyme preparation. A minor
contribution of phosphorylation of Ser-18 was also detected
(, peptides 2 and 3), and a few minor peaks did not yield
identifiable sequence (peptides 7, 8, and 9). However, the very low
phosphorylation of the 1-71 NTCB fragment
(Fig. 3 C) indicates that Ser-18 is, at best, a poor
substrate for casein kinase I.
Figure 5:
HPLC profile of tryptic phosphopeptides
from DARPP-32 and S137A-DARPP-32 phosphorylated by casein kinase I.
DARPP-32 ( A) and S137A-DARPP-32 ( B) were incubated in
the presence of [P]ATP and casein kinase I
and phosphorylated to a stoichiometry of
2 and
1 mol of
phosphate/mol of DARPP-32, respectively, as described under
``Experimental Procedures.'' Phosphoproteins were subjected
to tryptic digestion, and peptides corresponding to about 400,000 cpm
were resolved by reverse phase HPLC on a C
column. The
total recovery of the injected radioactivity was
95% in both cases.
Phosphopeptides were detected by direct measurement of the
radioactivity with an on line detector ( solid line).
Acetonitrile concentration gradient is indicated ( dashed
line). Peak numbers in A refer to Table
I.
Phosphorylation of Ser-137 in Striatal Slices and in Rat
Striatonigral Neurons in Vivo
DARPP-32 immunoprecipitated from
P-labeled striatal slices migrates as a doublet whose
lower band was shown to contain a specific acidic peptide absent from
the upper band
(10) . This acidic peptide (Fig. 6 A)
corresponded to the peptide containing phospho-Ser-137 (Fig. 6,
B and C) and was not phosphorylated by casein kinase
II (Fig. 6 D). The predicted sequence of this thermolitic
peptide contains only one phosphorylatable serine, indicating that
Ser-137 is phosphorylated in intact cells. Thus, for endogenous
DARPP-32 as for purified recombinant protein, phosphorylation of
Ser-137 results in a fast migration of DARPP-32 in SDS-PAGE and
accounts for the existence of a doublet. Since DARPP-32 is also
phosphorylated in intact cells by casein kinase II, we compared
thermolytic maps obtained from DARPP-32 immunoprecipitated from
P-prelabeled striatal slices (Fig. 6 A) and
from purified DARPP-32 phosphorylated in vitro by casein
kinase II and casein kinase I (Fig. 6 B). Despite some
differences in the relative intensities of the phosphopeptides labeled
in slices and in vitro, casein kinases I and II can account
for most of the phosphorylation of DARPP-32 in intact neurons.
Figure 6:
Phosphopeptide mapping of striatal
DARPP-32, recombinant DARPP-32, and S137A-DARPP-32. A,
two-dimensional thermolytic phosphopeptide map of DARPP-32
immunoprecipitated from P-prelabeled striatal slices.
B, thermolytic phosphopeptide map of DARPP-32 phosphorylated
by casein kinase I and casein kinase II in vitro using
[
P]ATP. C, as in B, but
with S137A-DARPP-32. D, as in B, but with casein
kinase II alone. Phosphopeptides, obtained and separated as indicated
in the legend to Fig. 4, were detected by autoradiography. Position of
the phosphopeptide containing Ser-137 is indicated by an
arrowhead; note that this peptide is absent in C and
D. The positions of the anode (+) and the cathode
(-) are indicated.
Since
accelerated electrophoretic mobility of DARPP-32 is due to its
phosphorylation on Ser-137, we used this property to estimate the
phosphorylation of this seryl residue in rats in vivo.
DARPP-32 is present in the cell bodies and dendrites of striatonigral
neurons, located in the striatum, and in the axon terminals,
innervating the substantia nigra
(32) . DARPP-32 from both areas
migrated as a doublet on SDS-PAGE (Fig. 7 A). The upper
and lower bands of the doublet comigrated with unphosphorylated
DARPP-32 and DARPP-32 phosphorylated by casein kinase I, respectively
(Fig. 7 B), confirming that, in intact cells as in
vitro, the lower band corresponds to DARPP-32 phosphorylated on
Ser-137. When tissue was incubated at 30 °C before homogenization,
the lower band disappeared, presumably due to dephosphorylation of
Ser-137 by endogenous phosphatases, and could be accounted for by an
increase in the amount of the upper band (Fig. 7 B).
Interestingly, the proportion of DARPP-32 contained in the upper and
the lower bands differed in the striatum and in the substantia nigra,
the lower band being more abundant in the latter
(Fig. 7 A). On immunoblots, the lower band represented 17
± 1% of the total DARPP-32 signal in the striatum and 33
± 2% in the substantia nigra (mean ± S.E., n = 4, Student's t test, t =
12.3, p = 0.0012). On the basis of the evidence
presented above, this indicates that the stoichiometry of
phosphorylation of Ser-137, in striatonigral neurons, is about 0.17 in
cell bodies and dendrites and about 0.33 in nerve terminals.
Figure 7:
Phosphorylation of DARPP-32 on Ser-137 in
striatonigral neurons, in vivo, as indicated by its mobility
shift. A, immunobloting detection of DARPP-32 in homogenized
rat striatum or substantia nigra. B, immunobloting detection
of DARPP-32 from substantia nigra ( lanes 1-4) incubated
for various amounts of time prior to homogenization (see
``Experimental Procedures''). Recombinant DARPP-32
non-phosphorylated ( lane 5), phosphorylated by casein kinase I
( lane 6) or phosphorylated by casein kinase I and then treated
with calf intestine alkaline phosphatase ( lane 7) were loaded
on the same gel. One-tenth of a substantia nigra ( lanes
1-4) or 250 ng of recombinant DARPP-32 ( lanes
5-7) were loaded on each lane. After transfer to
nitrocellulose, immunoblotting with monoclonal antibodies specific for
DARPP-32 was carried out as described under ``Experimental
Procedures.'' The quantification by densitometry of upper
band/lower band for lanes 1-4 is: 0 min, 55/45; 10 min,
90/18; 20 min, 87/7; 30 min, 93/4 (arbitrary
units).
for the phosphorylation of
DARPP-32 by casein kinase I was similar to that observed for another
PP-1c inhibitor, inhibitor-2, which is considered to be one of the best
substrates for this kinase in vitro (33, 34) .
Five proteins, implicated in very different cell functions, are known
to be phosphorylated in vivo on sites which are phosphorylated
by casein kinase I in vitro: the anti-oncogene p53
(35) , the oncogene SV40 large T antigen
(36) , glycogen
synthase
(28) , the lectin L-29
(37) , and inhibitor-2
(33) . Casein kinase I is considered to be an acidic
residue-directed kinase (I), the proposed consensus
sequence being A
- X-X-S where A
is
either an acidic amino acid (glutamate or aspartate) or a
phosphorylated residue
(28, 38) . Rat DARPP-32 sequence
contains three casein kinase I consensus sites, Ser-51, Ser-52, and
Ser-137. We show here that Ser-137 and Ser-189 are the main
phosphorylation sites for casein kinase I in rat DARPP-32, in
vitro. Ser-18 is also phosphorylated to a lesser extent, whereas
Ser-51 and Ser-52 are not phosphorylated to a detectable level.
Although Ser-189 is not located in a typical consensus sequence for
casein kinase I, phosphorylation of ``non-consensus'' sites
with highly purified casein kinase I has already been reported
(I). However, we have no evidence for the phosphorylation
of Ser-189 in intact cells, and this residue is not conserved among
species (). Studies on the kinetics of phosphorylation of
synthetic peptides by casein kinase I have stressed a role for the
amino-terminal side of the site to be phosphorylated. On the other
hand, the velocities of phosphorylation of peptides which differed in
their carboxyl-terminal domains, were shown to be very different
(39, 40) . Moreover, peptides which encompassed Ser-174
in inhibitor-2 were very poor substrates for the kinase as compared to
the whole protein
(33, 39) , and studies on SV40 large T
antigen have revealed that modification of Ser-677 greatly reduced the
phosphorylation of Ser-120 by casein kinase I
(41) . These
results and the present findings strongly support the importance of
residues other than the acidic residue at position -3 and,
possibly, of conformational factors in directing phosphorylation of
proteins by casein kinase I.
Table:
Sequencing of tryptic phosphopeptides from
DARPP-32 phosphorylated by casein kinase I
Table:
Sequence surrounding residues
phosphorylated by casein kinase I in rat DARPP-32 and comparison with
other species
Table:
Comparison of sequences surrounding
residues phosphorylated by casein kinase I in various protein
substrates
= 32,000;
TBAP, tetrabutyl ammonium phosphate; PAGE, polyacrylamide gel
electrophoresis; HPLC, high performance liquid chromatography; NTCB,
2-nitro-5-thiocyanobenzoic acid; dCTP
S, 2`-deoxycytosine
5`- O-(1-thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.