©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of DARPP-32, a Dopamine- and cAMP-regulated Phosphoprotein, by Casein Kinase I in Vitro and in Vivo(*)

Frédéric Desdouits (1)(§), David Cohen (1), Angus C. Nairn (2), Paul Greengard (2), Jean-Antoine Girault (1)

From the (1) From INSERM U114, Chaire de Neuropharmacologie, Collège de France, 75005 Paris, France and (2) Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

DARPP-32 ( dopamine- and c AMP- regulated phospho protein, M= 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.


INTRODUCTION

The activity of the catalytic subunit of protein phosphatase-1 (PP-1c)() 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) .

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.


EXPERIMENTAL PROCEDURES

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 Ccolumn (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) .


RESULTS

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 Vof 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.

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).

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 Ccolumn. 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).




DISCUSSION

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 Kfor 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 Ais 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.

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.

  
Table: Sequencing of tryptic phosphopeptides from DARPP-32 phosphorylated by casein kinase I

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.


  
Table: Sequence surrounding residues phosphorylated by casein kinase I in rat DARPP-32 and comparison with other species

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.


  
Table: Comparison of sequences surrounding residues phosphorylated by casein kinase I in various protein substrates

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.



FOOTNOTES

*
This work was supported in part by grants from the National Alliance for the Mentally Ill (Stanley Award), from the National Parkinson Foundation (to J.-A. G.), and by National Institute of Mental Health Grant MH-40899 (to A. C. N. and P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
F. D. was a recipient of doctoral fellowships from Direction des Recherches, Etude et Techniques and Rhône-Poulenc Rorer. To whom correspondence should be addressed: INSERM U114, Collège de France, 11, Place Marcelin Berthelot, 75005 Paris, France. Tel.: 33-1-44-27-12-61; Fax: 33-1-44-27-12-60.

The abbreviations used are: PP-1c, catalytic subunit of protein phosphatase-1; DARPP-32, dopamine- and cAMP-regulated phosphoprotein, M= 32,000; TBAP, tetrabutyl ammonium phosphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; NTCB, 2-nitro-5-thiocyanobenzoic acid; dCTPS, 2`-deoxycytosine 5`- O-(1-thiotriphosphate).


ACKNOWLEDGEMENTS

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.


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