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Phosphorylation of DARPP-32 and Protein Phosphatase Inhibitor-1 in Rat Choroid Plexus: Regulation by Factors Other Than Dopamine

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Phosphorylation of DARPP-32 and Protein Phosphatase Inhibitor-1 in Rat Choroid Plexus: Regulation by Factors Other Than Dopamine The Journal of Neuroscience, August 1992, V(8): 30713083 Phosphorylation of DARPP-32 and Protein Phosphatase Inhibitor-1 in Rat Choroid Plexus: Regulation by Factors Other Than Dopamine Gretchen L. Snyder,’ Jean-Antoine Girault, Iva Jonathan Y. C. Chen,2 Andrew J. Czernik,’ John W. Kebabian,2 James A. Nathanson, and Paul Greengardl ‘Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, 2Neuroscience Research, Abbott Laboratories, Abbott Park, Illinois 60064, and 3Department of Neurology and Program in Neuroscience, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 The molecular mechanisms underlying regulation of fluid main unclear. There is some evidence that CSF production is production by secretory epithelia such as the choroid plexus under neural and hormonal control and that some of the neu- are poorly understood. Two CAMP-regulated inhibitors of rotransmitters and hormones involved act through the regula- protein phosphatase-1, inhibitor-l (I,) and a dopamine- and tion of CAMP. For example, drugs that directly increaseCAMP CAMP-regulated phosphoprotein, M, = 32,000 (DARPP-32), levels by activating adenylyl cyclase (i.e., cholera toxin or for- are enriched in the choroid plexus. We show here that these skolin) have been reported to alter the rate of CSF production two phosphoproteins are colocalized in choroid plexus ep- in choroid plexus (Epstein et al., 1977; Feldman et al., 1979) ithelial cells. We have developed a novel method for study- and the rate of aqueoushumor production from the physiolog- ing the phosphorylation state of DARPP-32 and I, in intact ically analogousciliary process(Gregory et al., 1981; Caprioli cells, using a phosphorylation state-specific monoclonal an- et al., 1984). Several neurotransmitters and hormones,including tibody. Several drugs and hormones that are known to alter norepinephrine and vasoactive intestinal peptide (VIP), also fluid secretion and that increase CAMP levels (forskolin, iso- stimulate CAMP formation in choroid plexus and have been proterenol, vasoactive intestinal peptide) or cGMP levels hypothesized to influence CSF production by this mechanism (atrial natriuretic peptide) or that may use additional second (Nathanson, 1979, 1980; Crook et al., 1984; Lindvall et al., messenger pathways (5-HT), increase the phosphorylation 1985; Crook and Prusiner, 1986). However, at present, the of I, and DARPP-32 in rat choroid plexus. In contrast, do- mechanismsby which CAMP might alter CSF formation are pamine does not alter CAMP and cGMP levels, or I, and unknown. DARPP-32 phosphorylation. Our results indicate that DARPP- Neurotransmitters and hormones acting through CAMP are 32, known to be regulated by dopamine in a number of known to modulate cellular processesby the reversible phos- tissues, can be phosphorylated in response to non-dopa- phorylation ofproteins (Nestlerand Greengard, 1984). One class minergic factors, including hormones acting through non- of regulated phosphoproteins that has been postulated to play CAMP-dependent pathways. Our results also raise the pos- a role in the mediation of CAMP-associated hormone action is sibility that inhibition of phosphatase-1, as a result of I, and the protein phosphataseinhibitors, DARPP-32 (i.e., dopamine- DARPP-32 phosphorylation, might be part of a final common and CAMP-regulated phosphoprotein, A4, = 32,000) (Walaas et pathway in the action of several factors that are known or al., 1983a,b; Hemmings et al., 1984b; Walaas and Greengard, thought to alter cerebrospinal fluid production. 1984) and inhibitor-l (I,) (Ingebritsen and Cohen, 1983; Inge- britsen et al., 1983).Both proteins are phosphorylated by CAMP- Cerebrospinal fluid (CSF) is formed in and secreted from epi- dependent protein kinase on a single threonyl residue and, in thelial cells of choroid plexus (for review, see Cserr, 1971). phosphorylated form, are potent inhibitors of protein phospha- Although the physiological events underlying CSF production tase-I in vitro (Hemmings et al., 1984a).DARPP-32 is abundant are beginning to be understood (seeJohanson, 1989, for recent in brain neurons possessingthe dopamine D,-receptor (e.g., me- review), the mechanismsthat regulate this essentialprocess re- dium-sized striatonigral neurons) (Ouimet et al., 1984; Hem- mingsand Greengard, 1986) and is phosphorylated in thesecells in responseto dopamine acting through CAMP-dependent pro- Received Oct. 28, 1991; revised Mar. 12, 1992; accepted Mar. 16, 1992. tein kinase (Walaas et al., 1983a,b). DARPP-32 is also found We thank Drs. Angus Naim, Hugh Hemmings, Jr., and Atsuko Horiuchi for in high concentration in the choroid plexus (Hemmings and providing CAMP-dependent protein kinase catalytic subunit and antibodies against DARPP-32 and inhibitor- I, and Drs. Angus Naim and Robert MacKenzie for Greengard, 1986; Steardo and Nathanson, 1987). However, pri- helpful discussions of the experiments. The excellent technical assistance of Edwin or reports suggestthat dopamine is a poor activator of adenylyl Gomez and Michael Steffev in measurements of CAMP levels and Edward Hun- nicutt and Mary Rose De& in various aspects of the study is gratefully acknowl- cyclase in this tissue and there is no evidence for a distinct edged. We also thank Kathleen Sweadner for the K2 antiserum to Na,K-ATPase. dopamine-sensitive adenylyl cyclase (Nathanson, 1980). Be- G.L.S. was a postdoctoral fellow at Abbott Laboratories during a portion of this causeDARPP-32 phosphorylation has previously been shown investigation. This research was supported by Grant MH40899 to P.G. and U.S. PHS Grant EY05077 to J.A.N. to be regulated only by dopamine, this finding raisesthe question Corresoondence should be addressed to Gretchen L. Snyder. The Rockefeller of whether DARPP-32 is uniquely a dopamine-regulated pro- Universiiy, Department of Molecular and Cellular Neuroscience, 1230 York Av- tein and whether DARPP-32 and homologousphosphoproteins enue, Box 296, New York, NY 10021. = Present address: INSERM U114, College de France, Paris Cedex 05. might be involved in the action of other hormones and neu- Copyright 0 1992 Society for Neuroscience 0270-6474/92/l 2307 l-l 3$05.00/O rotransmitters. 3072 Snyder et al. * Regulation of DARPP-32 and Inhibitor-l Phosphorylation CAMP-KINASE Preparation of phosphopeptideantigens and phosphoproteins DARPP-32 The synthetic peptide chosen for immunization was a 1 O-residue peptide encompassing the cyclic AMP-dependent phosphorylation site<Thr,,) 20 40 of bovine DARPP-32 corresnondina to residues 28-37 (Hemmines et LDPRQVEMll RRRRPTPAM]LFR al., 1984~; Williams et al., 1986). The sequence of the peptide was NH,- Ile-Arg-Arg-Arg-Arg-Pro-Thr-Pro-Ala-Met-CONH1. This sequence is LDPRQVEMIRRRRPTPATLVL identical to that surrounding the cyclic AMP-dependent phosphoryla- 21 41 tion site in rat and rabbit I, with the exception of the methionine residue in DARPP-32, which is a threonine in I, (Aitken et al., 1982; Elbrecht INHIBITOR-1 et al., 1990; see Fig. 1). Note also that the Met,, in bovine DARPP-32 is replaced by a leucine in the rat sequence (Ehrlich et al., 1990). The Figure 1. Amino acid sequences surrounding the CAMP-dependent peptide was synthesized by Dr. J. Elliot at the Yale University Protein phosphorvlation sites of bovine DARPP-32 Cutmer\ 11 seauence) and rabbit and Nucleic Acid Chemistry Facility, by the solid-phase method (Hodg- or rai I, (her sequence). CAMP-dependent protein kinase phosphorv- es and Merrifield, 1975) using a model 430A Applied Biosystems Pep- lates a threonyl residue in a homologous region of both proteins; Thr3, tide Synthesizer, and was purified by preparative reverse-phase HPLC in DARPP-32 (Hemmines et al.. 1984~) and Thr,. in I. (Cohen et al.. using two Vydac C,, columns (25 x 250 mm) in series. Purity was 1977). The sequence of the synthetic peptide thatwas phosphorylated estimated to be >95% by analytical HPLC and amino acid analysis, and used for immunization is indicated by a box. One letter amino acid and was verified by mass spectrometry (data not shown). codes are used: A, alanine; D, aspartate; E, glutamate; F, phenylalanine; The synthetic peptide was phosphorylated in vitro by the catalytic I, isoleucine; L. leucine; M, methionine; P, proline; Q, glutamine; R, subunit of cyclic AMP-dependent protein kinase. A typical reaction arginine; T, threonine; V, valine. The sequences are from bovine caudate mixture contained 2 mM peptide, 5 mM y’*P-ATP (specific activity, 0.3 DARPP-32 (Williams et al., 1986) and rabbit or rat skeletal muscle I, Ci/mol), 10 &ml catalytic subunit, 50 mM HEPES, pH 7.4, 10 rnM (Aitken et al., 1982; Elbrecht et al., 1990). magnesium acetate, and 1 mM EGTA in a volume of 6 ml. Incubation was carried out for 18 hr at 30°C and stopped by addition of 3 ml of glacial acetic acid. Dowex AG I-XB (2 ml) was added, and the mixture The present study investigates the possibility that other neu- was stirred at 4°C for 1 hr and poured onto a small column. The flow- through and a 4 ml wash were pooled, lyophylized, and redissolved in rotransmitters and hormones known to regulate adenylyl cyclase H,O. The phosphorylated form of the peptide was separated from the activity in choroid plexus might regulate the phosphorylation remaining unphosphorylated peptide (< 5%) by HPLC on a preparative state of DARPP-32 and I, in intact tissue. In addition, we ex- Vydac C,, reverse-phase column. Elution was performed with an in- amine the possibility that other choroid plexus-enriched hor- creasing linear gradient of solvent B (70% CH,CN, 0.085% trifluoroac- etic acid in H,O) in solvent A (0.1% trifluoroacetic acid in H,O) as mone receptors known to activate CAMP-independent pathways follows: O-3 min, 0% B; 3-8 min, O-l 8% B; 8-10 min, 18-22% B. The may also participate in this regulation. To study the state of purified phosphopeptide was coupled to Limuh hemocyanin (Sigma), phosphotylation of DARPP-32 and I,, we have used a phos- which was shown to be devoid of contaminating phosphatase activity.
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