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Ca2+/Calmodulin-independent Activation of Calcineurin from Dictyostelium by Unsaturated Long Chain Fatty Acids

1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês

10.1074/jbc.274.53.37821

1083-351X

Ursula Kessen, Ralph SCHALOSKE, Annette Aichem, Rupert Mutzel,

Force Microscopy Techniques and Applications

This study describes a novel mode of activation for the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin fromDictyostelium discoideum we found a reversible, Ca2+/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca2+/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 μm with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca2+/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca2+/calmodulin action by binding to the calmodulin-binding site. This study describes a novel mode of activation for the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin fromDictyostelium discoideum we found a reversible, Ca2+/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca2+/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 μm with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca2+/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca2+/calmodulin action by binding to the calmodulin-binding site. p-nitrophenyl phosphate tetratricopeptide repeats The Ca2+/calmodulin-stimulated protein Ser/Thr phosphatase (protein phosphatase 2B; calcineurin) plays a pivotal role in various cellular processes. Its function in T-cell activation including the dephosphorylation and mobilization of the transcription factor NF-AT (nuclear factor ofactivated T-cells) and the enhanced expression of interleukin-2 has been extensively studied (for review see Refs. 1Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (627) Google Scholarand 2Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar). The structure of calcineurin has been conserved from yeast to man (3Kincaid R. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-23PubMed Google Scholar). The holoenzyme consists of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B). Calcineurin A varies in size from species to species (58–73 kDa) (4Guerini D. Biochem. Biophys. Res. Commun. 1997; 235: 271-275Crossref PubMed Scopus (140) Google Scholar). Calcineurin B is a 19-kDa Ca2+-binding protein homologous to calmodulin. High affinity binding of Ca2+ to calcineurin B at low Ca2+ concentrations (K d < 10−8m) leads to formation of the calcineurin A/B complex. Calcineurin B decreases the K m of calcineurin A for its protein substrates (5Stemmer P.M. Klee C.B. Biochemistry. 1994; 33: 6859-6866Crossref PubMed Scopus (244) Google Scholar). The holoenzyme is tightly regulated by Ca2+/calmodulin (for review see Refs. 6Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. 1988; 61: 149-200PubMed Google Scholar and 7Klee C.B. Ren H. Wang X. J. Biol. Chem. 1998; 273: 13367-13370Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar). Binding of Ca2+/calmodulin to a basic amphipathic α-helix (for review see Ref. 8James P. Vorherr T. Carafoli E. Trends Biochem. Sci. 1995; 20: 38-42Abstract Full Text PDF PubMed Scopus (347) Google Scholar) relieves inhibition by a C-terminal autoinhibitory domain and activates the enzyme by changing V max (5Stemmer P.M. Klee C.B. Biochemistry. 1994; 33: 6859-6866Crossref PubMed Scopus (244) Google Scholar). This suggests hydrophobic as well as ionic interactions between calcineurin A and Ca2+/calmodulin (Ref. 6Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. 1988; 61: 149-200PubMed Google Scholar and references therein).Dictyostelium calcineurin has previously been characterized in this laboratory (9Dammann H. Hellstern S. Husain Q. Mutzel R. Eur. J. Biochem. 1996; 238: 391-399Crossref PubMed Scopus (47) Google Scholar, 10Hellstern S. Dammann H. Husain Q. Mutzel R. Res. Microbiol. 1997; 148: 335-343Crossref PubMed Scopus (18) Google Scholar). The enzyme has some unusual features as compared with its counterparts from other species, e.g.N-terminal and C-terminal extensions (9Dammann H. Hellstern S. Husain Q. Mutzel R. Eur. J. Biochem. 1996; 238: 391-399Crossref PubMed Scopus (47) Google Scholar) resulting in its high molecular mass of 73 kDa. Expression of the single calcineurin A gene is developmentally regulated both at the mRNA and the protein level (9Dammann H. Hellstern S. Husain Q. Mutzel R. Eur. J. Biochem. 1996; 238: 391-399Crossref PubMed Scopus (47) Google Scholar). Biochemical characterization of the protein purified from recombinant bacteria and an overexpressing Dictyosteliumcell line revealed enzymatic properties that are fairly comparable with calcineurins from other sources (10Hellstern S. Dammann H. Husain Q. Mutzel R. Res. Microbiol. 1997; 148: 335-343Crossref PubMed Scopus (18) Google Scholar). Pharmacological evidence suggests at least three distinct roles for Dictyostelium calcineurin. Using the calcineurin inhibitors FK 506 and cyclosporin A, Horn and Gross (11Horn F. Gross J. Differentiation. 1996; 60: 269-275Crossref PubMed Google Scholar) have provided evidence that calcineurin is involved in both pathways of Dictyostelium differentiation. Moniakis et al. (12Moniakis J. Coukell M.B. Janiec A. J. Cell Sci. 1999; 112: 405-414Crossref PubMed Google Scholar) have shown by using cyclosporin A that the Ca2+ regulated expression of the Ca2+ ATPase, PAT1, is under the control of calcineurin. The calcineurin inhibitor deltamethrin, but not the inactive analog perimethrin, was shown to inhibit chemotaxis toward the attractant cAMP, suggesting a role in chemotaxis for calcineurin (13Polesky A.J.M. O'Day D.H. Mol. Biol. Cell. 1995; 6 (suppl.) (abstr.): 91Google Scholar). Ca2+ signaling in Dictyostelium is essential for chemotaxis toward cAMP (for review see Ref. 14Newell P.C. Malchow D. Gross J.D. Experientia. 1995; 51: 1155-1165Crossref PubMed Scopus (37) Google Scholar). The binding of cAMP to its cell surface receptors leads to Ca2+ influx across the plasma membrane and to an increase in the cytosolic Ca2+concentration (15Bumann J. Wurster B. Malchow D. J. Cell Biol. 1984; 98: 173-178Crossref PubMed Scopus (103) Google Scholar, 16Schlatterer C. Gollnick F. Schmidt E. Meyer R. Knoll G. J. Cell Sci. 1994; 107: 2107-2115Crossref PubMed Google Scholar, 17Yumura S. Furuya K. Takeuchi I. J. Cell Sci. 1996; 109: 2673-2678Crossref PubMed Google Scholar). It was shown that several inhibitors for phospholipase A2 blocked cAMP induced Ca2+influx to a great extent, suggesting a prominent role for phospholipase A2 in the signal transduction pathway leading to Ca2+ uptake (18Schaloske R. Malchow D. Biochem. J. 1997; 327: 233-238Crossref PubMed Scopus (29) Google Scholar). Phospholipase A2 releases fatty acids from phospholipids. It was demonstrated that long chain fatty acids mimic the action of cAMP by bypassing both cAMP-receptor stimulation and activation of phospholipase A2, directly inducing Ca2+ influx (18Schaloske R. Malchow D. Biochem. J. 1997; 327: 233-238Crossref PubMed Scopus (29) Google Scholar). Moreover long chain fatty acids liberate Ca2+ from internal stores and thus induce capacitative Ca2+ entry (19Schaloske R. Sonnemann J. Malchow D. Schlatterer C. Biochem. J. 1998; 332: 541-548Crossref PubMed Scopus (39) Google Scholar). Dictyostelium cells, which live as amoebae in the soil, are subject to large environmental changes, e.g. fluctuations in ion supply. Thus robust mechanisms supporting Ca2+signaling are required to maintain chemotactic capability. One group of candidates for signaling molecules are long chain fatty acids (18Schaloske R. Malchow D. Biochem. J. 1997; 327: 233-238Crossref PubMed Scopus (29) Google Scholar, 19Schaloske R. Sonnemann J. Malchow D. Schlatterer C. Biochem. J. 1998; 332: 541-548Crossref PubMed Scopus (39) Google Scholar). In this study we have investigated direct effects of long chain fatty acids on Dictyostelium calcineurin activity and found a novel, Ca2+/calmodulin-independent mode of activation for calcineurin. Arachidonic acid (free acid) was from Fluka (Buchs, Switzerland), arachidonic acid methyl ester, oleic acid, and bovine albumin (fatty acid-free) were from ICN (Eschwege, Germany). Arachidic acid and palmitic acid were from Sigma (Munich, Germany), and linoleic acid was from ICT (Bad Homburg, Germany). Calmodulin-Sepharose 4B was from Amersham Pharmacia Biotech (Freiburg, Germany). The Biomol Green reagent, the phosphate standard, and RII phosphopeptide were from Biomol (Hamburg, Germany).p-Nitrophenyl phosphate was from Merck (Darmstadt, Germany). Calmodulin (bovine brain) was from Alexis (San Diego, CA). Calcineurin A from Dictyostelium was purified from an overexpressing cell line as described previously (10Hellstern S. Dammann H. Husain Q. Mutzel R. Res. Microbiol. 1997; 148: 335-343Crossref PubMed Scopus (18) Google Scholar). Purification and characterization of recombinant Dictyostelium calcineurin B will be described elsewhere. 1A. Aichem and R. Mutzel, manuscript in preparation. Phosphatase activity assays with p-nitrophenyl phosphate (pNPP)2 as substrate were performed routinely in duplicate or triplicate according to Hellsternet al. (10Hellstern S. Dammann H. Husain Q. Mutzel R. Res. Microbiol. 1997; 148: 335-343Crossref PubMed Scopus (18) Google Scholar). Briefly, calcineurin A (final concentration, 140 nm) was preincubated in the absence or presence of calcineurin B (140 nm) and/or calmodulin (1 μm) in buffer A (50 mm Tris-HCl, 0.5 mm MnCl2, 0.5 mm dithiothreitol, 0.2 mm CaCl2, 20 mmMgCl2, pH 7.8) for 1 h. Subsequently fatty acid solutions or 1% ethanol (vehicle) as a control were added 1 min prior to addition of pNPP (20 mm). The reaction (total volume, 150 μl) was stopped after 1 h by adding an equal volume of 1m Na2CO3, 20 mm EDTA, 2 mm EGTA, pH 8.0. All incubations were performed at 30 °C. The absorbance of the dye was measured at 405 nm and corrected for blank absorbance. For determination of phosphatase activity with RII phosphopeptide as a substrate, the reaction was initiated by adding 35 μm of the phosphopeptide (total volume after addition, 50 μl). To terminate the reaction, 2 volumes of Biomol Green reagent were added after 40 min. The test was linear from 10 to 60 min. The dye was allowed to develop for 25 min before measuring the absorbance at 620 nm. In the experiments with calcineurin A alone, the final concentration of the phosphatase was 420 nm. In the presence of both calcineurin A and B, the concentration of each was 252 nm. The concentration of calmodulin was 1 μmthroughout. 1 unit is defined as the release of 1 nmol phosphate min−1 mg protein−1. Binding of calcineurin to calmodulin-Sepharose 4B in the absence or presence of arachidonic acid or arachidonic acid methyl ester was determined at 4 °C in a total volume of 250 μl containing 170 nmcalcineurin A, arachidonic acid, or arachidonic acid methyl ester (20 μm each) or 1% ethanol (vehicle), and 100 μl of a calmodulin-Sepharose 4B slurry equilibrated in buffer B (50 mm Tris-HCl, 150 mm NaCl, 2 mmCaCl2, 1 mm EDTA, 0.5 mmdithiothreitol, pH 7.8). The mixture was incubated under constant agitation for 30 min. The Sepharose was pelleted by centrifugation in an Eppendorf centrifuge for 2 s. 150-μl aliquots of the supernatant were withdrawn, and 100 μl was loaded onto SDS-polyacrylamide gels and subsequently subjected to Western blotting. For washing the resin the volume was adjusted to 250 μl with buffer B, resuspended, and recentrifuged. This procedure was repeated twice. After the second wash the supernatant was removed thoroughly. The protein was eluted by adjusting the volume to 250 μl with elution buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 20 mm EGTA, 0.5 mmdithiothreitol, pH 7.8), resuspending the resin, and incubation for 15 min. Elution was repeated once. Samples for SDS-polyacrylamide gel electrophoresis were withdrawn after each step as above. To elute calcineurin A with arachidonic acid, the phosphatase was bound to calmodulin-Sepharose 4B in buffer B as above. The resin was washed twice with the same buffer. Subsequently buffer B containing 20 μm arachidonic acid or 20 μm arachidonic acid methyl ester or 1% ethanol was added, and the samples were incubated for 15 min. The gel was washed once with buffer B, and the final elution was carried out with elution buffer as above. Aliquots for SDS-polyacrylamide gel electrophoresis were taken as described above. Proteins were chromatographed on SDS-polyacrylamide gels (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207123) Google Scholar), blotted onto nitrocellulose membranes (BA85, Schleicher & Schüll) (21Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2158) Google Scholar), and probed with a rabbit antiserum (dilution, 1:5000) against recombinant Dictyostelium calcineurin A as described previously (9Dammann H. Hellstern S. Husain Q. Mutzel R. Eur. J. Biochem. 1996; 238: 391-399Crossref PubMed Scopus (47) Google Scholar). The critical micellar concentration of fatty acids was determined as described by Chattopadhyay and London (22Chattopadhyay A. London E. Anal. Biochem. 1984; 139: 408-412Crossref PubMed Scopus (370) Google Scholar). Ethanolic stock solutions of arachidonic acid, linoleic acid, and arachidonic acid methyl ester (100 mm each) were stored under nitrogen at −80 °C. Diluted fatty acid solutions were prepared from stock aliquots immediately prior to the experiment and kept under nitrogen until use. All other fatty acid solutions were freshly prepared from solids or oils. Fig. 1 A shows a dose-response curve for activation of purified Dictyostelium calcineurin A by arachidonic acid (20:4) in the absence of calmodulin and calcineurin B. Half-maximal stimulation of the phosphatase activity with pNPP as substrate occurred between 5 and 10 μm of the fatty acid. Maximal stimulation was observed at 30 μm arachidonic acid and resulted in an approximately 4-fold activation. Arachidonic acid and calmodulin activated the phosphatase to the same extent. Calcineurin activities in the presence of 30 μmarachidonic acid and a saturating dose of calmodulin (1 μm) are not significantly different. Fig. 1 Bshows the activation of the holoenzyme by arachidonic acid. The dose-response relation is very similar to the one above, with half-maximal stimulation at 10 μm arachidonic acid and maximal stimulation at 30 μm resulting in a 3–3.5-fold stimulation. Again, there was no significant difference between the maximal stimulation by arachidonic acid and calmodulin. Half-maximal activation was observed well below the critical micellar concentration of 20 μm for arachidonic acid in the buffer system used for the phosphatase assays. Because there was no influence of calcineurin B on the activation process, subsequent experiments with pNPP as substrate were performed in the absence of the regulatory subunit. The synthetic nondecapeptide corresponding to the regulatory subunit of the cAMP-dependent protein kinase (type II) is a model peptide substrate for calcineurin (23Blumenthal D.K. Takio K. Hansen R.S. Krebs E.G. J. Biol. Chem. 1986; 261: 8140-8145Abstract Full Text PDF PubMed Google Scholar). As shown previously (10Hellstern S. Dammann H. Husain Q. Mutzel R. Res. Microbiol. 1997; 148: 335-343Crossref PubMed Scopus (18) Google Scholar), the basal activity of Dictyosteliumcalcineurin A toward the RII peptide in the absence of the regulatory subunit calcineurin B (Fig. 2) is lower than toward the nonpeptide substrate pNPP (0.4 ± 0.3 unitversus 152 ± 32 units for RII peptide (n = 4) and pNPP (n = 25), respectively). Similar observations have been reported for recombinantNeurospora crassa calcineurin A (24Higuchi S. Tamura J. Giri P.R. Polli J.W. Kincaid R.L. J. Biol. Chem. 1991; 266: 18104-18112Abstract Full Text PDF PubMed Google Scholar). Calcineurin B activates the catalytic subunit by lowering theK m (5Stemmer P.M. Klee C.B. Biochemistry. 1994; 33: 6859-6866Crossref PubMed Scopus (244) Google Scholar). This is reflected by the rise in calcineurin activity upon addition of calcineurin B. In the absence of calcineurin B, Ca2+/calmodulin activated calcineurin to approximately the same extent. Full activation was obtained in the presence of both calcineurin B and calmodulin. The activation by calmodulin in the absence of calcineurin B could be mimicked by the addition of 50 μm arachidonic acid. Addition of both calcineurin B and arachidonic acid again yielded full activation of the phosphatase. 50 μm arachidonic acid is a saturating dose for stimulation of the phosphatase activity. Half-maximal activity was obtained at 10 μm of the fatty acid (data not shown). Activation of calcineurin by arachidonic acid is therefore also valid for a peptide substrate. The unsaturated long chain fatty acids linoleic acid (18:2) and oleic acid (18:1) exerted effects on calcineurin similar to those of arachidonic acid with respect to half-maximal and maximal stimulating concentrations (Fig. 3). The maximal stimulation by linoleic acid shown in Fig. 3 seems to be lower than that of oleic acid or arachidonic acid, but there is no significant difference between the maximal stimulation by linoleic acid and by Ca2+/calmodulin (t test at the 0.05 level). To investigate the specificity of the effect of long chain unsaturated fatty acids on calcineurin and to obtain insight into the mechanism of stimulation, the methyl ester of arachidonic acid and the saturated fatty acids arachidic acid (20:0) and palmitic acid (16:0) were used. The methyl ester had no effect on the phosphatase activity (Table I). Moreover, both of the saturated fatty acids neither stimulated basal activity nor influenced the Ca2+/calmodulin-stimulated activity at concentrations that are saturating for the unsaturated fatty acids (50 μm). The data in Table I also show that the stimulation of the phosphatase activity by unsaturated fatty acids is not additive to Ca2+/calmodulin stimulation because the stimulation in the presence of both unsaturated fatty acids and Ca2+/calmodulin did not exceed the values in the presence of either compound. Taken together, these results suggest that both the carboxy group and the unsaturated character of the fatty acids are required for stimulation of calcineurin. Calmodulin and unsaturated fatty acids may exert their effects through a similar mechanism, possibly by binding to the same site.Table IEffect of fatty acids and arachidonic acid methyl ester on purified calcineurin A from DictyosteliumFatty acid (50 μm)Fatty acid alone (activity relative to control)nFatty acid plus calmodulin (activity relative to control)nAA (20:4)4.2 ± 0.484.9 ± 13Linoleic acid (18:2)3.0 ± 0.352.4 ± 0.82Oleic acid (18:1)4.4 ± 0.32NDArachidic acid (20:0)1.1 ± 0.132.5 ± 0.83Palmitic acid (16:0)1.0 ± 0.142.7 ± 0.43Arachidonic acid methyl ester (20:4)1.1 ± 0.122.8 ± 0.42 Open table in a new tab Next we analyzed whether the activation of calcineurin by arachidonic acid is reversible. Bovine serum albumin binds to arachidonic acid with high affinity (K d, 62 nm) (25Demant E.J.F. Anal. Biochem. 1999; 267: 366-372Crossref PubMed Scopus (16) Google Scholar) and should therefore effectively abolish activation by the fatty acid. Activation of calcineurin by 30 μm arachidonic acid resulted in an approximately 3-fold stimulation over basal activity (Fig. 4). Addition of 15 μm fatty acid-free bovine serum albumin reduced the phosphatase activity to basal levels, indicating that complexing the fatty acid stops the activation process. The enzyme was thereafter susceptible to stimulation by Ca2+/calmodulin as shown in Fig. 4. Activation by Ca2+/calmodulin after capturing the fatty acid was of the same magnitude as the response to arachidonic acid, demonstrating that the structure of calcineurin had not been disrupted irreversibly by arachidonic acid. Several lines of evidence suggested that unsaturated fatty acids and Ca2+/calmodulin exert their effects on calcineurin activity through similar mechanisms by binding to the same site. Therefore we tested whether arachidonic acid interferes with binding of purified Dictyosteliumcalcineurin to immobilized calmodulin. In a first set of experiments we examined binding of calcineurin to calmodulin in the presence of arachidonic acid (Fig. 5,A–C). Arachidonic acid inhibited binding of the phosphatase to calmodulin-Sepharose (Fig. 5 B), whereas with equal concentrations of arachidonic acid methyl ester (Fig. 5 C) most of the protein bound to the gel and could subsequently be eluted by buffer containing EGTA. In a second set of experiments we investigated whether a buffer containing arachidonic acid was able to elute prebound calcineurin from calmodulin-Sepharose (Fig. 5,D–F). This is indeed the case, as shown in Fig. 5 E. About half of the prebound protein could be displaced in a single elution step from calmodulin (Fig. 5 E, lane E*), whereas all the protein remained bound to calmodulin in the presence of arachidonic acid methyl ester (Fig. 5 F) and could only be eluted from calmodulin-Sepharose by incubation with EGTA. We show here that the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid activate calcineurin from Dictyostelium. The activation is independent of Ca2+/calmodulin. Half-maximal activation occurred at a concentration (5–10 μm) well below the critical micellar concentration (20 μm), indicating that it is not merely an effect of calcineurin binding to micelles. Politino and King (26Politino M. King M.M. J. Biol. Chem. 1987; 262: 10109-10113Abstract Full Text PDF PubMed Google Scholar) have described activation of calcineurin from bovine brain by binding to acidic phospholipids. This effect is clearly distinct from the one reported here because phospholipids bind to the regulatory subunit calcineurin B (27Politino M. King M.M. J. Biol. Chem. 1990; 265: 7619-7622Abstract Full Text PDF PubMed Google Scholar). Activation of Dictyostelium calcineurin by unsaturated fatty acids is independent of the presence of calcineurin B. Stimulation of enzymatic activity is specific for unsaturated fatty acids because saturated fatty acids were not active. Moreover, the negatively charged carboxy group is necessary for the activation process because the methyl ester derivative of arachidonic acid was inactive. Stimulation of calcineurin by fatty acids was demonstrated with chemically distinct substrates, the chromogenic substrate pNPP, and the RII phosphopeptide substrate corresponding to a phosphorylation site within the RII subunit of the cAMP-dependent protein kinase. The effect is readily reversible as shown by relieving the activation of calcineurin through capturing the fatty acids by albumin. Calcineurin activation by fatty acids could be a means for in vivo regulation. We suggest that stimulation by fatty acids or Ca2+/calmodulin are alternative mechanisms. What are the structural determinants for fatty acid binding to calcineurin A? The most likely candidates are the hydrophobic binding sites for calcineurin B and Ca2+/calmodulin. The following data exclude the calcineurin B-binding site as a candidate: (i) arachidonic acid activates the phosphatase both in the absence and in the presence of calcineurin B (Fig. 1). Even in the presence of low Ca2+ concentrations the binding of calcineurin B to the catalytic subunit is nearly irreversible (5Stemmer P.M. Klee C.B. Biochemistry. 1994; 33: 6859-6866Crossref PubMed Scopus (244) Google Scholar), and the calcineurin A/B complex is only dissociated in the presence of urea, SDS, or guanidine hydrochloride (Ref. 6Klee C.B. Draetta G.F. Hubbard M.J. Adv. Enzymol. 1988; 61: 149-200PubMed Google Scholar and references therein) and (ii) if arachidonic acid disturbed calcineurin B binding to calcineurin A, then the phosphatase activity would not increase upon addition of calcineurin B with RII phosphopeptide as a substrate (Fig. 2). Our results suggest that Ca2+/calmodulin and unsaturated fatty acids share the same binding domain and exert their effects through similar mechanisms: (i) arachidonic acid inhibits binding of calcineurin A to calmodulin and displaces calcineurin from immobilized calmodulin (Fig. 5); (ii) activation by fatty acids and by Ca2+/calmodulin is not additive (Table I); (iii) not only the full stimulation by Ca2+/calmodulin of the RII phosphopeptide phosphatase activity in the presence of calcineurin B could be mimicked by arachidonic acid but also the partial stimulation in the absence of calcineurin B (Fig. 2); and (iv) maximal activation by unsaturated fatty acids and Ca2+/calmodulin is in the same range (Figs. 1 and 2). Although these data argue strongly in favor of direct competition of Ca2+/calmodulin and arachidonic acid for binding to the same site, we cannot distinguish between fatty acid binding to the enzyme and to Ca2+/calmodulin in our competition experiments. Binding of Ca2+ to calmodulin results in exposure of hydrophobic and acidic amino acids on the protein surface that interact with a basic amphipathic α-helix within the target protein (28Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (598) Google Scholar). Therefore hydrophobic and ionic interactions might be required for binding of fatty acids to the basic amphipathic α-helix, which forms the Ca2+/calmodulin-binding site (8James P. Vorherr T. Carafoli E. Trends Biochem. Sci. 1995; 20: 38-42Abstract Full Text PDF PubMed Scopus (347) Google Scholar). Accordingly, not only the unsaturated hydrophobic character of the lipids is essential for proper activation but also the additional negatively charged carboxy residue. Therefore, the acidity of Ca2+/calmodulin renders an interaction with acidic lipids unlikely. Several calmodulin-binding proteins were found to be activated by long chain fatty acids. Human erythrocyte Ca2+ ATPase was activated by oleic and linoleic acid (29Niggli V. Adunyah E.S. Carafoli E. J. Biol. Chem. 1981; 256: 8588-8592Abstract Full Text PDF PubMed Google Scholar, 30Al-Jobore A. Roufogalis B.D. Can. J. Biochem. 1981; 59: 880-888Crossref PubMed Scopus (22) Google Scholar, 31Jarrett H.W. J. Biol. Chem. 1986; 261: 4967-4972Abstract Full Text PDF PubMed Google Scholar), bovine brain cyclic nucleotide phosphodiesterase was stimulated by oleic acid (31Jarrett H.W. J. Biol. Chem. 1986; 261: 4967-4972Abstract Full Text PDF PubMed Google Scholar, 32Pichard A.-L. Cheung W.Y. J. Biol. Chem. 1977; 252: 4872-4875Abstract Full Text PDF PubMed Google Scholar), and porcine cyclic nucleotide phosphodiesterase was activated by both unsaturated and saturated fatty acids (33Wolff D.J. Brostrom C.O. Arch. Biochem. Biophys. 1976; 173: 720-731Crossref PubMed Scopus (97) Google Scholar). Myosin light chain kinase from chicken gizzard was stimulated by arachidonic acid but not by arachidic acid (34Tanaka T. Hidaka H. J. Biol. Chem. 1980; 255: 11078-11080Abstract Full Text PDF PubMed Google Scholar). Activation of Ca2+/calmodulin-dependent enzymes by binding of fatty acids to their Ca2+/calmodulin-binding sites could therefore represent a more common, alternative mode of regulation of calmodulin-binding proteins. Two other protein Ser/Thr phosphatases were recently shown to be activated by unsaturated fatty acids, the phosphatases type 5 (35Skinner J. Sinclair C. Romeo C. Armstrong D. Charbonneau H. Rossie S. J. Biol. Chem. 1997; 272: 22464-22471Crossref PubMed Scopus (102) Google Scholar, 36Chen M.X. Cohen P.T.W. FEBS Lett. 1997; 400: 136-140Crossref PubMed Scopus (118) Google Scholar) and type 2C (37Klumpp S. Selke D. Hermesmeier J. FEBS Lett. 1998; 437: 229-232Crossref PubMed Scopus (50) Google Scholar). In both cases activation occurred at fatty acid concentrations greater than 100 μm, which was above the critical micellar concentration. In case of protein phosphatase 5 it was demonstrated that lipid vesicles consisting of fatty acids stimulate the enzymatic activity by binding to a motif consisting of 34 amino acids called tetratricopeptide repeat (TPR) domain. The TPR domain in protein phosphatase P5 is supposed to form a shield in front of the active site, and binding of lipid vesicles was suggested to result in displacement of the TPR domain allowing access of substrates to the active site (36Chen M.X. Cohen P.T.W. FEBS Lett. 1997; 400: 136-140Crossref PubMed Scopus (118) Google Scholar). We could not identify a TPR domain inDictyostelium calcineurin. Activation of calcineurin by binding of unsaturated fatty acids to the calmodulin-binding domain might, however, represent a similar mechanism by relieving inhibition of the C-terminal autoinhibitory domain. The physiological significance of calcineurin activation by unsaturated fatty acids is so far uncertain. No data on the release of fatty acids into the cytosol of Dictyostelium are available. However, in glucose-stimulated pancreatic islets, a rise in the mass of free arachidonic acid sufficient to yield an overall cellular concentration of 38–75 μm was measured (38Wolf B.A. Pasquale S.M. Turk J. Biochemistry. 1991; 30: 6372-6379Crossref PubMed Scopus (111) Google Scholar), making regulation of soluble proteins such as calcineurin possible. Unterweger and Schlatterer (39Unterweger N. Schlatterer C. Cell Calcium. 1995; 17: 97-110Crossref PubMed Scopus (28) Google Scholar) have shown that an increase in the cytosolic Ca2+ concentration is necessary for orientation and locomotion of Dictyostelium cells since clamping the cytosolic Ca2+ concentration by introducing the Ca2+ buffer [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] inhibited chemotaxis. In the presence of extracellular EGTA,Dictyostelium cells were able to protrude pseudopods and to migrate, albeit at lower velocity, indicating that liberation of Ca2+ from stores is sufficient for the chemotactic response (39Unterweger N. Schlatterer C. Cell Calcium. 1995; 17: 97-110Crossref PubMed Scopus (28) Google Scholar). The calcineurin inhibitor deltamethrin was reported to inhibit chemotaxis in Dictyostelium, suggesting a role for calcineurin (13Polesky A.J.M. O'Day D.H. Mol. Biol. Cell. 1995; 6 (suppl.) (abstr.): 91Google Scholar). Recently Persechini and Cronk (40Persechini A. Cronk B. J. Biol. Chem. 1999; 274: 6827-6830Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) have shown that the free cytosolic Ca2+ concentration necessary to produce sufficient Ca2+/calmodulin for in vivoactivation of high affinity Ca2+/calmodulin-binding proteins is in the range of several hundred nm, presuming that the K d of Ca2+/calmodulin binding to the calmodulin-binding protein is 2 nm. TheK d for Ca2+/calmodulin binding toDictyostelium calcineurin is in the low nanomolar range (41Winckler T. Dammann H. Mutzel R. Res. Microbiol. 1991; 142: 509-519Crossref PubMed Scopus (12) Google Scholar). At the low extracellular Ca2+ concentration in the range of 200 nm the free cytosolic Ca2+concentration cannot increase above this value merely by opening Ca2+ channels in the plasma membrane. Ca2+influx from the extracellular medium would thus not allow activation of calcineurin under these conditions. NeverthelessDictyostelium cells are able to perform chemotaxis at the extracellular Ca2+ concentration of 200 nm(42Malchow D. Böhme R. Gras U. Biophys. Struct. Mech. 1982; 9: 131-136Crossref PubMed Scopus (28) Google Scholar). 3R. Schaloske, unpublished results. Recently it was demonstrated that arachidonic acid releases Ca2+ from intracellular stores and thus induces capacitative Ca2+entry (18Schaloske R. Malchow D. Biochem. J. 1997; 327: 233-238Crossref PubMed Scopus (29) Google Scholar, 19Schaloske R. Sonnemann J. Malchow D. Schlatterer C. Biochem. J. 1998; 332: 541-548Crossref PubMed Scopus (39) Google Scholar). A direct effect of arachidonic acid on Ca2+- or H+-ATPases could be excluded (19Schaloske R. Sonnemann J. Malchow D. Schlatterer C. Biochem. J. 1998; 332: 541-548Crossref PubMed Scopus (39) Google Scholar). The mechanism of arachidonic acid-induced Ca2+ release remained unclear. The stimulatory effect of unsaturated fatty acids described here could mean that calcineurin is involved in Ca2+release. A testable model for the signal transduction pathway resulting in Ca2+ signaling would imply receptor occupation by cAMP, activation of phospholipase A2, and release of unsaturated fatty acids that activate calcineurin. Calcineurin might trigger liberation of Ca2+, which would be sufficient for the chemotactic response (39Unterweger N. Schlatterer C. Cell Calcium. 1995; 17: 97-110Crossref PubMed Scopus (28) Google Scholar). In addition, Ca2+ release leads to capacitative Ca2+ entry even at low extracellular Ca2+ concentrations (18Schaloske R. Malchow D. Biochem. J. 1997; 327: 233-238Crossref PubMed Scopus (29) Google Scholar). Taken together, this leads to an increase in the Ca2+/calmodulin concentration resulting in the alternative activation of calcineurin. As soon as the cytosolic Ca2+ concentration reaches resting levels through Ca2+ ATPase activity, calcineurin activation is relieved. We thank D. Malchow and J. Breed for critical reading of the manuscript.