Proc. Nati. Acad. Sci. USAVol. 77, No. 12, pp. 7039-7043, December 1980Biochemistry

Calcium-dependent protein kinase: Widespread occurrence invarious tissues and phyla of the animal kingdom and comparison ofeffects of phospholipid, calmodulin, and trifluoperazine

(cyclic nucleotides/diolein/phosphodiesterase)

J. F. KUO*, ROLF G. G. ANDERSSONt, BRADLEY C. WISE*, LUDMILA MACKERLOVAt,INGRID SALOMONSSONt, NANCY L. BRACKETT*, NORio KATOH*, MAMORU SHOJI*,AND ROBERT W. WRENN**Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322; and tDepartment of Pharmacology, Linkoping University,S-581 85 Link6ping, Sweden

Communicated by Irwin C. Gunsalus, August 14,1980

ABSTRACT A widespread occurrence of Ca +-dependentprotein kinase was shown in various tissues and phyla of theanimal kingdom. Phosphatidylserine appeared to be more ef-fective than calmodulin in supporting the Ca2+-dependentphosphotransferase activity. The phospholipid-sensitive Ca2+-dependent protein kinase activity, distributed in both the cy-tosolic and particulate fractions, was not inhibited by triflu-operazine, a specific inhibitor of calmodulin-sensitive, Ca2+_dependent reactions or processes. The enzyme activity levels,compared to those of cyclic AMP-dependent and cyclic GMP-dependent protein kinases, were exceedingly high in certaintissues (such as brain and spleen) and exhibited a much-greaterdisparity among tissues. The Ka for Ca2+ was about 100 gM inthe presence of phosphatidylserine; the value was as low as 2,gM in the presence of phosphatidylserine and diolein. It issuggested that phospholipid-sensitive Ca2+-dependent proteinkinase may mediate certain actions of Ca2+ in tissues, actingindependently or in a complementary manner with other pro-tein phosphorylation systems stimulated by calmodulin-Ca2+,cyclic AMP, or cyclic GMP.

Functional roles of Ca2 , cyclic AMP (cAMP), and cyclic GMP(cGMP) as intracellular second messengers and their interac-tions in regulating cellular functions are well recognized. Thesubject matter has been extensively reviewed (1). The diversityof actions of cAMP (2) and cGMP (3, 4) has been hypothesizedto be mediated via activation of cAMP-dependent protein ki-nase (A-PK) and cGMP-dependent protein kinase (G-PK), re-spectively. The importance of protein phosphorylation in bio-logical processes has been the topic of recent investigations (5).Despite the many physiological effects elicited by Ca2+, itsmolecular mechanisms remain largely obscure, with the notableexception of calmodulin (6), a Ca2+-binding protein activatingmany biological processes requiring Ca2+. The existence of aphospholipid-sensitive Ca2+-dependent protein kinase (Ca-PK)recently reported by Takai et al. (7) is of special significance.This enzyme not only provides hitherto unknown mechanismsfor the actions of Ca2+ and phospholipid but also further ex-pands conceptually the possibility of interactions of Ca2+ withcyclic nucleotides at the step of protein phosphorylation. Thepresent studies were undertaken to establish the widespreadoccurrence of this potentially important enzyme in animals andto demonstrate the requirement of phosphatidylserine, ratherthan calmodulin, for its activity.

EXPERIMENTAL PROCEDURESMaterials. Phosphatidylserine (bovine brain, in chloro-

form/methanol), cAMP, cGMP, lysine-rich histone (type III-S),and 1,3-diolein were obtained from Sigma. Homogeneouscalmodulin (bovine brain) was a gift from W. Y. Cheung (De-partment of Biochemistry, St. Jude Children's Research Hos-pital, Memphis, TN). Trifluoperazine-2HCI was obtainedthrough P. T. Ridley (Smith Kline and French). ['y-32P]ATP waseither purchased from New England Nuclear or prepared ac-cording to the method of Post and Sen (8). Cyclic [G-3H]AMPwas purchased from New England Nuclear.

Methods. Tissues from various phyla and species of animalswere homogenized in 3 vol of ice-cold 20 mM Tris-HCl, pH7.5/2 mM EDTA/50 mM 2-mercaptoethanol (extractionbuffer). The homogenates were centrifuged for 20 min at30,000 X g, and the supernatant fluids (extracts) thus obtainedwere appropriately diluted (3- to 90-fold) with the extractionbuffer and were used as the source of the soluble enzymes. Forexperiments concerning the subcellular distribution of enzymes(Table 2), the tissues were homogenized in 9 vol of ice-cold 25mM Tris-HCl, pH 7.5/0.25 M sucrose/2.5 mM MgCl2/2.5 mMethylene glycol bis(,B-aminoethyl ether)-N,N,N',N'-tetraacetate(EGTA)/50 mM 2-mercaptoethanol (homogenization buffer).The homogenates were first centrifuged for 5 min at 120 X gto remove cell debris, followed by centrifugation for 1 hr at100,000 X g to yield the cytosolic fractions. The pellets weresuspended in the original volume (as the homogenates) of thehomogenization buffer and were incubated in the presence of0.3% Triton X-100 for 1 hr in ice. The cytosolic and the par-ticulate fractions were diluted (3- to 100-fold) with the ex-traction buffer before assays. Ca-PK was purified from thebovine heart extract about 5000-fold by the following steps:ammonium sulfate precipitation, then chromatography onDEAE-cellulose, Sephadex G-200, controlled-pore glass, and,finally, phosphatidylserine-Affigel 102. Ca-PK from extractsof rat cerebral cortex, heart, lung, and spleen was purified about10- to 30-fold by chromatography on Sephadex G-200 columnslike, or similar to, those detailed in Fig. 3. The calmodulin-deficient phosphodiesterase from rat cerebral cortex was pre-pared by DEAE-cellulose chromatography as described (9)except that the crude extract was used in the present studies.A-PK and G-PK were assayed in the presence of 20,ug of the

purified stimulatory modulator of G-PK (10) and 40 Mg of ly-

Abbreviations: cAMP, cyclic AMP; cGMP, cyclic GMP; A-PK,cAMP-dependent protein kinase; G-PK, cGMP-dependent proteinkinase; Ca-PK, Ca2+-dependent protein kinase; EGTA, ethylene glycolbis(fl-aminoethyl ether)-N,N,N',N'-tetraacetate.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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FIG. 1. Effects of phosphatidylserine and Ca2+ on the proteinkinase activity in an extract (22 Mg of protein) of adult rat spleen.Assay conditions were the same as described in Experimental Pro-cedures except for (A) varying concentrations of phosphatidylserinein the presence or absence of added 0.5 mM CaCl2 and (B) varyingconcentrations of added CaCl2 in the presence or absence of 5 Mg ofphosphatidylserine. NoEGTA was added to chelate endogenous Ca2 .

The Ca2+ concentrations indicated represent the added CaCl2, notnecessarily the actual free Ca2+ present in the incubation. (A) 0, WithCa2+, Ka = 3 Mug/0.2 ml; 0, without Ca2+, Ka = 9 Mg/0.2 ml. (B) *,With phosphatidylserine, K. = 130 ,uM; 0, without phosphatidyl-serine, Ka = 200 MAM.

sine-rich histone by using 0.5 ,uM cAMP or cGMP to activatethe respective enzymes, as described elsewhere (11). The assaysystem for Ca-PK contained, in a final volume of 0.2 ml, Tris-HCl (pH 7.5), 5 ,umol; MgCl2, 2 ,mol; lysine-rich histone, 40rug; various amounts of CaCl2 and phosphatidylserine or cal-

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FIG. 2. Effects of 1,3-diolein on the affinity of phospholipid-sensitive Ca-PK for Ca2+. Assay conditions were the same as in Ex-perimental Procedures except for the inclusion of phosphatidylserine(5 Mg), various concentrations of added CaCl2, and, if present, diolein(2.5 ,ug). The enzyme sources were either (A) rat cerebral cortex ex-

tract (22 Mg of protein) or (B) purified bovine heart Ca-PK (2MUg) fromthe affinity gel step. The Ca2+ concentrations indicated represent theadded CaC12, not necessarily the actual free Ca2+ present in the in-cubation. 0, With diolein: (A) K. = 20 MM, (B) K. = 2 MM. 0,

Without diolein: (A) Ka = 100 AM, (B) K. = 50MuM.

Table 1. Levels of phospholipid-sensitive Ca-PK, A-PK, andG-PK in crude extracts of various rat tissues

Protein kinase,pmol 32P/min per g tissue

Tissue Ca-PK A-PK G-PK

Spleen 21,402 (213) 2627(250) 287 (82)Cerebral cortex 17,498 (281) 3820(362) 163 (65)Cerebellum 16,155 (260) 2615 (253) 1738 (265)Vas deferens 14,329 (178) 6295 (310) 144 (166)Midbrain 11,626 (223) ND NDBladder 6,432 (163) 2009 (230) 206 (122)Lung 3,021 (61) 3400(310) 1190 (210)Small intestine 2,692 (47) ND NDLiver 2,032 (88) 1500 (236) 155 (30)Ovary 1,089 (60) ND NDTestis 981 (69) ND NDUterus 932 (52) 2369(425) 520 (180)Adrenal 763 (46) ND NDKidney cortex 722 (49) 2422(385) 341 (58)Pancreas 576 (50) ND NDBlood cells (total) 575 (62) ND NDTrachea 506 (46) ND NDAorta 340 (56) 458(422) 154(182)Skeletal muscle 324 (28) 2730(385) 51 (5)Thymus 196 (14) ND NDHeart 173 (13) 1751(465) 479 (89)Epididymal fat 80 (19) 717 (282) 67 (52)

The crude extracts (containing 22-220 Mg of protein) were used toestimate the activity of protein kinases. Phospholipid-sensitive Ca-PKwas assayed in the presence and absence of added CaCl2 (0.5 mM) orphosphatidylserine (5 Mg), with lysine-rich histone as substrate. Thevalues presented are the Ca2+-stimulated activity seen in the presenceof phospholipid, which were corrected for the Ca2+-stimulated activityseen in its absence; the latter amounted to about 20-25% of the formerfor the brain tissues and about 10-15% for others. For A-PK andG-PK activity levels, the values presented are corrected for the basalvalues seen in the absence of added cAMP (0.5 MM) or cGMP (0.5,MM). The percent stimulation of protein kinases in response to theirrespective activators is indicated in parentheses. Results essentiallythe same as those shown were obtained when 50MgM EGTA was addedto the incubation mixture to chelate endogenous Ca2+. ND, not de-termined.

modulin; NaF, 8 ,umol (only for the particulate Ca-PK; Table2); [y-32P]ATP, 1 nmol (so0.8-1.3 X 106 cpm); and appropriateamounts of extract, cytosol, particulate, or purified enzymepreparations. The reaction was carried out for 2-10 min at300C, and the phosphorylated histone was precipitated with5% (wt/vol) trichloroacetic acid containing 0.25% tungstate,as described for A-PK and G-PK (2-4, 10, 11). Phosphodies-terase activity was assayed with 0.33 mM cyclic [G-3H]AMP(40,000 cpm) as substrate as described (12). CaCl2 (0.5 mM) wasalso included in the reaction mixture when stimulation of en-zyme activity by calmodulin was examined (Table 5). Proteinwas determined by the method of Lowry et al. (13).

RESULTSA protein kinase activity in an extract of adult rat spleen stim-ulated by Ca2+ was found to be largely dependent upon thepresence of phosphatidylserine (Fig. 1). The Ca2+-stimulatedactivity in the absence of added phospholipid, presumablycaused by calmodulin present in the extract, was much lowerthan in its presence. Phospholipid itself also stimulated theenzyme activity. Results similar to those for the rat spleen ex-tract were also noted for the extract of cerebral cortex from anewborn guinea pig (data not shown), confirming the originalobservation on the enzyme from rat brain reported by Takaiet al. (7). Addition of 2 ug of homogeneous calmodulin (anamount far in excess to maximally activate calmodulin-deficient

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Table 2. Subcellular distribution of phospholipid-sensitive Ca-PK, A-PK, and G-PK in the heart,spleen, and cerebral cortex of the guinea pig

Cytosolic fraction* Particulate fraction*Tissue Ca-PK A-PK G-PK Ca-PK A-PK G-PK

Heart 750 2055 45 280 510 30Spleen 17,630 3390 735 11,152 810 255Cerebral cortex 22,390 1900 100 31,907 4450 651

The cytosolic (containing 22-66 jig of protein) and the particulate (containing 2-50 jig of protein)fractions were used to estimate the protein kinase activity. The assay conditions and treatment of thedata were the same as for Table 1. The same results were obtained when 50 AM EGTA was added tothe incubation mixture.* Values for protein kinases are given as pmol of 32p per min per g of tissue.

phosphodiesterase, see Table 5) to the spleen and cerebral cortexextracts, in either the presence or absence of added phospho-lipid, did not increase the Ca2+-stimulated enzyme activity atany Ca2+ concentrations examined (data not shown).

Kishimoto et al. (14) recently reported that diolein not onlystimulates the activity of phospholipid-sensitive Ca-PK purifiedfrom the brain but also increases its affinity for Ca2+. In thepresent studies, the Ka for Ca2+ of the enzyme in rat cerebralcortex extract, assayed in the presence of phosphatidylserine,was decreased from about 100 ,uM in the absence of 1,3-dioleinto about 20 ,uM in its presence (Fig. 2A). The effect of dioleinon the Ka for Ca2+ in the presence of phospholipid, however,was much more pronounced (decreasing from 50 to 2 ,uM) forthe purified bovine cardiac enzyme (Fig. 2B). The resultsclearly indicate that the enzyme can be activated by Ca2+ atconcentrations presumably present intracellularly. Dioleinincreased the maximal activity of the purified cardiac Ca-PK(Fig. 2B), as reported for the purified brain enzyme by others(14). However, it was without significant effect on its maximalactivity in the cerebral cortex extract (Fig. 2A). As reported byothers (14), diolein per se had no effect on either the activityor the Ka for Ca2+ of the above Ca-PK preparations assayed inthe absence of added phospholipid (data not shown).The distribution of phospholipid-sensitive Ca-PK, compared

with that of A-PK and G-PK, in extracts of a number of adultrat tissues is shown in Table 1, with lysine-rich histone as a

common phosphate acceptor. The spleen, brain, and vas def-erens contained the highest levels of Ca-PK, whereas the epi-

didymal fat, heart, thymus, and skeletal muscle contained thelowest levels. It appears that there were no particular patternsof relationships among the tissue levels of Ca-PK, A-PK, andG-PK. The disparity of enzyme levels in rat tissues was far morepronounced for Ca-PK than for the other protein kinases.

Studies dealing with subcellular distribution of phospho-lipid-sensitive Ca-PK indicated that the majority of the enzymewas in the cytosolic fraction of the guinea pig heart, whereasit was distributed almost equally in both the cytosolic andparticulate fractions of the guinea pig spleen and cerebral cortex(Table 2). In comparison, the distributions of A-PK and G-PKin the same tissues were rather different.The widespread occurrence of phospholipid-sensitive Ca-PK

was further demonstrated by the presence of its activity intissues from four phyla and eight species of the animal kingdom(Table 3). The central nervous system of the nonmammals, as

in the case of the rat shown in Table 1, was a rich source of theenzyme. Significant amounts of the enzyme were also detectedin the peripheral nerves of the dog, such as the phrenic, sciatic,and vagus nerves. A much higher enzyme activity was foundin the stellate ganglion of the dog, suggesting that in the pe-ripheral nervous system the enzyme is concentrated in the nervebody and dendritic zone rather than in the axon. Interestingly,the egg shell gland of the egg-producing duck contained onlya low enzyme activity, suggesting that the enzyme may be moreintimately involved in the action rather than the metabolismof Ca2+.

Experiments were conducted in order to demonstrate further

Table 3. Phospholipid-sensitive Ca-PK in various tissues from several animal phyla

Protein kinase,pmol/min per g tissueWithout With

Phylum (class) Common name (genus) Tissue Ca2+ Ca2+

Annelida Earthworm (Lumbricus) Bodywall and nerve cord 1,471 2,947Mollusca Snail (Cepaea) Nerves 640 777Arthropoda (Crustacea) Lobster (Homarus) Brain 1,250 7,070

Heart 270 624Arthropoda (Insecta) Cockroach (Periplaneta) Ventral nerve 2,050 5,055

cord and gangliaHead (whole) 3,090 7,323

Chordata (Amphibia) Frog (Xenopus) Brain 3,900 13,540Heart 1,150 2,573

Chordata (Osteichthyes) Goldfish (Carassius) Brain 1,270 6,715Chordata (Aves) Duck (Anas) Brain 1,630 10,040

Egg shell gland 490 980Chordata (Mammalia) Dog (Canis) Spleen 23,520 50,750

Phrenic nerve 545 1,495Sciatic nerve 2,125 6,701Vagus nerve 1,430 5,015Stellate ganglion 5,332 19,935

The crude extracts (22-220 gg of protein) were assayed in the presence of phosphatidylserine (5 jg) as described in Ex-perimental Procedures and the legend of Table 1. When present, the concentration of CaCl2 was 0.5 mM. The same resultswere obtained when 50 jM EGTA was included in the assay.

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rebral cortex (A), lung (B), and heart (C). In each case, 1.2 ml of theextract obtained from 1 g of tissue was placed onto a Sephadex G-200-column (2.1 X 27 cm). The flow rate was 0.3 ml/min and the fractionsize was 0.8 ml. Fractions were assayed for Ca-PK activity with (0)or without (0) added CaCl2 (0.5 mM) in the presence of phosphati-dylserine (5Ag). & - - - &, Absorbance at 280 nm.

the disparity in tissue Ca-PK levels. Sephadex G-200 chroma-tography of extracts of the tissues (1 g) clearly indicated thatrelative levels of the enzyme activity were: cerebral cortex >>>lung > heart (Fig. 3), in line with the results obtained byassaying crude extracts shown in Table 1. A peak of Ca-PKactivity similar to the size of the peak of Ca-PK activity fromthe cerebral cortex was obtained when an extract of spleen (1g) was chromatographed on Sephadex under the same condi-tions (data not shown).The effects of calmodulin and phosphatidylserine on the

Ca-PK activity were directly compared (Table 4) by using thepeak enzyme fractions from the Sephadex G-200 chromatog-raphy such as shown in Fig. 3. As seen in studies using extracts,

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FIG. 4. Comparative effects of trifluoperazine on cAMP phos-phodiesterase and Ca-PK activities. (A) Rat cerebral cortex extract(22 Mg of protein) was assayed for phosphodiesterase activity with (0)or without (0) 0.5mM CaCl2 in the presence of various concentrationsof trifluoperazine with 0.33mM cAMP as substrate. (B) Rat cerebralcortex extract (22 Mg of protein) was assayed for Ca-PK activity in thepresence of added 0.5 mM CaCl2 and various concentrations of tri-fluoperazine with (0) or without (0) 5 ,ug of phosphatidylserine. Thenet activity stimulated by Ca2+ (about 150% in the absence and 350%in the presence of the phospholipid) is shown. (C) Same as B exceptthat purified bovine heart Ca-PK (2,ug) was used.

phosphatidylserine (5 ,g) was far more effective in supportingthe enzyme activity than was calmodulin (2 ,ug).The phenothiazine class of antipsychotic drugs inhibits

Ca2+-stimulated phosphodiesterase and adenylate cyclase fromthe brain by interfering with the binding of Ca2+ to calmodulin(15). In the present studies, trifluoperazine inhibited phos-phodiesterase in the cerebral cortex extract, with a half-maximalinhibitory concentration (IC5o) for the drug of about 10 IAM(Fig. 4A), in agreement with the value previously-reported forthe purified enzyme (15). Because Ca2+ and calmodulin arepresent in the extract, it can be considered that the drug wasacting on the calmodulin-requiring reaction. The Ca-PK ac-tivity in the same extract in the absence of added phosphati-dylserine was inhibited by the drug, also with an IC5o of about10 ,M, suggesting that the protein kinase activity involved mayrequire calmodulin (Fig. 4B). In the presence of added phos-pholipid, on the other hand, the elevated total Ca-PK activitywas inhibited to a lesser extent by the drug (ICo - 90 uM),suggesting the presence of another species of Ca-PK which maynot require calmodulin but may require phospholipid (Fig. 4B).This contention is supported by the observation that the drugwas without effect on the phospholipid-sensitive Ca-PK purifiedfrom bovine heart (Fig. 4C).The differential effects of calmodulin and phosphatidylserine

on phosphodiesterase and Ca-PK were further demonstrated(Table 5). The calmodulin-deficient phosphodiesterase frombrain was stimulated by Ca2+ in the presence of added cal-

Table 4. Effects of calmodulin and phosphatidylserine on Ca-PK from the Sephadex filtration ofvarious rat tissue extracts

No additions Calmodulin PhosphatidylserineSephadex G-200 Without With Without With Without With

fraction Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+

Exp. ACerebral cortex 0.90 3.05 0.84 3.42 2.45 52.2Lung 0.50 0.86 0.47 0.91 0.74 4.59Heart 0.46 0.52 0.56 0.59 0.77 1.23

Exp. BSpleen 1.97 2.90 2.04 2.87 2.75 37.1

The peak fractions from Sephadex G-200 chromatography of tissue extracts (Fig. 3 or similar ex-periments) were used as the source of Ca-PK. Rat tissues were cerebral cortex (7 ug of protein), lung(12 Mug), heart (11 jig), and spleen (12 Mg). The enzyme was assayed in the presence and absence ofphosphatidylserine (5Mg), calmodulin (2 Mg), or 0.5mM CaCl2. Values for Ca-PK are given as pmol of32p per min for 0.02 ml.

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7042 Biochemistry: Kuo et al.

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Table 5. Differential stimulation of cAMP phosphodiesterase (A-PDE) and Ca-PK activities byphosphatidylserine and calmodulin, and differential inhibition of their activities by trifludperazine

A-PDE, pmol cAMP/min Ca-PK, pmol 32P/minCaC12, Trifluo- With With With WithJAM perazine, MM Basal PS calmodulin Basal PS calmodulin

0 0 595 757 1008 0.27 0.30 0.23500 0 796 872 4575 0.33 1.32 0.26500 10 845 734 3035 0.33 1.32 0.26500 50 779 653 671 0.28 1.27 0.25

The calmodulin-deficient brain A-PDE (28 Mg) from the DEAE-cellulose step (9) and the cardiacCa-PK (0.5 Mg) from the affinity gel step (see Experimental Procedures) were used. The enzymes wereassayed in the presence and absence of phosphatidylserine (PS, 5 Mg); calmodulin (2 Ag), CaCl2 (500MM), and trifluoperazine (10 or 50 AM), as indicated. A much lower amount (0.5 Mg) of calmodulin wasfound to be sufficient to maximally stimulate the Ca2+-dependent A-PDE activity.

modulin, whereas phospholipid was without effect. The enzymewas directly stimulated by calmodulin, presumably due to theendogenous Ca2+. Its activity stimulated by Ca2+-calmodulinwas sensitive to trifluoperazine inhibition, as expected. Incomparison, purified cardiac Ca-PK required phospholipid,not calmodulin, for its activity and, furthermore, the stimulatedenzyme activity was not inhibited by the drug.

DISCUSSIONPhospholipid-sensitive Ca-PK, originally reported by Takai etal. (7), was shown to occur ubiquitously in various tissues andphyla of the animal kingdom. Its activity, which was stimulatedby micromolar concentrations of Ca2+ in the combined pres-ence of phospholipid and diolein (ref. 14 and Fig. 2), requiredphospholipid as a cofactor. Calmodulin was virtually inactive(Tables 4 and 5), in agreement with the findings of Takai et al.(7) on the enzyme from the brain. In spite of the foregoing, lowlevels of protein kinase activity stimulated by Ca2+ in the ab-sence of added phospholipid were noted in the crude extractsof the spleen (Fig. 1), cerebral cortex (Fig. 4B), and variousother tissues (legend to Table 1), and, moreover, this activitywas effectively inhibited by trifluoperazine (Fig. 4B) as forcAMP phosphodiesterase (Table 5 and Fig. 4A). It is not clear,however, whether the phospholipid-insensitive Ca-PK is relatedto the calmodulin-sensitive species of Ca-PK, such as myosinlight chain kinase (for example, see ref. 16).

Lysophosphatidylcholine (17, 18) stimulates brain phos-phodiesterase activity, whereas phosphatidylserine (ref. 17 andTable 5) and phosphatidylinositol (18) do not. Furthermore,it has been shown that phospholipids act as Ca2 -independentactivators of phosphodiesterase (17). These results are to becompared with the observations that both phosphatidylserineand phosphatidylinositol are the most effective phospholipidsin supporting the Ca-PK activity (7). It seems, therefore, thatthe structural determinants in phospholipids for Ca-PK andphosphodiesterase activation are not the same.

Several substrate proteins for the calmodulin-sensitive,Ca2+-dependent protein phosphorylation system have beenreported. These include phosphorylase (19), myosin light chain(16), tryptophan 5-monooxygenase (20), and membrane pro-teins (21, 22). Substrate proteins for the phospholipid-sensitivesystem, on the other hand, are practically unknown. Recently,however, we found that phospholipid was far more effectivethan calmodulin in supporting the Ca2+-stimulated phos-phorylation of endogenous proteins in the cytosol from the brain(23) and various tissues of the rat and guinea pig. The functionalsignificance of the two putative Ca2+-dependent proteinphosphorylation systems and their relationship with the cyclic

nucleotide-dependent systems remain to be defined. Given therapidly growing realization of Ca2+ as a central intracellularmediator, further investigations into these problems seem inorder.

Note Added in Proof. Mori et al. (24) have just reported that chlor-promazine inhibits the brain phospholipid-sensitive Ca-PK by com-petitively inhibiting the interaction of phospholipid with the enzyme.We have now observed that trifluoperazine (100 MM) also inhibitedthe enzyme from several tissues when it was assayed with a low con-centration (3 Mug/0.2 ml) of phosphatidylserine.

This work was supported by Swedish Medical Research Councilgrants 04V-5401-01-503905401, 04X-02080-14, and 04X-04498-06,by Swedish Medical Research Council Visiting Scientist Fellowship(J.F.K.), and by U.S. Public Health Service Grants HL-15696, CA-23391, T-32-GM-07594, and T-32-AM-07298.

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