calmodulin-dependent protein kinase II activity and autophosphorylation

Neurochem. lnt. Vol. 19, No. 3, pp. 271 279, 1991 Printed in Great Britain

0197-0186/91 $3.00+0.00 Pergamon Press plc

GANGLIOSIDE GM1 MODULATION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II ACTIVITY AND AUTOPHOSPHORYLATION FABIO BENFENATI, ~* KJELL FUXE 2 a n d LUIGI F. AGNATI ~ qnstitute of Human Physiology, University of Modena School of Medicine, Via Campi 287, 41100 Modena, Italy 2Department of Histology and Neurobiology, Karolinska lnstitutet, 104 01 Stockholm, Sweden (Received 12 November 1990 ; accepted 18 February 1991}

Abstract--Purified Ca2+/calmodulin-dependent protein kinase I1 (CaM kinase II) is functionally modulated by physiological concentrations of gangliosides. Ganglioside GM 1 has a dual effect on the Ca2+/cal modulin-dependent and independent activity and autophosphorylation of CaM kinase II. In the absence of Ca 2+ and calmodulin (CAM), GMI stimulates the autophosphorylation of the kinase and its activity evaluated using synapsin I as a substrate. This effect was maximal at 5 10 #M GM1, independent of the presence of Ca 2+ and relatively resistant to the inhibition by phenothiazines. The stimulation of the autophosphorylation involved predominantly the ~/~' subunit of the kinase. In contrast, in the presence of Ca 2+/CaM stimulation, ganglioside GM 1 caused a dose-dependent inhibition of the catalytic activity and autophosphorylation to values observed in the presence of gangliosides without Ca 2+/CAM. This inhibition (K~ = 40-45 pM~ was non-competitive with respect to CaM and effective on both subunits of CaM kinase 11. The lack of additive inhibition in the presence of trifluoperazine suggests that GMI interferes with the CaM binding site of CaM kinase II. Gangliosides, by inducing a blockade of the action of CaM and a simultaneous CaM-independent stimulation of CaM kinase II may represent a novel class of biological regulators of CaM-dependent enzymes.

as n e u r o t r a n s m i t t e r s or action potentials are mediated by changes in intracellular calcium concentration. C a l c i u m / c a l m o d u l i n - d e p e n d e n t protein kinase type II ( C a M kinase II) is one o f the most p r o m i n e n t Ca 2+activated protein kinases found in eukaryotes (Schulman, 1988 ; S c h u l m a n a n d Lou, 1989). In the brain it comprises up to 2 % o f total protein a n d is present as soluble a n d m e m b r a n e - a s s o c i a t e d forms in nerve terminals, cell bodies, dendrites a n d postsynaptic densities (Ouimet et al., 1984 ; Kelly et al., 1984). A variety of biochemical a n d physiological studies strongly suggest that this kinase plays a m a j o r functional role in signal t r a n s d u c t i o n at b o t h pre- and post-synaptic levels (Llinas et al., 1985; Willimund et al., 1986; Gorelick et al., 1988; M a l e n k a et al., 1989; Nichols et al., 1990). The brain holoenzyme is an oligomer of 650 k D a molecular weight c o m p o s e d o f variable n u m b e r s o f 50 k D a (~) and 60-58 k D a ( ~ / 3 ' ) subunits which b o t h exhibit catalytic activity ( M c G u i n n e s s et al., 1985). c D N A sequences show t h a t the various s u b u n ' t s are highly h o m o l o g o u s a n d share highly conserved domains. C a M kinase II requires Ca 2+ a n d cal-

P h o s p h o r y l a t i o n is an ubiquitous process o f posttranslational modification o f proteins which plays a regulatory role in a variety o f cellular functions (Nestler a n d G r e e n g a r d , 1984; H u n t e r a n d Cooper, 1985; Edelman et al., 1987). The highest concentrations of protein kinases a n d of their substrates are found in the nervous system, where they are implicated in the regulation of a variety of processes including neurotransmitter release, receptor-mediated responses, nerve growth a n d differentiation, short- a n d long-term memory. Several intracellular effects o f first messengers such

* Author to whom all correspondence should be addressed. Abbreviations: ATP, adenosine 5"-triphosphate, BSA, bov-

ine serum albumin; CaM, calmodulin; CaM kinase ll, calcium/calmodulin-dependent protein kinase type il; cAMP, cyclic AMP; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(,8-aminoethyl ether) N,N,N',N'-tetraacetic acid; kDa, kilodalton(s); IC50, half-maximal inhibitory concentration: PMSF, phenylmethylsulfonyl fluoride; SDS PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, standard error of the mean : TFP, trifluoperazine. 271

272

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modulin 1CAM) for initial activation. This event triggets an intramolecular a u t o p h o s p h o r y l a t i o n which temporally precedes the p h o s p h o r y l a t i o n of exogenous substrates and converts the enzyme in a C a ~ ' ~ C a M - i n d e p e n d e n t form, prolonging the biological effects of C a M kinase 11 beyond the transient elevation of intracellular calcium (Shields e t al., 1984 : Lai et al.. 1986: Miller and Kennedy, 1986). Gangliosides. a class of sialic acid-containing glycosphingolipids, are found in very high concentrations in the nervous system (Ledeen, 1985) where they consist of a large m e m b r a n e - b o u n d pool and of a smaller cytosolic pool ( S o n n i n o et a/., 1979). Biological effects ofgangliosides in the modulation of synaptic transmission, m e m b r a n e viscosity, receptor recognition processes, neuritogenesis as well as neuronal trophism and regeneration have been reported in the past few years (Ledeen, 1985: Skaper ~'t a/.. 1989). Recent studies have reported that gangliosides arc involved in the m o d u l a t i o n of protein phosphorylation processes in the nervous tissue. Several protein kinases in brain are modulated by physiological concentrations of gangliosides. Gangliosides inhibitcd the activity of protein kinasc C ( G o l d e n r i n g cl a/.. 1985: Kim el al., 1986: Cimino c t a l . . 1987: Kreulter c't a/.. 1987) and its glutamate-induced translocation from the cytosol to the plasma m e m b r a n e (Vaccarino {'; al.. 1987: F a v a r o n ~'t a/.. 1988 : M a n e v c t a l . , 1989). Gangliosides had a similar inhibitory effect on the catalytic activity and autophosphorylation of the c A M P - d e p e n d e n t protein kinase in the rabbit sciatic nerve, an effect which is further potentiated by a simultaneous stimulation of the cyclic nucleotide phosphodiesterase (Yates t'/ al., 1989). Moreover, protein kinase activities which arc selectively activated and inhibited by physiological conten{rations of gangliosides have been identified in brain tissue and partially purified (Chan. 1987, 1988). In previous papers from our and other groups it was d e m o n s t r a t e d that gangliosides have marked modulatory effects on Cae ~/CaM-stimulated protein p h o s p h o r y l a t i o n in synaptosomal preparations. In particular, GM1 had a biphasic eff'ect (stimulation at Io~. doses and inhibition at higher doses) of p h o s p h a t e incorporation in b a n d s comigrating with the ~- and [¢~fi'-subunits of (7aM kinase 11 and with C a M kinasc 11 substrates in thc presence of Ca ~~ and C a M (Goldenring el al.. 1985, Cimino el al.. 1987). This effect ~.as accompanicd by a stimulation of a C a " C a M independent p h o s p h o r y l a t i o n of a b a n d comigrating with the fl/fl'-subunit of C a M kinase II (Cimino eta/., 1987).

In the present work wc have investigated the efffects of G M I on the catalytic activity and on the autop h o s p h o r y l a t i o n of purified C a M kinasc 11 with the purpose of confirming the data obtained with synaptosomal preparations and of gctting new insight into the molecular mechanism of the ganglioside-induccd m o d u l a t i o n of this kinase. The results d e m o n s t r a t e thai GM1 stimulates the catalytic activity and the a u t o p h o s p h o r y l a t i o n of purified C a M kinasc I1 in the absence of Ca ~' C a M , whereas il inhibits Ihc Ca " ~ / C a M - s t i m u l a t c d ( ' a M kinasc 11 activity and a u t o p h o s p h o r y l a t i o n through a non-competitive inhibition of C a M binding to thc enzymc.

EXPERIMENTAl,

IJROCEI)U RES

A:latcria:~

[7-':P]ATP (3000 ('{mined was from New England Nuclear. P81 phosphocellulose paper was from Whalman. Calmodulin was prepared from bovine brain by tire method of Grand eta/. (1979). Synapsin I was purified from bovine brain by lhe detergent extraction procedure described b> Bfihler and Greengard (1987). CaM kinasc 11 was purilied from ral forebrain as described by McGuinness el al. (1985) through steps of DEAE cellulose chromatography, Seph acryl S-400 gel liltration and CaM-sepharo~ affinity chromalography. The purified enzyme {average specitic activity: 2 4 prnol ATP/min/mg prolein: average protein con cenlration:0.1 0.4mgml) was stored at 20 C i n a buff'er solution containing 25 mM Tris ltCI (Ph 7.5), 50%, glycerol i~ v). 0.5 mM EDTA, 10 mM 2-n'lercaploeihanol. ().1 mM PMSF. I t t g m l leupcptin without any apparent loss of aclivily. Pure ganglioside GMI Idisodium salt) was kindb supplied by Dr G. Toffano (Fidia Research Laboratories, Abano Terme, ltaly). Triffuoperazine hydrochloride was obtained from Research Biochemical Inc. All other chemicals (reagent grade) were obtained from standard commercial suppliers. •L~ay O! ( ' a M kinase 11 ~ala@tic acli~'izl

(.'aM kmase II activity was assayed by phosphorylation of purilied synapsin I as previously described (Kennedy et aL, 1983: McGuinness ~,t al., 1985). The reaction mixture (I00 /*1) contained 50 mM Tris 1t(71, pH 7.6, 10 mM MgCI-. 5 m M 2-mercaptoethanoh 5 pg BSA. 3 Hg Ca M, 10 jig synapsin 1, various amounts of gangliosidc GMI {dissolved in water) and either (t.4 mM EGTA or 0.4 mM EGTA/0.9 mM CaCI ,. Io Ihis mixture, the kinase {previously diluted into 5 mM Tris HCI pH 7.6.5 mM 2-mercaploethanol) was added and the samples preincubated at 30 (7 for 60 s. The reaction was initiated by the addition of [.,-~-'P]ATP (50 ItM. ca I 2 itCi:sample). All reactions were carried out for 15 s in order to ensure measurement of initial rates and were tcrnfinated by the addition of 10 pl of a mixture of 200 mM EDTA and 10 mM ATP (pH 7.0). Aliquots of the samples (55 t d) were spoiled onto phosphocellulose paper squares (0.75 × 1.5 cm), folk)wing the procedure of Wilt and Roskoski (1975). Thc squares ~ere washed with running deionized water for 20 rain, air dried and counted lk~r radioactivity by liquid scintillation spectrometry. ('aM kinase activity was linear wilh

273

Modulation of CaM kinase II by ganglioside GM 1 respect to enzyme concentration and time. The required enzyme dilution for the assay ranged between 200 and 1000fold. All samples were run in quadruplicate and the experiments repeated at least three times.

Measurement of CaM kinase H autophosphoD,lation The autophosphorylation of CaM kinase lI was assayed as previously described (Kennedy et al., 1983; McGuinness et al., 1985). CaM kinase Ii was diluted into 5 mM Tris HC1 pH 7.6, 5 mM 2-mercaptoethanol, 1 mg/ml BSA, 0.1 mM PMSF and 10/~g/ml leupeptin. Aliquots of the kinase (0.2 2.0/~g) were added to an assay mixture (final volume 100/~1) containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCI> 5 mM 2-mercaptoethanol, various amounts of ganglioside GMI (dissolved in water) and 0.4 mM EGTA in the absence or presence of 0.9 mM CaCI2 and 3.0/~g CaM, unless otherwise indicated. The reaction was initiated by the addition of [,,-32p]ATP (5 50 #M, ca I 2/JCi/sample) and was allowed to continue for 60 s at 0 C . The reaction was terminated by the addition of 50/d of SDS "stop" solution (Laemmli, 1970) and the mixture was boiled for 2 min and subjected to SDS polyacrylamide gel electrophoresis (SDS PAGE). Labeled fl/#' and ~ subunits of the kinase were localized by autoradiography of the dried gel the bands were excised, and the radioactivity was quantitated by liquid scintillation counting. Miscellaneous techniques and data analysis SDS PAGE was performed according to Laemmli (1970) using 9% acrylamide in the separating gel. The following proteins were used as molecular mass standards in SDS PAGE: phosphorylase b (94 kDa), bovine serum albumin (BSA; 67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and :¢-lactalbumin (14 kDa). The gels were stained with Coomassie Brilliant Blue R-250, destained, dried and subjected to autoradiography. Saturation and inhibition curves were analyzed using the computer program RECEPT (1984) performing both linear transformations and non-linear fitting analysis. Protein was determined according to Lowry et al. (1951) using BSA as a standard. Statistical analysis of the dos~response curves was carried out by means of one-way analysis of variance for repeated measurements.

RESULTS

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Fig. 1. Effects ofganglioside G M t on the activity of purified CaM kinase I1 evaluated as phosphorytation of purified synapsin I. The assay was carried out in the presence of EGTA (basal activity, ©) or of Ca2+/CaM (stimulated activity, 0 ) as described in Experimental Procedures. Phosphate incorporation into synapsin 1 under basal and stimulated conditions is expressed in percent of the respective value in the absence of gangliosides (0.06 /~mol ATP/min/mg enzyme and 3.6 #mol ATP/min/mg enzyme under basal and stimulated conditions, respectively). The points in the plot represent the mean (_+ SEM) of four separate experiments.

As shown in Fig. l, G M l exhibited a dose-dependent inhibition of the C a 2 + / C a M - s t i m u l a t e d kinase activity which started at 20 p M a n d reached a n almost complete inhibition of the kinase activity at 200 # M ( I C 5 0 = 4 5 pM). In contrast, G M I stimulated a C a 2 + / C a M - i n d e p e n d e n t activity o f the kinase in the absence o f a previous activation with C a 2 + / C a M which would have induced the p e r m a n e n t activation of the enzyme. This stimulation was already present at 2.5/~M G M 1 (the lowest c o n c e n t r a t i o n tested) and slowly declined at higher ganglioside c o n c e n t r a t i o n s (Fig. 1). In absolute terms the ganglioside-induced stimulation accounted only for the 4 - 5 % of the C a 2 + / C a M - s t i m u l a t e d activity o f C a M kinase II.

Effects o/ganglioside G M I on C a M kinase II activity

Effects o f G M I on C a M k&ase autophosphorvlation

To test the effect of G M 1 on C a M kinase catalytic activity, a standardized C a M kinase II assay system with synapsin I as a p h o s p h a t e acceptor was used. Synapsin I is by far the best substrate k n o w n for the kinase (Kin = 0.4 # M ) a n d is p h o s p h o r y l a t e d o n two sites (Ser-566 a n d Ser-603) present in the C O O H terminal region o f the molecule (De Camilli et al., 1990). T h e assay was carried o u t in the absence or presence o f C a 2 + / C a M to analyze b o t h the Ca2+/ C a M - d e p e n d e n t a n d the C a 2 + / C a M - i n d e p e n d e n t enzyme activities.

The a u t o p h o s p h o r y l a t i o n o f C a M kinase subunits is a process o f p a r a m o u n t i m p o r t a n c e in the regulation of its activity within the cell. The possibility that gangliosides affect this process was analyzed in the absence or presence o f Ca 2+ a n d C a M . Figure 2 shows the i n c o r p o r a t i o n o f p h o s p h a t e into the fl/[t' and ~ subunits of the kinase at various c o n c e n t r a t i o n s of ganglioside G M I (range: 2.5-250 /JM). In the absence o f ganglioside, a m a r k e d a u t o p h o s p h o r y l ation of b o t h subunits was o b t a i n e d only w h e n Ca 2+ / C a M were present. The presence of gangliosides

FABIO BENFENAT!et al.

274 GM

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had different effects on the Ca 2./CaM-dependent and independent kinase autophosphorylation. G M I induced a Ca2+/CaM-independent stimulation of the autophosphorylation of CaM kinase I1 which peaked at intermediate G M I concentrations and slowly declined at higher doses. This effect seemed to be rather selective for the fl/fl' subunit and affected the incorporation of phosphate into the :~ subunit only to a lesser extent (Fig. 2). In contrast, in the presence of the Ca3+/CaM, GM1 exhibited a dose-dependent inhibition of the autophosphorylation of both subunits at concentrations ranging between 5 and 250/~M. The quantitative analysis of phosphate incorporation into the fl/fl' and c{ subunits of the kinase as a function of the ganglioside concentration is reported in Fig. 3. As mentioned above, in the absence of Ca 2+ (Fig. 3, upper panel), GM1 stimulated phosphate incorporation into the fl/fl" subunit at all concentration tested. The peak effect (over 150% increase with respect to the basal values; ca 20 25% of the Ca2+/CaM-stimulated autophosphorylation) was between 5 and 25 #M, but phosphate incorporation stayed elevated up to 300 #M G M 1 (the highest tested dose). A similar pattern of stimulation, but a slighter effect, was observed for the c~subunit. In the presence of Ca ~+ but without CaM (Fig. 3, middle panel), the stimulating effect of G M I on the fi/fi' subunit phosphorylation was less intense than that observed in the absence of Ca 2+, and the effect on the ~ subunit phosphorylation was virtually absent. In the presence of Ca-'+/CaM (Fig. 3, lower panel), GM1 caused a marked inhibition of phosphate incorporation into both subunits of the kinase with a dose-dependent fashion. The amount of phosphate bound to the fi/fi" subunit at the highest dose of G M I in the presence of Ca2+/CaM approximately corresponded to that incorporated at the same G M I concentration in a Ca2+/CaM-independent fashion. The possibility that G M I antagonizes the activation of the kinase by Ca2+/CaM was investigated

by performing a kinetic analysis at various CaM concentrations (ranging between 0.02 to 2.0 #M) in the presence of three concentrations of GM1 (2.5, 12.5 and 62.5 ffM). The results of this experiment are reported in Fig. 4 (autoradiography of the phosphorylated kinase subunits) and Fig. 5 (doublereciprocal plots). The inhibitory effect of GM1 on phosphate incorporation into both fi fl' and ~ subunits was not counteracted by increasing the concentration ofsubstrate (CAM) (Fig. 4). When the data were analyzed by double-reciprocal (Lineweaver Burk) plots (Fig. 5), the computed lines of best fit performed I'or both subunits at various ganglioside concentrations intersected the X-axis indicating that the mechanism of inhibition of CaM kinase II autophosphorylation is not competitive with respect to CaM. The analysis of the same data using a Dixon plot (not shown) revealed a K~ value for G M I of 49.6 #M (fi/fi' subunit) and 44.8 ItM (c~ subunit). The demonstration that the inhibition of CaM kinase II by G M I is non-competitive with CaM, leaves open the question whether the ganglioside interacts with the CaM binding site in the regulatory domain of the kinase or with another site. To elucidate this aspect, the inhibitory effect of TFP, a competitive CaM antagonisk oil CaM kinase I1 autophosphorylation was analyzed in the presence or absence of G M I (Fig. 6). T F P induced a dose-dependent inhibition of the Ca-~+/CaM-stimulated autophosphorylation of both kinase subunits with an ICs{~ between I and 10 t~M (at 0.3 t~M CAM). However, when the C a ' ~ ! C a M - s t i m u l a t e d autophosphorylation of both kinase subunits was inhibited by the presence of 62.5 ttM GM1, no further inhibition by T F P was apparent up to a concentration o f l 0 1001~M, suggesting that the target o f t h e GM1induced inhibition may bc the CaM binding site in the regulatory domain of the kinase. Moreover. the Ca: ~/CaM-independent stimulation of the fl/fl' subunil autophosphorylation by GM 1 was also nnaffected by T F P concentrations tip to 100 ttM.

Modulation of CaM kinase II by ganglioside GM 1 300

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Fig. 3. Quantitative evaluation of the effects of ganglioside GMI on the autophosphorylation of purified CaM kinase II in the presence of EGTA (upper panel), Ca 2+ (middle panel) or Ca 2+ and CaM (lower panel). The autophosphorylation of CaM kinase II was assayed under the conditions described in Experimental Procedures, by counting the amount of phosphate incorporated into the ~ (O) and fl/fl' (O) subunits separated by SDS PAGE and the values expressed in percent of the respective value in the absence of gangliosides. The points in the plot represent the mean (+ SEM) of four separate experiments. The absolute values (mean + SEM) in the absence of ganglioside were : fl/fl'-subunit, 165 + 18 (EGTA), 186_+22 (Ca -'+) and 2250+90 (Ca2+/CaM) cpm [3:p]orthophosphate/min/l~g enzyme; ~-subunit, 145_+32 (EGTA), 163 _+28 (Ca 2+) and 2050 + 105 (Ca2+/CaM) cpm [nP]orthophosphate/min//~g enzyme.

DISCUSSION

Gangliosides are sialic acid-containing glycosphingolipids which consist of a hydrophobic domain (the ceramide) and of a polar, negatively charged domain (the oligosaccharide). They are particularly a b u n d a n t in neuronal cells, where they are distributed over a major portion of the neuronal surface and reach high concentrations at the synapse (Leeden, 1985). The ganglioside GM1 is one of the

275

most a b u n d a n t gangliosides endogenously present in the brain and its distribution mainly involves synaptic contacts in most brain areas (Fuxe et al., 1989). Gangliosides are involved in a variety of cellular functions (Leeden, 1985 ; Skaper et al., 1989). Protein phosphorylation, which represents a process of param o u n t importance in the transduction of extracellular signals and in the regulation of metabolic activity, is known to be modulated by physiological concentrations of gangliosides. Since both gangliosides and phosphorylation systems are particularly abundant in neurons, such an interaction may have an important functional role. Gangliosides have been shown to have modulatory effects on several brain protein kinases including CaM kinase II. Since CaM kinase II is a widespread enzyme which has been involved in a variety of brain functions such as neurotransmitter release and signal transduction at pre- and postsynaptic level (Llinas et al., 1985 ; Willimund et al., 1986 ; Gorelick et al., 1988 ; Malenka et al., 1989 ; Nichols et al., 1990), we decided to investigate the molecular interaction between gangliosides and CaM kinase II by using purified kinase and specific assays for its catalytic activity and autophosphorylation reaction. Ganglioside G M I had complex effects on purified CaM kinase II which can be summarized as follows : - - s t i m u l a t i o n of the Ca2+/CaM-independent autophosphorylation (20-25% of the Ca2÷/ CaM-stimulated autophosphorylation), an effect which mostly involves the fl/fl' subunit, is independent of the presence of Ca 2÷, is maximal at 5 10/tM G M 1 and relatively insensitive to T F P ; - - s t i m u l a t i o n of the Ca2+/CaM-independent catalytic activity (4-5% of the fully stimulated activity) ; --dose-dependent inhibition of the Ca2÷/CaM dependent autophosphorylation and catalytic activity of the kinase. This effect (Ka = 40-50 /~M GM1) affecting both subunits cannot be overcome by increasing the concentration of CaM nor potentiated by TFP. The above-mentioned effects cannot be simply ascribed to an interaction of the ganglioside with CaM or synapsin I. In fact, the binding of G M 1 to CaM is unlikely because of the net negative charge of both molecules and an interaction of G M I with synapsin I would not explain the effects on the kinase autophosphorylation as well as the opposite effects observed on the kinase activity under basal and stimulated conditions. Rather, the present results suggest

276

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Fig. 5. Kinetic analysis of the inhibition of C a M kinase II autophosphorylation by ganglioside G M I . Shown are double-reciprocal plots of the autophosphorylation of the fi/fl' (top panel) and ~ (bottom panel) subunits of C a M kinase 11 (velocity, [~-P]orthophosphate cpm incorporated per rain per sample) at various concentrations of substrate (CAM. /~M) in the presence of 0 (O), 2.5 (O), 12.5 ( A ) or 62.5 ( ~ ) I~M G M 1. Each assay point represents the average of triplicate determinations.

50 ',M)

Fig. 6. Inhibition of the autophosphorylatlon of CaM kinasc It by trifluoperazine. The autophosphorylation of ('aM kinase II was assayed by counting the a m o u n t of phosphate incorporated into the fi,,fl" (top panel) and :~ (bottom panel) subunits under the following conditions: EGTA (O), CaZ~/CaM (O), E G T A + 6 2 . 5 #M G M I (,~) and Ca -~, CAM+62.5 liM GM1 (A). ]'he values are expressed in percent of the value observed in the presence of Ca2 '/CAM and in lhe absence of gangliosides. The points in the plot repre,~nt the mean of three separate experiments. For further details, see Experimental Procedures and legend to Fig. 3.

Modulation of CaM kinase II by ganglioside GM 1 that GM1 may bind to the regulatory site of CaM kinase ii through hydrophilic and hydrophobic interactions involving the sialic acid and the ceramide moieties. When the kinase is not activated by Ca-'+/ CaM (resting state), a partial agonist activity of the ganglioside may bring about the Ca-'+/CaM-independent stimulation of kinase activity and autophosphorylation. However, when Ca-'+/CaM is present and fully activates CaM kinase II, the ganglioside displaces CaM from the regulatory site of the kinase in a non-competitive fashion, inducing inhibition of kinase activity and autophosphorylation. The absence of additive inhibitory effects between TFP (a competitive inhibitor of CaM) and GM1 in the presence of CaM suggests that the ganglioside inhibits CaM kinase I| by preventing the binding of CaM to the regulatory domain of the kinase. GM 1 can act as an atypical CaM antagonist. Classical CaM antagonists such as phenothiazines share common structural features represented by a hydrophobic moiety and a positively charged amino group and are able to competitively inhibit the actions of CaM by binding directly it in a Ca-'+-dependent manner (Wrenn et al.. 1981 : Prozialeck et al., 1982). Sphingosine, a long chain amino alcohol structural component of sphingolipids including gangliosides, has been found to be a competitive CaM antagonist, although structurally distinct from classical CaM antagonists (Bennett Jefferson and Schulman, 1988). However it shares with the latter compounds a hydrophobic character and a basic residue. In contrast, gangliosides, containing a hydrophobic moiety and a negative charged oligosaccharide may be able to partially mimic Ca-'+-activated CaM and to interact with the CaM site on the kinase. In the absence of CaM they may exhibit some degree of CaM-mimetic activity, inducing a Ca-'+/CaM-independent activation of CaM kinase II, whereas in the presence of CaM they act as non-competitive CaM antagonists. The data reported here are in agreement with the ganglioside-induced inhibition of CaM kinase |I autophosphorylation and catalytic activity in the presence of Ca -'+ and CaM and with the existence of a ganglioside-stimulated, Ca-'+/CaM-independent autophosphorylation of the [3/[~' subunit of CaM kinase II observed in synaptosomal preparations (Cimino et al., 1987). The Ca2+/CaM-independent autophosphorylation of CaM kinase II observed in the presence of gangliosides may involve, inter alia, Thr-287 of the [~/~' subunit, the major autophosphorylation site responsible for the permanent activation of the kinase (Lai

277

et al., 1987 ; Thiel et al., 1988 ; Fong et al., 1989), since the Ca-'+/CaM-independent autophosphorylation is accompanied by an increase of the kinase activity over basal values. It is still unclear why gangliosides are more active on the [~/[1" subunit rather than on the ~ subunit in the absence of Ca-'+/CaM. This phenomenon, which can explain the only partial activation of CaM kinase II in these conditions, appears to be functionally distinct from the inhibition of the Ca2+/CaM-activated kinase, and could be due to structural differences between the ~ and the 3/[1' subunits (Schulman, 1988 ; Schulman and Lou, 1989). Despite the high homology in the primary sequences of the two subunits, the major difference consists in two segments of 24 and 39 residues which are missing in the c~ subunit and which are located just after the end of the regulatory domain in the []/[~" subunit (Bennett and Kennedy, 1987; Lin et al., 1987). These amino acid segments may be involved in the selective activation of the [~/[~' subunit by GMI in the absence of Ca-'+/CaM. During the preparation of this work, a paper by Fukunaga et al. (1990) reported substantially similar effects of brain gangliosides on CaM kinase II. These Authors found, however, that the ganglioside-induced stimulation of CaM kinase II is sensitive to TFP and that the stimulation of CaM kinase II autophosphorylation involves both the ~ and 3/3' subunits. Moreover, the kinetic analysis of CaM antagonism by gangliosides was carried out on the phosphorylation of some CaM kinase |l substrates, but not on the kinase autophosphorylation and the results did not reveal any simple mechanism for the ganglioside-induced inhibition. In conclusion, gangliosides seem to act as biological regulators of CaM kinase II activity : they allow moderate levels of activity in resting conditions and keep under control the Ca2+/CaM-stimulated kinase by inhibiting its activity and autophosphorylation. By their involvement in the modulation of the activation of the major protein kinase activities in brain, gangliosides seem to act as endogenous regulators of signal transduction processes.

Acknowledgements--Thiswork has been supported by grants from the ltalian Research Council (CNR), the Swedish Medical Research Council and the Italian Ministry of University (MURST 60% and 40%). This work was presented at a scientificmeeting of the Fidia Research Laboratories (Abano Terme, Padova, Italy) on 24 November, 1989. REFERENCES B/ihler M. and Greengard P. (1987) Synapsin I bundles F-

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