Journal of h'eurochernlstry Raven P,Ltd New York

.

0 1990 International Society for Neurochernislry

Regulation of Ca*+/Calmodulin-DependentProtein Kinase I1 by Brain Gangliosides Koji Fukunaga, *Eishichi Miyamoto, and Thomas R. Soderling Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee, U.S.A.;and *Department of Pharmacology, Kumamoto University Medical School. Kumamoto. Japan

Abstract: Purified rat brain Caz+/calmodulindependent

protein kinase I1 (CaM-kinase 11) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT I b was the most potent, giving half-maximal activation at 25 p M . Gangliosides G D l a and GMI also gave activation, but asialo-GM I was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase I1 on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase I1 was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 p M ) of GTlb, GDla, and GM 1 also inhibited CaM-kinase II activity. This inhibition appears to be substratedirected, as the extent of inhibition is very dependent on the substrate used. The molecular

mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 28 1-309). which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase 11. Using purified brain CaM-kinase I1 in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 p M ) . G T l b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that G T l b can partially mimic the effects of CaZt/CaM on native CaM-kinase I1 and on peptide CaMK 281-309. Key Words: Protein kinase-Protein phosphorylation-Calcium-Calmodulin-Gangliosides. Fukunaga K. et al. Regulation of Ca'+/calmodulin-dependent protein kinase I1 by brain gangliosides. J. Neurochem. 54, 102-109 (1990).

Gangliosides, which are sialic acid-containing glycosphingolipids, are found in high concentrations in neural cells. Previous studies have reported that gangliosides may be involved in the modulation of cell growth, determination of antigenic sites, and recognition of various bioeffectors such as bacterial toxins and hormones (Hakomori, 1981, 1984). In the nervous system, gangliosides have been shown to modulate receptor functions and release of neurotransmitters (Cumar et al., 1978; Dreyfus et al., 1984; Hollmann and Seifert, 1986) and promote cell proliferation and/or neurite formation (Tsuji et al., 1983; Katoh-Semba et a]., 1984; Ledeen, 1984). Although the exact mechanisms by which gangliosides may influence these diverse systems are not defined, recent evidence suggests the involvement of protein kinases, including tyrosine kinases associated with epidermal growth factor and

platelet derived growth factor receptors (Bremer et al., 1984; Hanai et al., 1988a,b), protein kinase C (Kreutter et al., 1987), and undefined protein kinases (Tsuji et al., 1985; Chan, 1987a, 1988), some of which are activated by Ca2+/calmodulin (CaM). Ca*+/calmodulin-dependent protein kinase I1 (CaMkinase 11) is a multifunctional enzyme which exists in particularly high concentration in the central nervous system. It is thought to be involved in Ca'+-dependent neuronal processes such as the biosynthesis and exocytosis of neurotransmitters, regulation of metabolic reactions, and perhaps alterations of synaptic efficacy (reviewed in Schulman, 1988; Colbran et al., 1989). Immunohistochemical studies have shown localization of CaM-kinase I1 at the synaptic junction, especially the postsynaptic density where it comprises approximately 30%of the protein (Quimet et al., 1984; Erondu

Received January I I, 1989; revised manuscript received May 3, 1989: accepted May 24. 1989. Addrss comswndence and reprint requests to Dr. T. R.Soderling at Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 372324295, U S A .

Abbreviations used: CaM, calmodulin; CaM-kinase 11, Ca2+/calmodulindependent protein kinase 11; kDa, kilodalton; MAP-2, microtubule-associated protein 2; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

~

I02

~~~

REGULATION OF CaM-KINASE II BY GANGLIOSIDES and Kennedy, 1985; Fukunaga et al., 1988). Recently, Goldenring et al. (1985) and Cimino et al. (1987) reported that a Ca’+/CaM-dependent protein kinase was regulated by gangliosides. This study was initiated t o determine whether this kinase was CaM-kinase I1 and t o investigate the mechanism of regulation by gangliosides.

MATERIALS AND METHODS Materials TrisialogangliosideGT 1b, disialoganglioside GD 1a, monosialoganglioside GM 1, and a bovine brain ganglioside mixture containing these three gangliosidesplus GDl b were obtained from Biosynth AG. Asialo-GM I , cerebroside, sialic acid, and casein were from Sigma; histone H,from Boehringer Mannheim; phosphocellulose PI 1 paper from Whatman; [y”PIATP from New England Nuclear (Dupont); and protein A-Sepharose CL4B from Pharmacia. Syntide-2 and the peptide CaMK 28 1-309 were synthesized as described previously (Basset al., 1987;Colbran et al., 1988). Synapsin I was purified from rat brain (Schiebler et al., 1986);glycogen synthase from rabbit muscle (Schworer and Soderling, 1983); microtubuleassociated protein 2 (MAP-2) and tau factor from bovine brain (Yamamoto et al., 1985); myosin light chain from chicken gizzard (Miyamoto et al., 1981); and CaM (Gopalakrishna and Anderson, 1982)and myelin basic protein from bovine brain (Deibler et al., 1972). The CaM-kinase 11, purified from rat brain (Hashimoto et al., 1987), was kindly provided by Dr. Roger Colbran (Vanderbilt University). The completely CaZf-independent form of CaM-kinase I1 was prepared by partial proteolysis with chymotrypsin as described (Colbran et al., 1988). Affinity purified polyclonal antibodies against brain CaM-kinase I1 were prepared as described (Fukunaga et al., 1988). CaMdeficient synaptic membranes from rat forebrain were prepared as previously described (Fukunaga et al., 1984).

Methods CaM-kinase II assay. The standard assay for CaM-kinase

103

terminated after 30 sec by addition of sodium dodecyl sulfate (SDS) sample buffer (Laemmli, 1970). After boiling for 2 min, the samples were subjected to SDS/polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide and then autoradiography. Standard proteins included carbonic anhydrase [29 kilodalton (kDa)], ovalbumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (93 kDa), and myosin heavy chain from chicken gizzard (200 kDa). Immunoprecipitation. Four hundred micrograms of synaptic membrane was phosphorylated for 1 min in 200 *I, as described above. The sample was centrifuged at 10,OOO g for I min, and the pelleted membrane solubilized in buffer containing 50 mM Tris (pH 7.9, 0.5 M NaCI, 0.5% Triton X100, 0.5% deoxycholate, I mM EDTA, I mM NasV04, 30 mM sodium pyrophosphate, 50 mM NaF, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride,.5 mg/L leupeptin, and 20 mg/ L soybean trypsin inhibitor. After centrifugation at 10,OOO g for 5 min, the supernatant was incubated with 25 pg of the polyclonal antibody for 20 min at 4°C and then with 50 pl of a 50% suspension of protein A-Sepharose CL4B for an additional 20 min. The precipitate obtained by centrifugation was washed four times using the solubilization buffer. The final pellet was resuspended in SDS sample buffer, boiled, and subjected to SDS/PAGE and then autoradiography. Other methods. SDSJPAGE was performed by the method of Laemmli ( 1970). Identification of 32P-labeledamino acids in proteins was determined by partial acid hydrolysis (6 M HCI for 2 h at 100°C) and high voltage paper electrophoresis (2,500 V for 1 h). Protein was determined by the method of Bradford (1976) using bovine serum albumin as standard.

RESULTS Stimulation of brain CaM-kinase I1 by gangliosides T h e effects of gangliosides on purified brain CaMkinase I1 were investigated using either synapsin I or the synthetic peptide syntide-2 as substrate. In the presence of EGTA, CaM-kinase I1 was stimulated by gangliosides in a dose-dependent manner (Fig. 1). Halfmaximal stimulation was observed at -25 pM for

11, based on the phosphorylation of synthetic peptide syntide-

2 (analogous to site 2 in glycogen synthase; Hashimoto and Soderling, 1987), contained 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mMEGTA, 0.1 mM [y-”P]ATP (3,000-5,OOO cpm/pmol), 1 mg/ml bovine serum albumin, 20 p M syntide-2, or 1 p M synapsin I (or other substrates as indicated) and various concentrations of gangliosides in a total volume of 20 pl. Assays for total kinase activity included 0.2 mM CaCI2 and 0.6 pM CaM, whereas assays for Ca2+independent activity contained 1 mM EGTA. The assays were preincubated at 30°C for 2 min, the reaction initiated by addition of kinase (4 nM subunit), and, after a 2-min assay, a 10-pl aliquot was spotted on phosphocellulose paper squares and processed as described (Roskoski, 1983). Protein phosphorylation in synaptic membranes. For endogenous protein phosphorylation, the reaction mixture contained, in a volume of 80 pl, 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 0.1 mM [y-3ZP]ATP(3,0005,000 cpm/pmol), 80 pg of synaptic membrane protein, and either 1 mM EGTA (basal phosphorylation), 1 mM EGTA plus 125 p M GT lb (gangliosidedependentphosphorylation), or 0.2 mM CaC12and 0.6 pM CaM (CaMdependent phosphorylation). Following a I-min preincubation at 30°C the reaction was initiated by addition of the [T-~~P]ATP and was

, 50

100

150

200

250

0

GANGLIOSIDES (pM) FIG. 1. Effects of gangliosides on CaM-kinase II activity. The purified CaM-kinase l l (4 nM subunit) was assayed as in Materials and Methods in the presence of Ca*+/CaMplus GT1b (O), EGTA plus GTl b (0) EGTA . plus GDla (o), or EGTA plus GM1 (A) using 20 ,.IM syntide-2 a s substrate. Proteolyzed CaM-kinase II (5 nM subunit) was assayed in the presence of EGTA plus GT1b (m). The results represent the means for two separate experiments.

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K. FL'KUNAG.4 ET AL.

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GT I b and at 50 p.M for GDI a and GM 1. Polysialogangliosides such asCT I b and G D l a were more potent activators than GM I . whereas asialo-GM I and cerebroside. which contain the ceramide hydrophobic portion and sugar residues, and sialic acid were without effect (Table I). CaM-kinase I1 activity assayed in the presence of CaZt and CaM was partially inhibited at higher concentrations of GTI b (Fig. I ) , as well as at 250 jdf cerebroside (not shown), suggesting that the inhibition may be related to the hydrophobic ceramide portion of the ganglioside. The initial rate of syntide-2 phosphorylation by CaM-kinase I1 was linear in the presence of EGTA and GTlb (not shown), suggesting a direct interaction of the ganglioside with the kinase. This conclusion was tested by using a proteolytic fragment of CaM-kinase 11 that does not bind Ca"/CaM and is fully active (Colbran et al.. 1988). Low concentrations of G T l b had no effect on this form of CaM-kinase 11, but concentrations above I 0 0 p M gave inhibition equal to that observed with native kinase. The stimulation of native CaM-kinase 11 by 125 p M G T l b was completely reversed by trifluoperazine (Ki = 30 p M , not shown). Kinetic analysis (Table 2) revealed that the K,,, values for syntide-2 and synapsin I were about the same in the presence of EGTA/GTlb or Ca2+/CaM,but the VI, was only about f as great in the presence of EGTA/GTI b as in the presence of Ca*'/CaM.

TABLE 2. Kinetic analysis of CaM-kinase I1 in the presence of gangliosides or Caz+/CuM ~~~~~

Assay conditions EGTA

CaZ++ CaM

+ GT I b '-

K,

vmax

Substrates

(pM)

(rmol/ min/mg)

Syntide-2 Svnawinl

7.5 2 0.6 1.3r0.3

13.9 t 0.8 5.1r0.5

KGl

(pM) 10.6 t 0.5

1.420.3

(rmol/

min/mg) 40.7 2 2.9 15.1?0.8

Purified CaM-kinase 11 was assayed using various concentrations of syntide-2 (2-14 p M ) or synapsin I (0.3-1.2 aM) in the presence of EGTA ( I m M )and G T l b (I25 pM)or Ca2' and CaM (0.6 p M ) , Kinetic constants were determined by least squares fitting ofthe data 10 the Lineweaver-Burk equation. Values are expressed as the means 2 SEM for three separate experiments.

f

-

Autophosphorylation of CaM-kinase 11 The autophosphorylation of the 50- and 60-kDa subunits ofCaM-kinase I1 was also stimulated by GTI b in the presence of EGTA to about 22 and 35%, respectively, of the levels obtained in the presence of CaZ+/CaM.Phosphoamino acid analyses of the 32Plabeled kinase subunits, after separation by gel electrophoresis in the presence of SDS, yielded only phosphoserine in the 50-kDa subunit, with predominantly phosphoserine but also some phosphothreonine in the 60-kDa subunit (Fig. 2). This finding contrasts with autophosphorylation in the presence of Ca2+/CaM,

A

50 kDa subunit

P-Scr

P-Thr 0

1

2

3

4

3

4

TIME (min)

80 u 0

.

< 0

TABLE 1. EMpcts of various gangliosides and sialic acid on purified CaM-kinase II activity Effectors None GTlb GDla GM I BGM Asialo-GM I Cerebroside Sialic acid

CaM-kinase I1 activity (pmol/min) 0.6 2 0.1 17.9f 1.5 10.9 5 0.3 4.6 f 0. I 6.5 2 0.2 0.6 f 0. I 0.5 f 0.I 0.5 5 0.1

Purihed C a M - k i w I1 was assayed in the presence of I mM EGTA and the indicated gangliosida ( I 25 PM)or sialic acid ( I25 pM)using syntide-2 as substrate. Values shown are means ? SEM for three separate experiments. BGM stands for bovine brain ganglioside mixture.

J Nmuwhrm. Vd 54. No 1.1990

0

1

2

TIME (min) FlG. 2. Time course of autophosphorylation of threonine and serine residues in CaM-kinase II. Purified CaM-kinase II (2 pg) w a s in-

cubated at 0 ° C in the presence of Ca2'/CaM ( 0 , O )or EGTA plus 125 f l G T l b (A. A). The autophosphorylated kinase subunits were separated by SDS/PAGE, and the a (A) and 3, (8)subunits were Subjected to phosphoamino acid analysis (see Materials and Methods)to quantitate phosphothreonine (0,A) and phosphoserine (0. A). Results a r e expressed as a percentage of the cpm in phosphoserine a t 4 rnin.

REGULATION OF CaM-KINASE I I BY GANGLIOSIDES

105

which gives significant phosphothreonine, especially at very early times (Fig. 2 and Lai et al., 1987). Reversephase HPLC analysis of CNBr 32P-peptides derived from the kinase subunits showed little (less than 5% of the total radioactivity) of the CB, fragment [this fragment contains the threonine-2861287 autophosphorylation site (Schworer et al., 1988)l for the 50-kDa subunit and 8%CBI for the 60-kDa subunit. The ganglioside-stimulated autophosphorylation resulted in formation of little Ca'+-independent kinase activity (less than lo%), whereas autophosphorylation in the presence of Ca2+/CaMgave 40-50% Ca2+-independent activity (not shown). The initial rate of gangliosidestimulated autophosphorylation was not affected by dilution up to 10-fold, indicating an intramolecular reaction (not shown).

FIG. 4. lmmunoprecipitation of 3ZP-labeledCaM-kinase II from synaptic membranes. Purified CaM-kinase II (1 pg) (lane 1) and synaptic membranes (400 pg) (lanes 2-5) were incubated under various phosphorylation conditions (see Materials and Methods), subjected to immunoprecipitation. and then SDS/PAGE and autoradiography. Lane 1, Ca2+ plus CaM; lane 2, EGTA; lane 3, EGTA plus GTl b; lane 4, same as lane 3, but using nonimmune immunoglobulin G; lane 5.Ca" plus CaM. Concentrations of various additions were as in Fig. 3.

Ganglioside stimulation of CaM-kinase I1 in synaptic membranes Ganglioside-stimulated autophosphorylation of CaM-kinase 11 was also investigated using synaptic membranes purified from rat brain. Addition of 125 p M G T l b to the synaptic membranes resulted in increased phosphorylation of polypeptides of apparent molecular mass of 160, 76, 74, and 60 kDa (Fig. 3). The 60-kDa polypeptide was identified as a subunit of CaM-kinase I1 as it was specifically immunoprecipitated by antibody to the kinase (Fig. 4, lanes 2 and 3). Analysis by immunoprecipitation also revealed ganglioside stimulation of the 50-kDa subunit of the kina%, which was not apparent in the one-dimensional gel electrophoretic separation. The polypeptides at 74 and 76 kDa were probably synapsin I, because addition of exogenous synapsin I enhanced the amount of 32P

labeled at these positions. The addition of synapsin I also enhanced phosphorylation of proteins in the 4464-kDa range. These are probably proteolytic degradation products derived from synapsin I as they were also observed when the synapsin I was phosphorylated by purified CaM-kinase 11. In addition to G T l b, GD la and GM1 were potent stimulators of synaptic membrane phosphorylation, but asialo-GM 1, cerebroside, and sialic acid were ineffective (not shown). The stimulatory effects of G T 1b did not require addition of Ca" (not shown). Addition of Ca2+/CaM to the synaptic membrane phosphorylation mixture enhanced phosphorylation of the same set of polypeptides as was stimulated by EGTA/GTIb (Fig. 3, lanes 5 and 6 ) .

FIG. 3. Endogenousproteinphosphorylationin the synaptic membrane. Synaptic membrane (80 pg), incubated in an 80-pl volume at 30°C for 30 sec in the presence of various effectors, was analyzed by SDS/PAGE and autoradiography. A Coomassie Blue stain of membrane proteins(35pg) alone (lane 1) or in the presence of exogenous synapsin I (1 pg) (lane 2). B Autoradiographs of phosphorylation in the presence of 1 mM EGTA (lane 1); EGTA GT1b (lane 2); EGTA plus exogenous synapsin I (1 plus 125 pg) (lane 3); EGTA plus GTl b and synapsin I (lane 4); 0.2mM Ca2' (lane 5); Ca" plus CaM (3 pM) (lane 6); CaZ+plus synapsin I (lane 7);and Ca2+/CaMplus synapsin I (lane 8).

Substrate specificity of ganglioside-stimulated CaM-kinase I1 The ability of G T 1b to stimulate CaM-kinase I1 was assessed using several different protein substrates of this kinase. As shown in Table 3, G T 1b in the presence of EGTA stimulated the phosphorylation of syntide-2, synapsin I, and glycogen synthase, but had little effect on the phosphorylation of MAP-2, tau factor, smooth muscle myosin light chain, myelin basic protein, casein, and histone H I . Mechanism of the ganglioside stimulatory effect The lack of stimulation by gangliosides with certain substrates of CaM-kinase 11 could mean that the stimulatory effect was due to interaction of the ganglioside with the substrate. This would be somewhat surprising for syntide-2 as it is a small peptide. To address this question and further analyze the mechanism of ganglioside stimulation, a synthetic peptide corresponding to residues 28 1-309 and containing the CaM-binding, inhibitory, and autophosphorylation sites (Colbran et al., 1988; Kelly et al., 1988; Payne et al., 1988) was utilized. It has been shown that binding of Ca*+/CaM to this peptide removes its inhibitory property and stimulates phosphorylation of threonine-286 in the peptide. A completely Ca"/CaM-independent form of CaM-kinase 11, generated by autophosphorylation and partial proteolysis (Levine and Sahyoun, 1987; Colbran J . Neurmhem.. Vol. 54, No. 1. 1990

K . FUKUNAGA ET AL.

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TABLE 3. Effect of G T l b on CaM-kinase II activity using different substrates Kinax activity (pmol/min) EGTA + GT 1b (% stimulation)

Concentration Substrate

(WW

13.9 (41) 20.4 (39) 4.1 (46) 0.2 (5) 0.2 (2) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0)

1

Synapsin I Syntide-2 Glycogen synthase MAP-2 Tau factor MLC MBP Casein Histone H,

Ca2++ CaM

10 5 1

I 5 10 10 10

Ca2'

+ CaM + GTlb

(% inhibition)

34.1 52.5 8.8 4.1 8.9 3.5 3.8

31.7 (7) 47.2 (10) 8.8 (0) I .3 (68) 4.6 (48) 1.0 (71) 0.6 (84) 0.3 (73) 0.3 (57)

1.1

0.7

Purified CaM-kinase I1 was assayed using the indicated substrates with the indicated additions (EGTA, 1 mM, GT I b, 125 p M CaM, 3 p M ; a'+ 0.2 , W ) Values . are given as means for three separate experiments. MLC, myosin light chain; MBP, myelin basic protein. Numbers in parentheses are percentage stimulation or percentage inhibition relative to kinase activity assayed in the presence of Ca2' + CaM.

et al., 1988), was used for these studies to avoid complications due to interactions of the activators with the kinase. As shown in Fig. 5, GTlb has no direct effect on this proteolyzed form of CaM-kinase 11, but, like Ca2+/CaM,did completely reverse the inhibitory effect of peptide 281-309. Unlike Ca2+/CaM,GTlb did not stimulate the phosphorylation of threonine-286 in peptide 28 1-309 (data not shown). Inhibitory effects of gangliosides on CaM-kinase I1 In the presence ofCaZ+/CaM,GTlb strongly inhibited the phosphorylation of most of the proteins in Table 3, except for glycogen synthase, synapsin I, and syntide-2. As with the stimulatory effects, GDla (125 p M ) and GM 1 (125 p M ) also gave strong inhibition, but asialo-GM 1, cerebroside, and sialic acid gave less I

I

than 15% inhibition even at 250 pM (Table 4). Even though gangliosides are known to bind Ca2+,the inhibitory effects could not be reversed when Caz+was increased to 1 mM or when CaM was increased to 15 &(not shown). When tau factor was used as substrate, kinetic analysis indicated the inhibition was by a competitive mechanism, but when MAP-2 was the substrate, noncompetitive kinetic data were obtained (Fig. 6). Effects of gangliosides on other CaM-stimulated enzymes To determine whether the effects of gangliosides were specific for CaM-kinase 11, two other CaM-stimulated enzymes were tested. In the presence of EGTA, GT 1 b (50 p M ) gave partial stimulation (50% of the activity

TABLE 4. Effects of various gangliosides on CaM-kinase II activity in the presence ofCa'+/CaM CaM-kinase I1 activity Concentration Addition None GTlb GDla

0'

0

1

2

3

caw

U)"~TION OF 1281-391

4

5

I

lml

FIG. 5. Effects of CaZ+/CaMand EGTA/GTl b on the inhibitionof CaM-kinase II by peptide CaMK 281-309. Protedyzed CaM-kinase II (5 nM subunit) was assayed with 20 pM syntide-2 and the indicated concentrations Of CaMK 281-309 for 2 rnin at 30°C in the presence of 1 mM EGTA (0).0.5 mM Ca2' plus 15 pM CaM (A), or 1 mM EGTA plus 125 phf GT1b (0)Kinase . activity is expressed as a percentage of activity determined in the absence of CaMK 281-309 under each assay condition (60.6 pmol/rnin for 0; 46.7 pmol/rnin for A; 43.9 pmdlmin for 0).

J. Neurochem.. Vol. 54, No. 1. 1990

GM 1 Asialo-GM 1 Cerebroside Sialic acid

(PM)

125

250 125 250 125 250 125 250 125 250 I25 250

(pmol/min)

(90)

5.22 5 0.10 1.58 5 0.06 1.04 f 0.04 1.60 2 0.04 0.96 f 0.02 2.89 IT 0.09 1.87 ? 0.06 4.50 5 0.05 4.48 f 0.09 5.02 ? 0.12 4.51 f 0.19 4.79 ? 0.20 4.44 f 0.09

100

30 20 31 18 55

36 86 86 96 86 92 85

Purified CaM-kinase 11 was assayed in the presence of Ca2+(0.2 mM) and CaM (3 p M ) plus the indicated concentrations of gangliosides using I p M tau factor as substrate. Values are means ? SEM for three experiments.

REGULATION OF CUM-KINASE II BY GANGLIOSIDES

A

107

.c 3 -

E .-.

z r

1 2 -

/

L

E

v

>

1 -

> I

I _ .

4

L

-3

-2

A A 0

-1

2

1

1/s

-

3

4

(pM)-*

B

l/C

(pm-1

FIG. 6. Inhibition pattern of tau factor and MAP-2 phosphorylation by ganglioside CaM-kinase I1 activities were assayed in the absence (0)or presence of either 50 pM (0) or 125 p M GT1 b (A)using various concentrations of tau factor (A) or MAP-2 (B) as substrates Data

are presented as Lineweaver-Burk plots

in the presence of Ca’+/CaM) of calcineurin (p-nitrophenyl phosphate as substrate), but was without effect on myosin light chain kinase at concentrations from 25 to 250 p M . In the presence of Ca2+/CaM, G T l b gave potent inhibition of both CaM-stimulated enzymes.

DISCUSSION Previous studies have reported that the activities of several protein kinases, including protein kinase C (Kreutter et al., I987), epidermal growth factor-receptor kinase (Hanai et al., 1988u,b), and unidentified protein kinases (Tsuji et al., 1985; Chan, 1987~1,1988), were modified by gangliosides. Goldenring et al. (1985) and Cimino et al. ( 1987) suggested that CaM-kinase I1 activity in isolated synaptic membranes was regulated by gangliosides. The results reported in this paper confirm these reports, using not only synaptic membranes but also the purified brain CaM-kinase 11. With GTIb, GD 1a, and GM 1 in the presence of EGTA, stimulation could be attained to about 30%of the CaM-kinase I1 activity obtained in the presence of Ca2+/CaM.In contrast with the results of Goldenring et al. ( 1985), stimulation by gangliosides did not require the presence of Ca’+, even when the gangliosides were exhaustively di-

alyzed against EGTA to remove most of the bound Ca2+ (Abramson et al., 1972; Behr and Lehn, 1973). Trisialo ganglioside was the most potent activator. and asialo-GM I , sialic acid, and cerebroside were without effect. Stimulation of CaM-kinase 11 by gangliosides was dependent on the protein substrate utilized. With GT Ib, large stimulation was obtained using the proteins synapsin I and glycogen synthase or the synthetic peptide syntide-2. Little or no stimulation was observed with MAP-2, tau factor, myosin light chain, myelin basic protein, casein, or histone H , as substrates. This substrate selectivity could suggest that the stimulation is due to binding of the gangliosides to the substrate, thereby stimulating their phosphorylation. Three observations, however, would tend to argue against this mechanism. First, substantial evidence indicates that a n inhibitory domain within CaM-kinase I1 interacts with and suppresses the basal activity of the kinase in the absence ofCa*+/CaM (Colbran et al., 1988. 1989). Thus, interaction of gangliosides with just the protein substrates would not be expected to stimulate the inactive form of CaM-kinase 11. Second. removal of the regulatory domain from CaM-kinase I1 by limited proteolysis resulted in loss of the stimulatory effect of GTI b. If gangliosides were stimulating kinase activity

108

K . FUKUNAGA ET AL.

through interaction with the substrate, one would have expected stimulation of the constitutively active form of CaM-kinase 11. Third, gangliosides must directly interact with CaM-kinase I1 because they stimulate autophosphorylation of the kinase. Furthermore, Fig. 5 documents that G T 1b does interact with the regulatory domain of CaM-kinase I1 and reverse its inhibition. These results strongly support the conclusion that stimulation of CaM-kinase 11 is by direct interaction with gangliosides. In addition to directly stimulating CaM-kinase 11, it is likely that gangliosides also bind certain proteins and inhibit their phosphorylation, as suggested by Chan (19876) for myelin basic protein. Such a mechanism could explain the inhibition of phosphorylation of certain proteins in the presence of Ca2+/CaM (Table 3). In fact, the same proteins whose phosphorylations are inhibited in the presence of Ca2+/CaM show no stimulation by gangliosides in the presence of EGTA. It would be interesting to determine whether the phosphorylation of some of these proteins, such as MAP2, casein, and histone H I ,by cAMPdependent protein kinase is inhibited by gangliosides. This inhibition by gangliosides in the presence of Ca2+/CaM was not reversed by increasing the concentrations of Ca2+ or CaM, suggesting that it is not competitive with these activators. Kinetic analyses with various protein substrates did not reveal any simple mechanism for the inhibition (Fig. 6). It is possible that interaction ofgangliosides with certain proteins, such as myelin basic protein, to inhibit their phosphorylation has physiological significance. Gangliosides are known to stimulate several other CaM-sensitive enzymes including cyclic nucleotide phosphodiesterase (Davis and Daly, 1980) and adenylate cyclase (Partington and Daly, 1979). Thus, it is possible that gangliosides might mimic Ca2+/CaMand interact with the CaM-binding domains of proteins. We found that gangliosides could also stimulate the CaM-dependent protein phosphatase calcineurin, but not smooth muscle myosin light chain kinase. This is not too surprising as it appears that different CaMbinding proteins interact with different portions of CaM (Ni and Klee, 1985; Putkey et al., 1986). Interaction ofgangliosides with the CaM-binding domain of CaMkinase I1 was directly examined using the synthetic peptide corresponding to residues 28 1-309 (CaMK 28 1-309) of the a-subunit of CaM-kinase 11. CaMK 28 1-309, which contains the CaM-binding domain and the inhibitory domain of CaM-kinase 11, is a potent inhibitor of a Ca2+-independent, proteolytic form of CaM-kinase I1 (Colbran et al., 1988). With both peptide 28 1-309 and native CaM-kinase 11, binding of GTlb, like Ca2'/CaM, reversed the potency of the inhibitory domain, but, unlike Ca2'/CaM (Colbran et al., 1988), did not stimulate phosphorylation of threonine-286. Our studies on the stimulation of CaM-kinase I1 in rat brain synaptic membranes by gangliosides generally confirm previous reports (Goldenring et al., 1985; J. Neurochem.. Vol. S4, No. 1. 1990

Cimino et al., 1987). In agreement with Chan (1987u), but in contrast with Goldenring et al. (1989, we did not observe a requirement for Ca2+.However, it is not clear that a single protein kinase in the synaptic membrane is involved. The ganglioside-stimulated protein kinase studied by Chan ( 1 9 8 7 ~showed ) different properties on diethylaminoethyl-cellulose and gel filtration than did CaM-kinase 11. Likewise, the physiological role of ganglioside stimulation of CaM-kinase I1 is not clear. A specific ganglioside is thought to be involved in regulation of cell growth by modulating the phosphorylation of growth factor receptor in some tumor cell lines (Bremer et al., 1984). Gangliosides (Wiegandt, 1967; Hansson et al., 1977; Ledeen, 1978) and CaM-kinase I1 (Quimet et al., 1984; Fukunaga et al., 1988) are both localized at synaptic junctions and may therefore interact. In fact, gangliosides comprise up to 5- 10%of the total lipid of some membranes in the central nervous system (Ledeen, 1978), suggesting that their concentrations could be high enough to be physiological regulators of certain enzymes such as CaM-kinase 11. If CaM-kinase I1 is stimulated by gangliosides in vivo, the process would not result in formation of the Ca'+-independent form of CaM-kinase 11, as occurs with activation by Ca*+/ CaM. These different mechanisms of stimulating CaMkinase I1 may be important for different neuronal functions mediated by this multifunctional kinase.

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