79

Neuroscience Letters, 131 (1991)79-82 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100568R NSL 08065

Calretinin, a neuronal calcium binding protein, inhibits phosphorylation of a 39 kDa synaptic membrane protein from rat brain cerebral cortex T o m o k o Yamaguchi*, Lois Winsky and David M. Jacobowitz Laboratory of Clinical Science, NIMH, Bethesda MD 20892 (U.S.A.) (Received 13 May 1991; Revised version received 19 June 1991; Accepted 21 June 1991)

Key words: Calretinin; Calcium binding protein; Protein phosphorylation; Brain protein; Synaptic membrane protein The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin. At the same concentration, calretinin produced a slight increase in the phosphorylation of several other synaptic membrane proteins which appeared additive with the stimulation produced by either calmodulin or phosphatidylserine in the presence of calcium.

Recent investigations have disclosed the identification and characterization of the calcium binding protein calretinin [11, 12, 14, 17]. Calretinin is present in subsets of neurons throughout the brain [1, 7] and is densely localized in sensory cells including, for example, the retina, organ of Corti and sensory ganglia [5, 6, 13]. Calretinin is a soluble protein with a molecular mass of 31.5 kDa (29 kDa by gel electrophoresis) and isoelectric point of 5.3 and shares 50-60% amino acid sequence homology with calbindin D-28k, its closest relative in the calcium binding protein family [11, 12, 14, 17]. As with calbindin D-28k, the role of calretinin in brain has not been determined. The influences of some other EF hand calcium binding proteins such as calmodulin and calcineurin on neural transmission are known to be mediated in large part by specific effects on phosphoproteins [4, 9]. The present study examined whether calretinin might also affect protein phosphorylation. Synaptic membranes were prepared by sucrose density centrifugation using the P2 fraction obtained by hypotonic treatment of a synaptosomal fraction from rat cerebral cortex (3-4 g) homogenates using buffer A (20 mM NaC1, 2 mM EDTA, 0.5 mM EGTA, 20 mM HEPES,

*Present address: Department of Biochemistry, Tokyo Women's Medical College, 8-1 Kawada-Cho, Shinjuku-Ku, Tokyo, Japan. Correspondence: L. Winsky, Laboratory of Clinical Science, NIMH, Bldg. 10, Rm. 3D-48, Bethesda, MD 20892 U.S.A. Fax: (1) (301) 402-0188.

pH 7.4 with or with out sucrose) and procedures essentially as previously described [3] except that the P2 pellet was washed only once. The synaptic membrane fraction was resuspended in 2 ml of buffer A and stored at - 8 0 ° C until use. Phosphorylation reactions were in a total of 80-120 pl comprised of 12 mM HEPES (pH 7.4), 10 mM MgCI2, 0.1 mM DTT, 10-35 pM ATP, 10 pCi/ 100 pl 32p-ATP (New England Nuclear) containing 5 mM EGTA or 200 pM CaC12 with or without calretinin (10 or 100 nM) prepared from guinea pig brain [17]. In some cases, bovine calmodulin (10 pM, Calbiochem), phosphatidylserine (100 pg/ml, Suppelco Inc.), or a protein kinase C inhibitor (100 mM peptide PKC 19-36, Peninsula Labs.) were included in the reaction mixtures. Synaptic membranes (80 pg/10-30 pl) were added after a 90 s preincubation (30°C) of tubes containing buffer and other reaction components. The reaction was stopped after 60 s by the addition of 80-100 pl sample buffer (75 mM Tris, pH 8.0, 2% SDS, 15% glycerol, 5% 2-mercaptoethanol, 0.005% Bromophenol blue). Samples were then heated to 95°C for 5 rain. Proteins were separated by one-dimensional SDS polyacrylamide (11%) gel electrophoresis and stained with silver, then dried and placed on autoradiographic film (XOmat AR, Kodak). The amount of radiolabeled phosphate incorporation into individual protein bands was estimated by liquid scintillation counting of radioactive bands cut from the gels. The appearance of a phosphorylated band at 39 kDa

80

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Fig. I. Effect of calretinin on the phosphorylation pattern of synaptic membrane proteins. Samples were incubated in phosphorylation buffer (see text) containing 100/aM calcium plus 0 nM (A), 10 nM (B) or 100 nM (C) calretinin. Left side shows silver stain of protein bands in rat synaptic membrane samples and migration of protein molecular weight standards (Biorad). Right figure shows autoradiogram developed from the dried gel. Arrow indicates the 39 kDa phosphoprotein band. The decrease in the appearance of the 39 kDa phosphorylation band by 100 nM calretinin has also been observed using synaptic membranes prepared from guinea pig brain (data not shown).

in the synaptic membrane fraction was inhibited in a dose related manner by calretinin (Fig. 1). At 10 nM, calretinin inhibited the incorporation of 32p-ATP by the 39 kDa phosphoprotein by 12% and produced 63% inhibition at 100 nM as estimated by liquid scintillation counting of radioactivity associated with this band cut from gels. Phosphorylation of the 39 kDa band was stimulated by calcium (200 pM) as shown in Fig. 2. The incorporation of 32p-ATP by the 39 kDa band was variable between assays (compare Figs. 1, 2 and 3). Indeed, the 39 kDa phosphoprotein appeared as a sharp band on autoradiograms in only 3 of the 5 separate assays whose data comprise this study. While the factors responsible for this variability remain to be determined, an inhibitory effect of calretinin on the 39 kDa phosphoprotein has been observed in all cases where the band was visible in control (calcium) conditions. Several incubation conditions were introduced to differentiate the effects of calretinin from those of other effectors of synaptic membrane phosphorylation. Phosphorylation of the 39 kDa band and the inhibition produced by 100 nM calretinin were unaffected by either phosphatidylserine (PS), a protein kinase C (PKC) inhibitor peptide (PKC 19-36) or calmodulin at concentra-

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Fig. 2. Effect of calretinin and some effectors of protein kinase C on phosphorylation of synaptic membrane proteins. Synaptic membranes were incubated in the presence of 200/aM calcium ( + ) or 5 mM EGTA ( - ) . Molecular weights of some prominant phosphoprotein bands were calculated from migration of standards and are indicated on the right. CR, 100 nM calretinin; PS, 100/ag/ml phosphatidylserine; PKCI, I00 mM PKC-inhibitor (peptide PKC 19 36).

tions producing clear effects on other phosphoprotein bands (Figs. 2 and 3). Calretinin (100 nM) produced a slight increase (2431% vs 200 pM calcium alone condition) in the phosphorylation of bands at 51, 44, 16 and 12 kDa as determined by liquid scintillation counting of bands cut from dried gels. Phosphorylation of an 80 kDa band also appeared to be increased by 100 nM calretinin (Figs. 2 and 3) although this band could not be reliably isolated for quantitation. The phosphorylation of many of these proteins was also stimulated by either calmodulin or PS and combinations of calretinin with either calmodulin or PS appeared to produce additive effects on phosphate incorporation into proteins (Figs. 2 and 3). For example, calmodulin increased the CPM of the 51 kDa band by 20%, calretinin by 24% and the combination resulted in a 47% increase in CMP associated with this band vs the CPM of calcium alone. The CMP associated with a 44 kDa band was increased 26% by calretinin, 38% by calmodulin and 52% by the combination. Phosphorylation of a 16 kDa band was increased 25% by calretinin, 20% by calmodulin and 73% by the combination of both and the CMP associated with a 12 kDa band were increased 31% by calretinin, 48% by calmodulin and 96% by a corn-

81 bination of calretinin plus calmodulin. Finally, the PKC inhibitor peptide inhibited the phosphorylation of the 80, 16 and 12 kDa protein bands in both the presence and absence of calretinin (100 nM) (Fig. 3). The identity of the 39 kDa protein or the kinase(s) mediating phosphorylation of this protein was not determined. Data indicated that the 39 kDa phosphoprotein is not a calmodulin kinase substrate. In addition, phosphorylation of this band was stimulated by calcium but little or no effect of PS or the PKC inhibitor peptide were observed, indicating that phosphorylation may not be dependent on protein kinase C. Identity as a casein kinase II (CK II) substrate also having a similar molecular weight was ruled out since heparin (5/~g/ml), an inhibitor of CK II (for review see ref. 15), had no effect on this phosphoprotein band when introduced into the reaction mixture (data not shown). Additional studies of the 39 kDa protein, and the kinases affecting it, will be needed to determine the identity and function of this protein. Calretinin appeared to affect radiolabeled phosphate incorporation into proteins in both the presence or absence of EGTA. It should be noted that calmodulin also produced a slight increase in the phosphorylation of some proteins in the EGTA condition (data not shown) suggesting that not all calcium was sequestered in this condition. Thus, the present results cannot address the degree to which the calretinin effects were calcium de-

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Fig. 3. Effect of calretinin and calmodulin on phosphorylation of synaptic membrane proteins. Synapticmembranes were incubated in the presence of 200/~M calcium (+) or 5 mM EGTA (-). CaM, 10 /~Mbovinecalmodulin;CR, 100 nM calretinin;PKC-I, 100 mM PKCinhibitor (peptide PKC 19-36).

pendent. The mechanism by which calretinin produced the decrease in the phosphorylation of the 39 kDa protein band, or the increase in phosphorylation of other proteins, is unknown. For the stimulatory effect of calretinin on other phosphoprotein bands, the data suggest an additive effect with calcium/calmodulin dependent protein kinase II and protein kinase C stimulation but no specific interaction with these kinases. The possibility remains that calretinin effects are mediated through actions on some other kinase system. Alternatively, calretinin could have produced the stimulatory effects on protein phosphorylation by modulating the actions of other enzymes. For example, inhibition of a protein phosphatase by calretinin could increase the appearance of several phosphoprotein bands and produce additive effects with kinase activation by other substances. Calretinin and calbindin D-28k have been identified as high affinity calcium binding proteins, with calculated or proposed affinities of 0.1-1.0 pM for calcium and containing 4-6 active calcium binding sites [11, 12, 16]. For calbindin D-28k, the high affinity and capacity of calcium binding and large concentrations of this protein has led to a hypothesized role of calbindin D-28k in calcium buffering, particularly in neurons having large calcium fluxes or localized calcium currents [2, 8]. Others, however, have argued against this buffering action based on affinity studies indicating that calbindin D-28k may be saturated under normal resting conditions in neurons, thus preventing any physiological buffering action by this protein during depolarization [10]. The results presented in this report provide the first evidence for a more active role of calretinin in neuronal biochemical processes. Many questions remain to be answered regarding the mechanism by which calretinin produced these multiple effects on the appearance of phosphoproteins. Nonetheless, it is hoped that the results presented here will provide a first step towards determining a function of this neuronal calcium binding protein. Finally, it will be interesting to determine whether other calcium binding proteins such as calbindin D-28k produce similar effects on protein phosphorylation.

1 Arai, R., Winsky,L., Arai, M. and Jacobowitz,D.M., Immunohistochemicallocalizationof calretinin in the rat hindbrain, J. Comp. Neurol., in press. 2 Baimbridge,K.G., Miller, J.J. and Parkes, C.O., Calciumbinding protein distribution in the rat brain, Brain Res., 239 (1982) 519525. 3 Chan, K.-F.J., Ganglioside-modulatedprotein phosphorylation,J. Biol. Chem.,262 (1987) 5248-5255. 4 Cohen, P., The calmodulin-dependentmultiprotein kinase. In P. Cohen and C.B. Klee (Eds.), Calmodulin, Elsevier, Amsterdam, 1988, pp. 145-194.

82 5 Dechesne, C.J., Winsky, L., Kim, H.N., Goping, G., Vu, T.D., Wenthold, R.J. and Jacobowitz, D.M., Identification and ultrastructural localization of a calretinin-like calcium binding protein (protein 10) in the guinea pig and rat inner ear, Brain Res., in press. 6 Ichikawa, H., Jacobowitz, D.M., Winsky, L. and Helke, C.J., Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents, Brain Res., in press. 7 Jacobowitz, D.M. and Winsky, L., Immunocytochemical localization of calretinin in the forebrain of the rat, J. Comp. Neurol., 304 (1991) 198-218. 8 Jande, S.S., Maler, L. and Lawson, E.M., Immunohistochemical mapping of vitamin D-dependent calcium-binding protein in brain, Nature, 294 (1981) 765-767. 9 Klee, C.B., Draetta, G.F. and Hubbard, M.J., Calcineurin, Adv. Enzymol. Relat. Areas Mol. Biol., 61 (1988) 149-200. I0 Leathers, V.L., Linse, S., Forsen, S. and Norman, A.W., Calbindin-D28k, a 1 alpha, 25-dihydroxyvitamin D3-induced calcium binding protein, binds five or six Ca 2 + ions with high affinity, J. Biol. Chem., 265 (1990) 9838-9841. 11 Parmentier, M. and Lefort, A., Structure of the human brain calcium-binding protein calretinin and its expression in bacteria, Eur. J. Bioehem., 196 (1991) 79-85.

12 Rogers, J.H., Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons, J. Cell Biol., 105 (1987) 13431353. 13 Rogers, J.H., Two calcium-binding proteins mark many chick sensory neurons, Neuroscience, 31 (1989) 697-709. 14 Rogers, J.H., Kahn, M. and Ellis, J., Calretinin and other CaBPs in the nervous system. In R. Pochet, D.E.M. Lawson and C.W. Heizmann (Eds.), Calcium Binding Proteins in Normal and Transformed Cells, Plenum, New York, 1989, pp. 195-203. 15 Tuazon, P.T. and Traugh, J.A., Casein kinase I and II - - multipotential serine protein kinases: structure, function and regulation. In P. Greengard and G.A. Robison (Eds.), Advances in Second Messenger and Phosphoprotein Research, Vol. 23, Raven, New York, 1991, pp. 123-164. 16 Wasserman, R.H. and Taylor, A.N., Vitamin D3-induced calciumbinding protein in chick intestinal mucosa, Science, 152 (1966) 791793. 17 Winsky, L., Nakata, H., Martin, B.M. and Jacobowitz, D.M., Isolation, partial amino acid sequence and immunohistochemical localization of a brain-specific calcium binding protein, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 10139-10143.