Journal of Natrochernis~y Raven Press, Ltd., New York 0 1992 International Society for Neurochemistty
Monoclonal Antibody Analysis of Phosphatidylserine and Protein Kinase C Localizations in Developing Rat Cerebellum Atsuo Miyazawa, Hiroko Inoue, Tohru Yoshioka, *Tetsuro Horikoshi, *Keiji Yanagisawa, TMasato Umeda, and tKeizo Inoue Department of Human Basic Sciences, School of Human Sciences, Waseda University, Tokorozawa; *Department of Physiolou, School of Dental Medicine, Tsurumi University, Yokohama; and ?Department of Health Chemistry, Faculty of Pharmaceutical Science, The University of Tokyo, Tokyo, Japan
Abstract: Understanding the topographical relationships between phosphatidylserine (PS) and protein kinase C (PKC) within neurons can provide clues about the mechanism of translocation and activation of PKC. For this purpose we applied monoclonal antibodies (Abs) of PS and PKC to sections ofdeveloping rat cerebellum. The anti-PKC Ab immunohistochemical pattern showed homogeneous staining of Purkinje cells over various postnatal ages, whereas the antiPS Ab staining showed a heterogeneous localization over these ages. Purkinje cells did not stain well betwcen postnatal day 14 (PND 14) and PND 2 1, suggestingthat the PS was
lost from the membrane during preparation of the sections during this period. These data imply that interactions between PS and PKC vary in Purkinje cells during postnatal development. Key Words: Developing Purkinje cell-Antiprotein kinase C antibody-Anti-phosphatidylserine antibody-Immunohistochemical study-Phosphatidylserineprotein kinase C interaction-Cerebellum. Miyazawa A. et al. Monoclonal antibody analysis of phosphatidylserine and protein kinase C localizations in developing rat cerebellum. J. Neurochem. 59, 1547- 1554 ( 1992).
The study of lipid-protein interaction is important for the analysis of protein kinase and phospholipase function. The structural and dynamic aspects of such lipid-protein interactions have previously been investigated only by physical methods such as spin-labeled electron spin resonance. These methods, however, are applicable only to artificial membrane systems (Marsh, 1990). Recently we made monoclonal antibodies (Abs) to various types of phospholipids (Miyazawa et al., 1988; Umeda et al., 1989) and have applied these Abs to developmental analysis of signal transduction systems (It0 et al., 1991). In the course of our studies, we found we could localize phosphatidylinositol4,5-bisphosphate (PIP,) and phosphatidylserine (PS) on the developingnerve cell by immunohistochemistry. Considering the known lateral diffusion of phospholipid molecules in the membrane, such a localization of phospholipids seems very unlikely. Because the phos-
pholipid molecule itself cannot be fixed by fixative, we believe that the localization of phospholipid obtained with the immunostaining by anti-phospholipid Ab may represent a degree of interaction between phospholipid and protein. In this article, we describe an analysis ofthe localization of PS in the developing cerebellum using monoclonal Ab for PS. The localization of y-type protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37), the dominant PS-binding protein, was also examined at the same ages by using an anti-PS Ab.
Received January 16, 1992; revised manuscript received April 6, 1992; accepted April 13, 1992. Address correspondence and reprint requests to Dr. T. Yoshioka at Department of Human Basic Sciences, School of Human Sciences, Waseda University, 2-579-1 5 Mikajima, Tokorozawa, Saitama 359, Japan. Abbreviafions used: Ab, antibody; BSA, bovine serum albumin;
MATERIALS AND METHODS
Monoclonal Abs The preparation and characterization ofanti-PS monoclonal Ab, designated M A 7 [immunoglobulin M (IgM)], have been described in detail prcviously by Umeda et al. (1989). PS4A7 is highly specific for PS and has shown no
ELISA, enzyme-linkedimmunosorbcnt assay; IgM, immunoglobulin M; PIP,, phosphatidylinositol4,5-bisphosphate;PKC, protein kinase C; PMA, phorbol 12-myhtate 13-acetate;PND, postnatal day; PS,phosphatidylserine;TBS, 50 mMTris-HCI, 150 mMNaCl (pH 7.4);TBS, 10 mM Tris-HC1, 150 mM NaCl (pH 7.4);TCA, trichloroacetic acid.
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A . MIYAZAWA E?' AL.
cross-reaction with other acidic phospholipids. The Ab was purified further by HPLC. Anti-y-PKC monoclonal Ab (clone CKI 97-5) was kindly provided by Prof. Nishizuka of Kobe University. The preparation and characterization of anti-y-PKC monoclonal Ab have been described previously by Kitano et al. (1 987) and Hashimoto et al. ( 1988). This Ab reacted strongly and preferentially with the y-subspecies of PKC.
Immunohistochemistry Wistar rats of the ages indicated were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and killed by perfusion with fixative containing 3% paraformaldehyde and 1% glutaraldehyde in 0.1 Msodium phosphate buffer at pH 7.4 via the left ventricle and washing out from the right atrium. The fixed brain was dissected and placed in the same fixative for more than 24 h at 4°C. Next, the cerebellum was embedded in 5% agar in Tris-buffered saline (TBS; containing 50 mM Tris-HCI, pH 7.4, I50 mM NaCl) and was sectioned sagittally into 50-pm thick slices with a microslicer (Dosaka, Japan). The prepared slices were then washed in TBS. After being pretreated with 1% bovine serum albumin (BSA) in TBS ( 1% BSA-TBS) for 2 h at room temperature, the sections were incubated with the monoclonal Ab (PS4A7, 36 pg/ml) in 1% BSA-TBS for more than 24 h at 4°C. Afler the sections were washed with TBS, they were incubated with horseradish peroxidase conjugated rabbit anti-mouse IgM Ab (Zymed Laboratories, South San Francisco, CA, U.S.A.) in 1% BSA-TBS for 2 h at room temperature. After the sections were washed with TBS again, the staining procedure was performed using a solution containing 0.1% diaminobenzidine tetrahydrochloride and 0.22% hydrogen peroxide in 50 mMTris-€ICI at pH 7.4. In the case of immunohistochemistry for y-PKC, the procedures were essentially the same as described in previous articles with a slight modification (Hashimoto et al., 1988). In brief, rats of various ages were perfused through the aorta with a fixative containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. The fixed cerebellum was dissected and washed with 30% sucrose in TBS for several days at 4°C. Sagittal sections (1 3 pm thick) were cut on a cryostat (Leitz, Wetzlar, F.R.G.) and collected on coverslips. The sections were incubated overnight at 4°C with anti-y-PKC monoclonal Ab (CKI 97-5, 1 :200 dilution), washed with TBS, and were then stained using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, U.S.A.).
Preparation of PS-liposome to absorb anti-PSAb The small unilamellar liposomes of PS were prepared by the sonication of the multilamellar liposome. The dried lipid film containing 2.5 pmol of PS was swollen in TBS and was sonicated for 5 min at 4°C under a nitrogen flow. This solution was centrifuged at 10,000 g for 10 min at 4°C. Next, the supernatant was used for absorption of antiPS Ab.
Assay of PKC activity Wistar rats of the ages indicated were anesthetized and their cerebella were dissected and sectioned sagittally into 2 0 0 - ~ mthick slices with a microslicer in artificial CSF containing 1 13 mM NaCI, 3 m M KCI, 10 Mglucose, I m M NaHPO,,, 1 mM MgCI,, 2 mM CaCI,, 25 m M NaHCO,, and gassed with 0,/C02 (95%/5%). Under a binocular mi-
J. Neurochem.. Vol. 59, No. 4, 1992
croscope, white matter including the granular layer was manually removed by razor from each slice, then residual parts including molecular layer were collected and frozen in liquid nitrogen. The cortices of the cerebellum were homogenized with buffer A (20 mMTris-HC1, pH 7.2; 10 mM MgCI,; 2 m M EDTA; 10 mM EGTA; 50 pM phenylmethylsulfonyl fluoride; 50 pg/ml of leupeptin; and 2.5 pg/ml of pepstatin) and centrifuged at 100,000g for 60 rnin at 2°C. The supernatant was decanted, and the pellet was resuspended with buffer A containing 0.1% Triton X-100. PKC activity was determined in the reaction medium containing 20 m M Tris-HC1, pH 7.4; 10 mMMgC1,; 0.01%Triton X-100; 50 pg of histone; 10 p M [32P]ATP;0.5 mA4 CaCI,; 20 pg of PS, 2 pg of diolein; and enzyme in 100 p1 for 15 rnin at 25°C. The Ca2'/PS-independent kinase activity was measured under the same conditions without Ca2+,PS, and diolein, but with 1 mMEGTA. The reaction was terminated by addition of 1 ml of 25% trichloroacetic acid (TCA) and acid-precipitable materials were collected by a filter. The filter was washed three times with 5% TCA and radioactivity was counted.
Enzyme-linked immunosorbent assay (ELISA) for inhibition of Ab binding Wistar rats were anesthetized with sodium pentobarbital and perfused through the left ventricle. The blood was washed out with 50 ml of Ringer solution. The perfused cerebellum was dissected and cut into blocks, which were then frozen in liquid nitrogen. The frozen cerebellar blocks were homogenized with 50 mM Tris-HC1 buffer, pH 7.4, containing 1 mM EGTA, 200 mM phenylmethylsulfonyl fluoride, and 0.3% mercaptoethanol. The homogenate was centrifuged at 100,000g for 60 rnin at 2"C, then the supernatant containing cytosolic proteins was submitted to ELISA. ELISA for the lipid haptens was performed as described previously by Umeda et al. (1986). The wells of the microtiter plates were coated with 50 pl of PS in ethanol (10 p M ) by evaporation overnight at room temperature. The nonspecific binding sites on the wells were blocked by 10 M T r i s HCI, 150 mM NaCl (pH 7.4) buffer (TBS) containing 30 mg/ml of BSA for 60 rnin at room temperature. After the plates were washed with T'BS, the wells were incubated with 100 pl of the cytosolic proteins at room temperature. The plates were washed once again, and 50 pI of the anti-PS monoclonal Ab (PS4A7) diluted with T'BS containing 10 mg/ml of BSA was poured into the wells, which were allowed to stand for 2 h at room temperature. The bound Ab was detected by biotinylated anti-mouse IgM Ah (Zymed Laboratories), followed by incubation with peroxidase-conjugated streptavidin (Zymed Laboratories). Optical density at 492 nm was measured by addition of o-phcnylencdiamine as substrate.
RESULTS Developmental changes of PS localization in cerebellum Immunoreactivity of anti-PS Ab was demonstrated on germinal cells (except for the external layer) at postnatal day 0 (PND 0). When Purkinje cells start to align on PND 3, the anti-PS immunoreactivity was
PKC AND PS IN CEREBELLAR DEVELOPMENT
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FIG. 1. lmmunostainingof rat cerebellum at various ages with anti-PS monoclonal Ab. Sagittal sections of rat cerebellumat various ages were stained with anti-PS monoclonalAb PS4A7. The samples were from (A) PND 7, (B) PND 14, (C)PND 21, and (D) adult rat cerebellum. E: Absorption of anti-PS monoclonalAb with PS-liposome in adult rat cerebellum. The imrnunoreactivityof anti-PS monoclonalAb (0.4 pM) was markedly reduced by absorption with an excess amount of PS-liposome (500 pM PS). Calibration bar 50 pn. 2
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extended to the cytoplasm. On PND 5, Purlcinje cells showed strong immunoreactivity in their cytoplasm as well as nucleus. In this case weak immunoreactivity of anti-PS was observed in the molecular layer of rat cerebellum. In the external germinal layer, no anti-PS immunoreactivity was observed in the premigratory granule cells. In the internal granular layer, some cells showed strong immunoreactivity in their cytoplasm and nucleus, but the others showed only weak staining in their cytoplasm. By PND 7, immunoreactivities were well observed in Purkinje cells, stellate and basket cells, and also in fully migrated granule cells (Fig. 1A). The pattern of anti-PS Ab immunostaining on PND 10 was almost the same as that of PND 7, but immunoreactivity was markedly decreased in the granule cells (data not shown). On PND 14 and 2 1, the anti-PS Ab immunostaining pattern became ambiguous and faded but the pattern was the same as that of thc adult cxcept for the existence of premigratory granule cells (Fig. 1B and C). This differs from the case of the anti-PIP, Ab staining at the distal part of the Purkinje dendrite, which transiently disappears around PND 14 (Ito et al., 1991). The anti-PS Ab immunoreactivity on the Purkinje dendrites was always present during PND 14-2 I , although the amount of PS seemed very low. When we treated these preparations with chloroform/ methanol solution, we could extract the same amount of PS from fixed preparations at different developmental stages (data not shown). In the case of adult rat cerebellum, most of the Purkinje cell was stained intensely except cell body (Fig. 1D). To examine if this staining is due to antigen (PS)-Ab interaction, anti-PS Ab was absorbed by PSliposome in an adult rat cerebellum, and sections were sliced and stained. As shown in Fig. 1E, no staining pattern was observed. The staining pattern data
for PS in developing rat cerebellum is summarized in Table 1. Developmental changes in the immunohistochemical localization of yPKC in cerebellar slices As the immunoreactivity of both anti-cy- and anti0-PKC Abs was found to be poor in Purkinje cells (data not shown), we concentrated at anti-y-PKC Ab in the following experiments. Results obtained in this study agreed well with those in Nishizuka’s laboratory, but we obtained some additional new findings in the developing cerebellum. Anti-y-PKC Ab immunoreactivity appeared in Purkinje cells from PND 3 through the adult stage, and it was also found to be present in basket and stellate cells from PND 14 to PND 21, but not in the adult stage. The fine dendritic arborizations of the Purkinje cells were clearly detected by the anti-y-PKC Ab immunostaining from PND 7 to the adult stage (Fig. 2A-D). Strong immunoreactivity was also seen in the axonal tracts of Purkinje cells from PND 7 to PND 14, but not clearly in axons from PND 2 1 (Fig. 2C) to the adult stage. At PND 10 and 14, a number of small varicosities were stained on the Purkinje cell axons. Purkinje cell nuclei stained weakly from PND 3 to the adult stage. The immunoreactivity of basket cells was stronger than that of stellate cells throughout the stage after PND 14. In these cells immunoreactivity of anti-y-PKC Ab was observed both in the nucleus and the cytoplasm. We can estimate that the quantity of PKC in the adult rat may be smaller than that of PND 21 rat, because the staining density shown in Fig. 2D seems faded compared with that of Fig. 2C. We confirmed this by direct measurement of PKC activity in the molecular layer of developing rat cerebellum. Results are shown in Fig. 3. As was expected from immunohistochemical data shown in Fig. 2, PKC activity in the
TABLE 1. Localization and intensity of anti-PS immunoreactivity in developing rat cerebellum PND Region External germinal layer Germinal cell Glial fiber Molecular layer Stellate and basket cell Bergmann fiber Purkinje cell Dendrite Perikaryon Axon Granular layer Granule cell Golgi cell Germinal cell
0 -
3
5
7
-
-
-
+
+
n n
n n
+ n -
+ +++
+
+
n
n
t+
+-i-+
n
n
n
n
n
n n
+
+++
n
n
++
+t
n
-t+
++t
+++
++ ++
10
++ + +++ +++ n
+ + ++
n, not identified; -, no reactivity; t,weak reactivity; ++, medium reactivity; +++, strong reactivity.
J. Neurochem.. Vol. S9, No. 4. 1992
21
Adult
n n
n n
++
1-
+ ++ +-
++ +++ -
n
n
n
14
++ ++
++ -
+ n
-
+ n
PKC AND PS IN CEREBELLAR DEVELOPMENT
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FIG. 2. lmmunostaining of rat cerebellum at various ages with anti-y-PKC monoclonal Ab. Sagittal sections of rat cerebellum at various ages were stained with anti-y-PKC monoclonalAb CKI 97-5. The samples were from (A) PND 7, (B) PND 14, (C)PND 21, and (D) adult rat cerebellum. Arrows indicate some interneurons such as basket and stellate cells in the molecular layer of the cerebellum. Calibration bar 50 pm. 2:
molecular layer reached a maximum value on PND 21 and decreascd markedly in adulthood, although the activity of PKC shown in Fig. 3 is the summation of all types of isozyme. Inhibition of the interaction between anti-PS Ab and PS by cytosolic protein The inhibitory effect of cytosol fraction obtained from rat cercbellum on the interaction of PS with anti-PS Ab was examined on ELISA. When the cytosol fraction was introduced into the reaction prior to the reaction of PS with the anti-PS Ab, the interaction between the two was inhibited in a concentration-dc-
pendent manner. To determine the optimum reaction period at room temperature, we performed a time course analysis for the inhibition reaction. As shown in Fig. 4A, an incubation period of 10 min was found to be enough. Inhibition of the interaction between PS and anti-PS Ab by the cytosol fraction was examined using cerebellar blocks of PND 7 and PND 2 1, because the PKC activity was quite different between the two. The results are shown in Fig. 4B. The amount of antigen that reacted with anti-PS Ab was expressed by the optical density of reaction medium. When the cytosol fraction was heated at 60°C for 1 h, the inhibitory effect disappeared. As shown in Fig. 4,
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A . MIYAZAWA ET AL.
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r
I :
3 0
0.8
0.6
tt
3
5" i
0.4 0.2 0 7
14
21
Adult
Postnatal Day
FIG. 3. Developmentalchanges of PKC activity in the molecular layer of rat cerebellum. The molecular layer of various ages was homogenized, and PKC activity was assayed as described in Materials and Methods.
the antigen-Ab reaction was largely inhibited by soluble protein extracted from cerebellar blocks of PND 21 specimens. These results suggest that PS binding molecules in extracted protein can protect the epitope of the PS molecule. PKC might be a potential candidate for the protective effect in the interaction between PS and its antibody. Immunohistochemical examination of PMA-treated cerebellum For validation of the above assumption, we perfused adult rat brain with physiological buffer solution containing 200 n M of phorbol 12-myristate 13-acetate (PMA) for 10 min, followed by fixation. The PMA-treated adult cerebellum was stained by monoclonal Abs to PS and PKC. Results are shown in Fig. 5 . After PMA treatment, the staining pattern created by anti-PS Ab changed dramatically. Comparing data in Fig. 5A to the pattern shown in Fig. lD, it is seen that Purkinje cell bodies and the granular layer stained well but the molecular layer did not. Some parts of the thick trunks of Purkinje cells were stained to some extent. Unexpectedly, the staining pattern created by anti-y-PKC Ab faded entirely compared with the data shown in Fig. 2D. The fading is remarkable in the Purkinje cell body. On the other hand, small spots corresponding to stellate and/or basket cells appeared after PMA treatment. In conclusion, the effect of PMA on the staining pattern suggests that PMA can regulate the degree of interaction between PS and probably PKC, i.e., if PMA can translocate only PKC.
found to be different from that of PKC as shown in Fig. 1. Figure 4A and B shows that the reaction between PS and anti-PS Ab was largely blocked by the addition of cytosolic protein obtained from cerebellar slices. These results could explain why PS seemed to be localized in Purkinje cells as shown in Fig. 1. We suggest that staining density docs not necessarily reflect the amount of PS in the tissue, but rather the degree of interaction between PS and PS-binding proteins. It was reported by many authors that PS-binding proteins could interrupt an antigen-anti-PS Ab reaction (Wolf and Sahyoun, 1986; Rybicki et al., 1988; Kaetzel et al., 1989). The most obvious candidate for a PS-binding protein is PKC. Given this, the staining pattern in Fig. 1 could be explained if the interaction in situ between PS and PKC changed with developing stages. A site densely stained by the anti-PS Ab could be a site where PKC was translocated to the membrane and activated in thc presence of membrane PS. Conversely, an unstained part could indicate that
-0 0
5
10
20
Incubation T h e
BI
0.0 0.6 (Y
3 0.4
0.2
-
I
0 .I 0.0 8.0 16.0
CytosoYc Protein
(pgW
DISCUSSION Figures 2 and 3 indicate that immunohistochemicalstaining density is proportional to enzymatic activity. It is likely that staining density is proportional to the amount of PKC, even if the antibody used in this experiment can stain only the y-type PKC. Interestingly, the immunohistochcmical staining of PS was
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FIG. 4. A Time course of inhibition of anti-PS monoclonal Ab binding to PS. The wells were preincubatedwith c y t o s d i protein extract from PND 21 rat [5pg/ml (A); 10pg/rnl (O)], and then were incubated with anti-PS monoclonal Ab (36 pglml). 6:Inhibition of anti-PS monoclonal Ab binding to PS by cytosolic protein extract from PND 7 (A)and PND 21 (0).The wells were prelncubated with cytosolic protein for 10 min at room temperature, and then were incubated with anti-PS Ab (36 pglml).
PKC AND PS IN CEREBELLAR DEVELOPMENT
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FIG. 5. lmmunostainingof adult rat cerebellumtreated with PMA. Sagittal sections of adult rat cerebellumtreated with PMA were stained with anti-PS monoclonal Ab (A) and anti-y-PKC monoclonal Ab (B). The rat cerebellum was perfused with physiological buffer solution containing 200 nM PMA via the left ventricle for 10 min before fixation. Compare data in A and B to control conditions in Figs. 1D and 2D, respectively. Calibration bar = 50 pm.
PKC bound to PS in the membrane and interrupted the interaction between PS and anti-PS Ab. This explanation is supported by several experimental results obtained by other workers. Newton and Koshland ( 1989) indicated that the rates of autophosphorylation and substrate phosphorylation were specifically regulated by PS content in the reaction system. Bazzi and Nelsestuen (1987) reported that PKC showed C22f-dependentbinding to phcspholipid vesic!es containing PS. Finally, Huang and Huang (1 990) found that PS interacted preferentially with the regulatory domain of PKC and resulted in activation of the kinase. This explanation is strongly supported by the results obtained in Fig. 5. When PMA was introduced into the adult rat cerebellum, PKC staining faded markedly, especially in the soma region of Purkinje cells and their surroundings as shown in Fig. 5B. This might be due to down-regulation of PKC by PMA as reported by Nishizuka’s laboratory (Ase et al., 1988a). Although it is not confirmed by biochemical analysis in the experiment presented here, this decrease in staining may represent a reduction in PKC content of Purkinje cells. In this case, the staining by anti-PS Ab of soma was found to be good compared with the data shown in Fig. 1D. All data presented here strongly suggest that anti-PS Ab and PKC recognize competitively the same site of the PS molecule. Granule cells
were also stained by anti-PS Ab after PMA treatment. Although different types of PKC are known to be present in granule cells (Ase et al., 19886; Huang et al., 1988),PS in the granule cells may coexist closely with PKC from PND 14 onward. Therefore, we can estimate the local activity of PKC by the localization of area not stained by anti-PS Ab. According to the above discussion we can conclude that the weak staining by mti-PS Ab represents 2 strong icteraction between PS and PKC, whereas intense anti-PS Ab staining expresses weak interaction between PS and PKC. Therefore, PKC activity can be estimated only by staining intensity with anti-PS Ab but not with antiPKC Ab. Our interpretation of data shown in Fig. 1 (staining of PS) is that PKC activity in the dendrite of the Purkinje cell is activated after PND 14, reaches its maximum at PND 2 1, and is decreased in adulthood. In the soma, however, PKC activity may increase from PND 14 until adulthood as shown in Table 1. Granule cells also showed PND-dependent staining density of PS as shown in Fig. 1 and Table 1. A gradual decrease in PS staining may represent a gradual increase in PKC activity. Acknowledgment: We arc grateful to Prof. Y . Nishizuka, Kobe University, who gave us several types of monoclonal antibodies for PKC and continuous encouragement. We are
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also grateful to Drs. H. Gainer an d R. F. Irvine for critical reading and correction of the manuscript. This work was supported partially by a Grant-in-Aid for Scientific Research o n Priority Areas of “Impulse Signaling” (No. 0 1659002) from the Japanese Ministry of Education, Science, and Culture, and Beckman Japan Co., Ltd.
REFERENCES Ase K., Beny N., Kikkawa U., Kishimoto A., and Nishizuka Y. (19884 Differential down-regulation of protein kinase C s u b species in KM3 cells. FEBS Ideft.236, 396-400. Ase K., Saito N., Shearman M. S., Kikkawa U., Ono Y., Igarashi K., Tanaka C., and Nishizuka Y. (19886) Distinct cellular expression of PI- and SII-subspecies of protein kinase C in rat cerebellum. J. Neurosci. 8, 3850-3856. h i M. D. and Nelsestuen G. L. (1987) Association of protein kinase C with phospholipid vesicles. Biochemistry 26, 1 15122. Garcia-Ladona F. J., Palacios J. M., Girard C., and Combos G. (199 1) Autoradiographic characterization of [3H]-~-glutamate binding sites in developing mouse cerebellar cortex. Neuroscience 41,243-255. Hashimoto T., Ase K., Sawamura S., Kikkawa U., Saito N., Tanaka C., and Nishizuka Y. (1988) Postnatal development ofa brainspecific subspecies of protein kinase C in rat. J. Neurosci. 8, 1678- 1683. Huang K.-P. and Huang F. L. ( 1990) Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation. J. Biol. Chem. 265,738-744. tluang F. L., Yoshida Y., Nakabayashi H., Young W. S., 111, and Huang K.-P. (1988) Immunocytochemical localization of protein kinase C isozymes in rat brain. J. Neurosci. 8,4734-4744. Ito E., Miyazawa A., Takagi H., Yoshioka T., Horikoshi T., Yana-
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gisawa K., Nakamura T., Kudo Y., Umeda M., Inoue K., and Mikoshiba K. (199 1) Developmental assembly ofcalcium-mobilizing systems for excitatory amino acids in rat cerebellum. Neurosci. Res. 11, 179-188. Kaetzel M. A., Hazarika P., and Dedman J. R. (1989) Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins. J. Biol. Chem. 264, 1446314470. Kitano T., Hashimoto T., Kikkawa U., Ase K., Saito N., Tanaka C., Ichimori Y., Tsukamoto K., and Nishizuka Y. (1987) Monoclonal antibodies against rat brain protein kinase C and their application to immunocytochemistry in nervous tissues. J. Neurosci. I, 1520- 1525. Marsh D. (1990) Lipid-protein interactions in membranes. I T B S Lett. 268, 371-375. Miyazawa A., Umeda M., Horikoshi T., Yanagisawa K., Yoshioka T., and lnoue K. (1988) Production and Characterization of monoclonal antibodies that bind to phosphatidylinositol 4,5bisphosphate. Mol. Immunol. 25, 1025-1031. Newton A. C. and Koshland D. E. Jr. (1989) High cooperativity, specificity and multiplicity in the protein kinase C-lipid intcraction. J. Biol. Chem. 264, 14909-14915. Rybicki A. C., Heath R., Lubin B.. and Schwartz R. S . (1988) Human erythrocyte protein 4.1 is a phosphatidylserine binding protein. J. Clin. Invest. 81, 255-260. Umeda M., Diego I., Ball E. D., and Marcus D. M. ( 1986) Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine. J. Immunol. 136,2562-2567. Umeda M., Igarashi K., Nam K. S., and Inoue K. (1989) Effective production of monoclonal antibodies against phosphatidylserine: stereo-specificrecognition of phosphatidylserineby monoclonal antibody. J. Immunol. 143,2273-2279. Wolf M. and Sahyoun N. (1986) Protein kinasc C and phosphatidylserine bind to M, I 1 0,000/lI5,000 polypeptides enriched in cytoskcletal and postsynaptic density preparations. J. Biol. Chem. 261, 13327-13332.
Monoclonal Antibody Analysis of Phosphatidylserine and Protein Kinase C Localizations in Developing Rat Cerebellum Atsuo Miyazawa, Hiroko Inoue, Tohru Yoshioka, *Tetsuro Horikoshi, *Keiji Yanagisawa, TMasato Umeda, and tKeizo Inoue Department of Human Basic Sciences, School of Human Sciences, Waseda University, Tokorozawa; *Department of Physiolou, School of Dental Medicine, Tsurumi University, Yokohama; and ?Department of Health Chemistry, Faculty of Pharmaceutical Science, The University of Tokyo, Tokyo, Japan
Abstract: Understanding the topographical relationships between phosphatidylserine (PS) and protein kinase C (PKC) within neurons can provide clues about the mechanism of translocation and activation of PKC. For this purpose we applied monoclonal antibodies (Abs) of PS and PKC to sections ofdeveloping rat cerebellum. The anti-PKC Ab immunohistochemical pattern showed homogeneous staining of Purkinje cells over various postnatal ages, whereas the antiPS Ab staining showed a heterogeneous localization over these ages. Purkinje cells did not stain well betwcen postnatal day 14 (PND 14) and PND 2 1, suggestingthat the PS was
lost from the membrane during preparation of the sections during this period. These data imply that interactions between PS and PKC vary in Purkinje cells during postnatal development. Key Words: Developing Purkinje cell-Antiprotein kinase C antibody-Anti-phosphatidylserine antibody-Immunohistochemical study-Phosphatidylserineprotein kinase C interaction-Cerebellum. Miyazawa A. et al. Monoclonal antibody analysis of phosphatidylserine and protein kinase C localizations in developing rat cerebellum. J. Neurochem. 59, 1547- 1554 ( 1992).
The study of lipid-protein interaction is important for the analysis of protein kinase and phospholipase function. The structural and dynamic aspects of such lipid-protein interactions have previously been investigated only by physical methods such as spin-labeled electron spin resonance. These methods, however, are applicable only to artificial membrane systems (Marsh, 1990). Recently we made monoclonal antibodies (Abs) to various types of phospholipids (Miyazawa et al., 1988; Umeda et al., 1989) and have applied these Abs to developmental analysis of signal transduction systems (It0 et al., 1991). In the course of our studies, we found we could localize phosphatidylinositol4,5-bisphosphate (PIP,) and phosphatidylserine (PS) on the developingnerve cell by immunohistochemistry. Considering the known lateral diffusion of phospholipid molecules in the membrane, such a localization of phospholipids seems very unlikely. Because the phos-
pholipid molecule itself cannot be fixed by fixative, we believe that the localization of phospholipid obtained with the immunostaining by anti-phospholipid Ab may represent a degree of interaction between phospholipid and protein. In this article, we describe an analysis ofthe localization of PS in the developing cerebellum using monoclonal Ab for PS. The localization of y-type protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37), the dominant PS-binding protein, was also examined at the same ages by using an anti-PS Ab.
Received January 16, 1992; revised manuscript received April 6, 1992; accepted April 13, 1992. Address correspondence and reprint requests to Dr. T. Yoshioka at Department of Human Basic Sciences, School of Human Sciences, Waseda University, 2-579-1 5 Mikajima, Tokorozawa, Saitama 359, Japan. Abbreviafions used: Ab, antibody; BSA, bovine serum albumin;
MATERIALS AND METHODS
Monoclonal Abs The preparation and characterization ofanti-PS monoclonal Ab, designated M A 7 [immunoglobulin M (IgM)], have been described in detail prcviously by Umeda et al. (1989). PS4A7 is highly specific for PS and has shown no
ELISA, enzyme-linkedimmunosorbcnt assay; IgM, immunoglobulin M; PIP,, phosphatidylinositol4,5-bisphosphate;PKC, protein kinase C; PMA, phorbol 12-myhtate 13-acetate;PND, postnatal day; PS,phosphatidylserine;TBS, 50 mMTris-HCI, 150 mMNaCl (pH 7.4);TBS, 10 mM Tris-HC1, 150 mM NaCl (pH 7.4);TCA, trichloroacetic acid.
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A . MIYAZAWA E?' AL.
cross-reaction with other acidic phospholipids. The Ab was purified further by HPLC. Anti-y-PKC monoclonal Ab (clone CKI 97-5) was kindly provided by Prof. Nishizuka of Kobe University. The preparation and characterization of anti-y-PKC monoclonal Ab have been described previously by Kitano et al. (1 987) and Hashimoto et al. ( 1988). This Ab reacted strongly and preferentially with the y-subspecies of PKC.
Immunohistochemistry Wistar rats of the ages indicated were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and killed by perfusion with fixative containing 3% paraformaldehyde and 1% glutaraldehyde in 0.1 Msodium phosphate buffer at pH 7.4 via the left ventricle and washing out from the right atrium. The fixed brain was dissected and placed in the same fixative for more than 24 h at 4°C. Next, the cerebellum was embedded in 5% agar in Tris-buffered saline (TBS; containing 50 mM Tris-HCI, pH 7.4, I50 mM NaCl) and was sectioned sagittally into 50-pm thick slices with a microslicer (Dosaka, Japan). The prepared slices were then washed in TBS. After being pretreated with 1% bovine serum albumin (BSA) in TBS ( 1% BSA-TBS) for 2 h at room temperature, the sections were incubated with the monoclonal Ab (PS4A7, 36 pg/ml) in 1% BSA-TBS for more than 24 h at 4°C. Afler the sections were washed with TBS, they were incubated with horseradish peroxidase conjugated rabbit anti-mouse IgM Ab (Zymed Laboratories, South San Francisco, CA, U.S.A.) in 1% BSA-TBS for 2 h at room temperature. After the sections were washed with TBS again, the staining procedure was performed using a solution containing 0.1% diaminobenzidine tetrahydrochloride and 0.22% hydrogen peroxide in 50 mMTris-€ICI at pH 7.4. In the case of immunohistochemistry for y-PKC, the procedures were essentially the same as described in previous articles with a slight modification (Hashimoto et al., 1988). In brief, rats of various ages were perfused through the aorta with a fixative containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. The fixed cerebellum was dissected and washed with 30% sucrose in TBS for several days at 4°C. Sagittal sections (1 3 pm thick) were cut on a cryostat (Leitz, Wetzlar, F.R.G.) and collected on coverslips. The sections were incubated overnight at 4°C with anti-y-PKC monoclonal Ab (CKI 97-5, 1 :200 dilution), washed with TBS, and were then stained using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, U.S.A.).
Preparation of PS-liposome to absorb anti-PSAb The small unilamellar liposomes of PS were prepared by the sonication of the multilamellar liposome. The dried lipid film containing 2.5 pmol of PS was swollen in TBS and was sonicated for 5 min at 4°C under a nitrogen flow. This solution was centrifuged at 10,000 g for 10 min at 4°C. Next, the supernatant was used for absorption of antiPS Ab.
Assay of PKC activity Wistar rats of the ages indicated were anesthetized and their cerebella were dissected and sectioned sagittally into 2 0 0 - ~ mthick slices with a microslicer in artificial CSF containing 1 13 mM NaCI, 3 m M KCI, 10 Mglucose, I m M NaHPO,,, 1 mM MgCI,, 2 mM CaCI,, 25 m M NaHCO,, and gassed with 0,/C02 (95%/5%). Under a binocular mi-
J. Neurochem.. Vol. 59, No. 4, 1992
croscope, white matter including the granular layer was manually removed by razor from each slice, then residual parts including molecular layer were collected and frozen in liquid nitrogen. The cortices of the cerebellum were homogenized with buffer A (20 mMTris-HC1, pH 7.2; 10 mM MgCI,; 2 m M EDTA; 10 mM EGTA; 50 pM phenylmethylsulfonyl fluoride; 50 pg/ml of leupeptin; and 2.5 pg/ml of pepstatin) and centrifuged at 100,000g for 60 rnin at 2°C. The supernatant was decanted, and the pellet was resuspended with buffer A containing 0.1% Triton X-100. PKC activity was determined in the reaction medium containing 20 m M Tris-HC1, pH 7.4; 10 mMMgC1,; 0.01%Triton X-100; 50 pg of histone; 10 p M [32P]ATP;0.5 mA4 CaCI,; 20 pg of PS, 2 pg of diolein; and enzyme in 100 p1 for 15 rnin at 25°C. The Ca2'/PS-independent kinase activity was measured under the same conditions without Ca2+,PS, and diolein, but with 1 mMEGTA. The reaction was terminated by addition of 1 ml of 25% trichloroacetic acid (TCA) and acid-precipitable materials were collected by a filter. The filter was washed three times with 5% TCA and radioactivity was counted.
Enzyme-linked immunosorbent assay (ELISA) for inhibition of Ab binding Wistar rats were anesthetized with sodium pentobarbital and perfused through the left ventricle. The blood was washed out with 50 ml of Ringer solution. The perfused cerebellum was dissected and cut into blocks, which were then frozen in liquid nitrogen. The frozen cerebellar blocks were homogenized with 50 mM Tris-HC1 buffer, pH 7.4, containing 1 mM EGTA, 200 mM phenylmethylsulfonyl fluoride, and 0.3% mercaptoethanol. The homogenate was centrifuged at 100,000g for 60 rnin at 2"C, then the supernatant containing cytosolic proteins was submitted to ELISA. ELISA for the lipid haptens was performed as described previously by Umeda et al. (1986). The wells of the microtiter plates were coated with 50 pl of PS in ethanol (10 p M ) by evaporation overnight at room temperature. The nonspecific binding sites on the wells were blocked by 10 M T r i s HCI, 150 mM NaCl (pH 7.4) buffer (TBS) containing 30 mg/ml of BSA for 60 rnin at room temperature. After the plates were washed with T'BS, the wells were incubated with 100 pl of the cytosolic proteins at room temperature. The plates were washed once again, and 50 pI of the anti-PS monoclonal Ab (PS4A7) diluted with T'BS containing 10 mg/ml of BSA was poured into the wells, which were allowed to stand for 2 h at room temperature. The bound Ab was detected by biotinylated anti-mouse IgM Ah (Zymed Laboratories), followed by incubation with peroxidase-conjugated streptavidin (Zymed Laboratories). Optical density at 492 nm was measured by addition of o-phcnylencdiamine as substrate.
RESULTS Developmental changes of PS localization in cerebellum Immunoreactivity of anti-PS Ab was demonstrated on germinal cells (except for the external layer) at postnatal day 0 (PND 0). When Purkinje cells start to align on PND 3, the anti-PS immunoreactivity was
PKC AND PS IN CEREBELLAR DEVELOPMENT
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FIG. 1. lmmunostainingof rat cerebellum at various ages with anti-PS monoclonal Ab. Sagittal sections of rat cerebellumat various ages were stained with anti-PS monoclonalAb PS4A7. The samples were from (A) PND 7, (B) PND 14, (C)PND 21, and (D) adult rat cerebellum. E: Absorption of anti-PS monoclonalAb with PS-liposome in adult rat cerebellum. The imrnunoreactivityof anti-PS monoclonalAb (0.4 pM) was markedly reduced by absorption with an excess amount of PS-liposome (500 pM PS). Calibration bar 50 pn. 2
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extended to the cytoplasm. On PND 5, Purlcinje cells showed strong immunoreactivity in their cytoplasm as well as nucleus. In this case weak immunoreactivity of anti-PS was observed in the molecular layer of rat cerebellum. In the external germinal layer, no anti-PS immunoreactivity was observed in the premigratory granule cells. In the internal granular layer, some cells showed strong immunoreactivity in their cytoplasm and nucleus, but the others showed only weak staining in their cytoplasm. By PND 7, immunoreactivities were well observed in Purkinje cells, stellate and basket cells, and also in fully migrated granule cells (Fig. 1A). The pattern of anti-PS Ab immunostaining on PND 10 was almost the same as that of PND 7, but immunoreactivity was markedly decreased in the granule cells (data not shown). On PND 14 and 2 1, the anti-PS Ab immunostaining pattern became ambiguous and faded but the pattern was the same as that of thc adult cxcept for the existence of premigratory granule cells (Fig. 1B and C). This differs from the case of the anti-PIP, Ab staining at the distal part of the Purkinje dendrite, which transiently disappears around PND 14 (Ito et al., 1991). The anti-PS Ab immunoreactivity on the Purkinje dendrites was always present during PND 14-2 I , although the amount of PS seemed very low. When we treated these preparations with chloroform/ methanol solution, we could extract the same amount of PS from fixed preparations at different developmental stages (data not shown). In the case of adult rat cerebellum, most of the Purkinje cell was stained intensely except cell body (Fig. 1D). To examine if this staining is due to antigen (PS)-Ab interaction, anti-PS Ab was absorbed by PSliposome in an adult rat cerebellum, and sections were sliced and stained. As shown in Fig. 1E, no staining pattern was observed. The staining pattern data
for PS in developing rat cerebellum is summarized in Table 1. Developmental changes in the immunohistochemical localization of yPKC in cerebellar slices As the immunoreactivity of both anti-cy- and anti0-PKC Abs was found to be poor in Purkinje cells (data not shown), we concentrated at anti-y-PKC Ab in the following experiments. Results obtained in this study agreed well with those in Nishizuka’s laboratory, but we obtained some additional new findings in the developing cerebellum. Anti-y-PKC Ab immunoreactivity appeared in Purkinje cells from PND 3 through the adult stage, and it was also found to be present in basket and stellate cells from PND 14 to PND 21, but not in the adult stage. The fine dendritic arborizations of the Purkinje cells were clearly detected by the anti-y-PKC Ab immunostaining from PND 7 to the adult stage (Fig. 2A-D). Strong immunoreactivity was also seen in the axonal tracts of Purkinje cells from PND 7 to PND 14, but not clearly in axons from PND 2 1 (Fig. 2C) to the adult stage. At PND 10 and 14, a number of small varicosities were stained on the Purkinje cell axons. Purkinje cell nuclei stained weakly from PND 3 to the adult stage. The immunoreactivity of basket cells was stronger than that of stellate cells throughout the stage after PND 14. In these cells immunoreactivity of anti-y-PKC Ab was observed both in the nucleus and the cytoplasm. We can estimate that the quantity of PKC in the adult rat may be smaller than that of PND 21 rat, because the staining density shown in Fig. 2D seems faded compared with that of Fig. 2C. We confirmed this by direct measurement of PKC activity in the molecular layer of developing rat cerebellum. Results are shown in Fig. 3. As was expected from immunohistochemical data shown in Fig. 2, PKC activity in the
TABLE 1. Localization and intensity of anti-PS immunoreactivity in developing rat cerebellum PND Region External germinal layer Germinal cell Glial fiber Molecular layer Stellate and basket cell Bergmann fiber Purkinje cell Dendrite Perikaryon Axon Granular layer Granule cell Golgi cell Germinal cell
0 -
3
5
7
-
-
-
+
+
n n
n n
+ n -
+ +++
+
+
n
n
t+
+-i-+
n
n
n
n
n
n n
+
+++
n
n
++
+t
n
-t+
++t
+++
++ ++
10
++ + +++ +++ n
+ + ++
n, not identified; -, no reactivity; t,weak reactivity; ++, medium reactivity; +++, strong reactivity.
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21
Adult
n n
n n
++
1-
+ ++ +-
++ +++ -
n
n
n
14
++ ++
++ -
+ n
-
+ n
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FIG. 2. lmmunostaining of rat cerebellum at various ages with anti-y-PKC monoclonal Ab. Sagittal sections of rat cerebellum at various ages were stained with anti-y-PKC monoclonalAb CKI 97-5. The samples were from (A) PND 7, (B) PND 14, (C)PND 21, and (D) adult rat cerebellum. Arrows indicate some interneurons such as basket and stellate cells in the molecular layer of the cerebellum. Calibration bar 50 pm. 2:
molecular layer reached a maximum value on PND 21 and decreascd markedly in adulthood, although the activity of PKC shown in Fig. 3 is the summation of all types of isozyme. Inhibition of the interaction between anti-PS Ab and PS by cytosolic protein The inhibitory effect of cytosol fraction obtained from rat cercbellum on the interaction of PS with anti-PS Ab was examined on ELISA. When the cytosol fraction was introduced into the reaction prior to the reaction of PS with the anti-PS Ab, the interaction between the two was inhibited in a concentration-dc-
pendent manner. To determine the optimum reaction period at room temperature, we performed a time course analysis for the inhibition reaction. As shown in Fig. 4A, an incubation period of 10 min was found to be enough. Inhibition of the interaction between PS and anti-PS Ab by the cytosol fraction was examined using cerebellar blocks of PND 7 and PND 2 1, because the PKC activity was quite different between the two. The results are shown in Fig. 4B. The amount of antigen that reacted with anti-PS Ab was expressed by the optical density of reaction medium. When the cytosol fraction was heated at 60°C for 1 h, the inhibitory effect disappeared. As shown in Fig. 4,
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A . MIYAZAWA ET AL.
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r
I :
3 0
0.8
0.6
tt
3
5" i
0.4 0.2 0 7
14
21
Adult
Postnatal Day
FIG. 3. Developmentalchanges of PKC activity in the molecular layer of rat cerebellum. The molecular layer of various ages was homogenized, and PKC activity was assayed as described in Materials and Methods.
the antigen-Ab reaction was largely inhibited by soluble protein extracted from cerebellar blocks of PND 21 specimens. These results suggest that PS binding molecules in extracted protein can protect the epitope of the PS molecule. PKC might be a potential candidate for the protective effect in the interaction between PS and its antibody. Immunohistochemical examination of PMA-treated cerebellum For validation of the above assumption, we perfused adult rat brain with physiological buffer solution containing 200 n M of phorbol 12-myristate 13-acetate (PMA) for 10 min, followed by fixation. The PMA-treated adult cerebellum was stained by monoclonal Abs to PS and PKC. Results are shown in Fig. 5 . After PMA treatment, the staining pattern created by anti-PS Ab changed dramatically. Comparing data in Fig. 5A to the pattern shown in Fig. lD, it is seen that Purkinje cell bodies and the granular layer stained well but the molecular layer did not. Some parts of the thick trunks of Purkinje cells were stained to some extent. Unexpectedly, the staining pattern created by anti-y-PKC Ab faded entirely compared with the data shown in Fig. 2D. The fading is remarkable in the Purkinje cell body. On the other hand, small spots corresponding to stellate and/or basket cells appeared after PMA treatment. In conclusion, the effect of PMA on the staining pattern suggests that PMA can regulate the degree of interaction between PS and probably PKC, i.e., if PMA can translocate only PKC.
found to be different from that of PKC as shown in Fig. 1. Figure 4A and B shows that the reaction between PS and anti-PS Ab was largely blocked by the addition of cytosolic protein obtained from cerebellar slices. These results could explain why PS seemed to be localized in Purkinje cells as shown in Fig. 1. We suggest that staining density docs not necessarily reflect the amount of PS in the tissue, but rather the degree of interaction between PS and PS-binding proteins. It was reported by many authors that PS-binding proteins could interrupt an antigen-anti-PS Ab reaction (Wolf and Sahyoun, 1986; Rybicki et al., 1988; Kaetzel et al., 1989). The most obvious candidate for a PS-binding protein is PKC. Given this, the staining pattern in Fig. 1 could be explained if the interaction in situ between PS and PKC changed with developing stages. A site densely stained by the anti-PS Ab could be a site where PKC was translocated to the membrane and activated in thc presence of membrane PS. Conversely, an unstained part could indicate that
-0 0
5
10
20
Incubation T h e
BI
0.0 0.6 (Y
3 0.4
0.2
-
I
0 .I 0.0 8.0 16.0
CytosoYc Protein
(pgW
DISCUSSION Figures 2 and 3 indicate that immunohistochemicalstaining density is proportional to enzymatic activity. It is likely that staining density is proportional to the amount of PKC, even if the antibody used in this experiment can stain only the y-type PKC. Interestingly, the immunohistochcmical staining of PS was
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FIG. 4. A Time course of inhibition of anti-PS monoclonal Ab binding to PS. The wells were preincubatedwith c y t o s d i protein extract from PND 21 rat [5pg/ml (A); 10pg/rnl (O)], and then were incubated with anti-PS monoclonal Ab (36 pglml). 6:Inhibition of anti-PS monoclonal Ab binding to PS by cytosolic protein extract from PND 7 (A)and PND 21 (0).The wells were prelncubated with cytosolic protein for 10 min at room temperature, and then were incubated with anti-PS Ab (36 pglml).
PKC AND PS IN CEREBELLAR DEVELOPMENT
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FIG. 5. lmmunostainingof adult rat cerebellumtreated with PMA. Sagittal sections of adult rat cerebellumtreated with PMA were stained with anti-PS monoclonal Ab (A) and anti-y-PKC monoclonal Ab (B). The rat cerebellum was perfused with physiological buffer solution containing 200 nM PMA via the left ventricle for 10 min before fixation. Compare data in A and B to control conditions in Figs. 1D and 2D, respectively. Calibration bar = 50 pm.
PKC bound to PS in the membrane and interrupted the interaction between PS and anti-PS Ab. This explanation is supported by several experimental results obtained by other workers. Newton and Koshland ( 1989) indicated that the rates of autophosphorylation and substrate phosphorylation were specifically regulated by PS content in the reaction system. Bazzi and Nelsestuen (1987) reported that PKC showed C22f-dependentbinding to phcspholipid vesic!es containing PS. Finally, Huang and Huang (1 990) found that PS interacted preferentially with the regulatory domain of PKC and resulted in activation of the kinase. This explanation is strongly supported by the results obtained in Fig. 5. When PMA was introduced into the adult rat cerebellum, PKC staining faded markedly, especially in the soma region of Purkinje cells and their surroundings as shown in Fig. 5B. This might be due to down-regulation of PKC by PMA as reported by Nishizuka’s laboratory (Ase et al., 1988a). Although it is not confirmed by biochemical analysis in the experiment presented here, this decrease in staining may represent a reduction in PKC content of Purkinje cells. In this case, the staining by anti-PS Ab of soma was found to be good compared with the data shown in Fig. 1D. All data presented here strongly suggest that anti-PS Ab and PKC recognize competitively the same site of the PS molecule. Granule cells
were also stained by anti-PS Ab after PMA treatment. Although different types of PKC are known to be present in granule cells (Ase et al., 19886; Huang et al., 1988),PS in the granule cells may coexist closely with PKC from PND 14 onward. Therefore, we can estimate the local activity of PKC by the localization of area not stained by anti-PS Ab. According to the above discussion we can conclude that the weak staining by mti-PS Ab represents 2 strong icteraction between PS and PKC, whereas intense anti-PS Ab staining expresses weak interaction between PS and PKC. Therefore, PKC activity can be estimated only by staining intensity with anti-PS Ab but not with antiPKC Ab. Our interpretation of data shown in Fig. 1 (staining of PS) is that PKC activity in the dendrite of the Purkinje cell is activated after PND 14, reaches its maximum at PND 2 1, and is decreased in adulthood. In the soma, however, PKC activity may increase from PND 14 until adulthood as shown in Table 1. Granule cells also showed PND-dependent staining density of PS as shown in Fig. 1 and Table 1. A gradual decrease in PS staining may represent a gradual increase in PKC activity. Acknowledgment: We arc grateful to Prof. Y . Nishizuka, Kobe University, who gave us several types of monoclonal antibodies for PKC and continuous encouragement. We are
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also grateful to Drs. H. Gainer an d R. F. Irvine for critical reading and correction of the manuscript. This work was supported partially by a Grant-in-Aid for Scientific Research o n Priority Areas of “Impulse Signaling” (No. 0 1659002) from the Japanese Ministry of Education, Science, and Culture, and Beckman Japan Co., Ltd.
REFERENCES Ase K., Beny N., Kikkawa U., Kishimoto A., and Nishizuka Y. (19884 Differential down-regulation of protein kinase C s u b species in KM3 cells. FEBS Ideft.236, 396-400. Ase K., Saito N., Shearman M. S., Kikkawa U., Ono Y., Igarashi K., Tanaka C., and Nishizuka Y. (19886) Distinct cellular expression of PI- and SII-subspecies of protein kinase C in rat cerebellum. J. Neurosci. 8, 3850-3856. h i M. D. and Nelsestuen G. L. (1987) Association of protein kinase C with phospholipid vesicles. Biochemistry 26, 1 15122. Garcia-Ladona F. J., Palacios J. M., Girard C., and Combos G. (199 1) Autoradiographic characterization of [3H]-~-glutamate binding sites in developing mouse cerebellar cortex. Neuroscience 41,243-255. Hashimoto T., Ase K., Sawamura S., Kikkawa U., Saito N., Tanaka C., and Nishizuka Y. (1988) Postnatal development ofa brainspecific subspecies of protein kinase C in rat. J. Neurosci. 8, 1678- 1683. Huang K.-P. and Huang F. L. ( 1990) Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation. J. Biol. Chem. 265,738-744. tluang F. L., Yoshida Y., Nakabayashi H., Young W. S., 111, and Huang K.-P. (1988) Immunocytochemical localization of protein kinase C isozymes in rat brain. J. Neurosci. 8,4734-4744. Ito E., Miyazawa A., Takagi H., Yoshioka T., Horikoshi T., Yana-
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gisawa K., Nakamura T., Kudo Y., Umeda M., Inoue K., and Mikoshiba K. (199 1) Developmental assembly ofcalcium-mobilizing systems for excitatory amino acids in rat cerebellum. Neurosci. Res. 11, 179-188. Kaetzel M. A., Hazarika P., and Dedman J. R. (1989) Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins. J. Biol. Chem. 264, 1446314470. Kitano T., Hashimoto T., Kikkawa U., Ase K., Saito N., Tanaka C., Ichimori Y., Tsukamoto K., and Nishizuka Y. (1987) Monoclonal antibodies against rat brain protein kinase C and their application to immunocytochemistry in nervous tissues. J. Neurosci. I, 1520- 1525. Marsh D. (1990) Lipid-protein interactions in membranes. I T B S Lett. 268, 371-375. Miyazawa A., Umeda M., Horikoshi T., Yanagisawa K., Yoshioka T., and lnoue K. (1988) Production and Characterization of monoclonal antibodies that bind to phosphatidylinositol 4,5bisphosphate. Mol. Immunol. 25, 1025-1031. Newton A. C. and Koshland D. E. Jr. (1989) High cooperativity, specificity and multiplicity in the protein kinase C-lipid intcraction. J. Biol. Chem. 264, 14909-14915. Rybicki A. C., Heath R., Lubin B.. and Schwartz R. S . (1988) Human erythrocyte protein 4.1 is a phosphatidylserine binding protein. J. Clin. Invest. 81, 255-260. Umeda M., Diego I., Ball E. D., and Marcus D. M. ( 1986) Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine. J. Immunol. 136,2562-2567. Umeda M., Igarashi K., Nam K. S., and Inoue K. (1989) Effective production of monoclonal antibodies against phosphatidylserine: stereo-specificrecognition of phosphatidylserineby monoclonal antibody. J. Immunol. 143,2273-2279. Wolf M. and Sahyoun N. (1986) Protein kinasc C and phosphatidylserine bind to M, I 1 0,000/lI5,000 polypeptides enriched in cytoskcletal and postsynaptic density preparations. J. Biol. Chem. 261, 13327-13332.
