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Developmental expression of the protein kinase C family in rat hippocampus
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 78 (1994) 291-295
Short Communication
Developmental expression of the protein kinase C family in rat hippocampus Xiaolan Jiang a, Meghna U. Naik a, Jan Hrabe b, Todd Charlton Sacktor ~'* " Laboratory of Molecular Neuroscience, Departments of Pharmacology and Neurology, Box 29, SUNY Health Science Center at Brooklyn, Brooklyn, N Y 11203, USA, b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, N Y 10461. USA (Accepted 30 November 1993)
Abstract
Protein kinase C (PKC) is a heterogeneous family of ten or more isoforms which plays an important role in neuronal signal transduction. Isoforms from all subclasses are prominently expressed in the rat hippocampus, as demonstrated by immunoblot with isozyme-specific antisera: Ca2+-dependent (a, /3I, /3II and y), CaZ+-independent (& e and a newly characterized PKC related to 7) and atypical (~'). In addition, the ~" isoform is also found as the free, constitutively active catalytic domain, protein kinase M ~" (PKM~). Two distinct patterns of expression of PKC isozymes in rat hippocampus are found during development from El8 to P28. PKC~', PKM( and PKCa are present at birth and their expression does not increase postnatally. In contrast, the other isoforms are expressed only at low levels at birth and then increase in the first 4 weeks postnatally. These two patterns of expression suggest distinct functions for PKC isozymes during development. Key words: Protein kinase M; Zeta; Synaptic plasticity; Phosphorylation; Long-term potentiation
The PKC family of s e r i n e / t h r e o n i n e protein kinases is thought to regulate both neuronal development and synaptic transmission. Nishizuka [9] has recently classified the isozymes of PKC into three groups: 'conventional', CaZ+-dependent PKC's (a, /3I, /3II, y), 'new', Ca2+-independent PKC's (6, e, rt, 0) and 'atypical' PKC's (~', A). These groups are activated by different signal transduction mechanisms (reviewed in ref. 9). Conventional PKC's possess a region (C2), which binds Ca 2+ in the presence of lipid. The new PKC's lack C2, do not require Ca 2+ for activation and share with the conventional PKC's a diacylglycerol/phorbol ester binding site (C1), containing two cysteine-rich regions. Atypical PKC's contain only a single cysteine-rich region, do not appear to bind phorbol esters [10] and may be activated by products of phosphatidylinositol 3-kinase [8]. In addition to these species, a high-molecular weight PKC [17] has recently been characterized. This PKC, which is expressed in brain [17], is recognized by antiserum to the C4-V5 region of ft. The
* Corresponding author. Fax: (1) (718) 270-2241. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 3 ) E 0 2 1 1 - 3
latter isozyme is expressed predominantly in lung and skin [11], but negligibly in neural tissue [17]. In addition to the heterogeneity produced by multiple genes for PKC, the ~" isozyme is expressed in brain as both PKC~" and PKM~', the free, constitutively active catalytic domain of the isozyme [14]. While PKM may be produced from several PKC isozymes in vitro by proteolytic removal of the amino-terminal regulatory domain [6], the endogenous formation of PKM in hippocampus appears to be largely specific to the ,~ isozyme. PKM~" increases in the maintenance phase of LTP in the CA1 region of the hippocampus [14], suggesting a specific role for this constitutively-active form of PKC in long-term modifications of synaptic efficacy. In order to delineate further the potential functional roles of PKMg" and the individual isoforms of PKC, we examined their expression during rat hippocampal development. PKC isozyme-specific antisera were made as previously described [14] against carboxy-terminal sequences of each of the PKC isoforms: a: PQFVHPILQSAV, /3I: SYTNPEFVINV, /3II: SFVNSEFLKPEVKS, -/: PDARSPTSPVPVPVM, 6: N P K Y E Q F L E , e: Y F G E D L M P , ~: EYINPLLLSAEESV, rt: QTSTKQK-
292
X. Jiang et aL / Decelopmental Brain Research 78 (1994) 291-295
T N K P T Y N E E F C . The rabbit polyclonal antisera were obtained by injection of the peptides conjugated to maleimide-activated bovine serum albumin (Pierce) and emulsified in Titermax (Vaxcel). All antisera were affinity-purified with SulfoLink Coupling Gel (Pierce). The postnatal levels of PKC isozymes were determined by four experiments, each consisting of 13 rats of different ages from a single litter. After anaesthesia with ketamine (20 m g / m l , 0.2-0.3 ml/rat), the hippocampi were dissected immediately after decapitation, frozen and stored in liquid nitrogen. The hippocampi were homogenized by hand (40 strokes at 4°C) in buffer containing Tris-HCl, pH 7.5 (50 raM), E D T A (1 mM), E G T A (1 mM), 2-mercaptoethanol (5 mM), phenylmethylsulfonyl fluoride (0.1 mM), aprotinin (16.7 kallikrein units/rot), benzamidine (5 mM) and leupeptin (0.1 mM) and centrifuged at 4000 g for 15 min. All chemicals, unless specified, were from Sigma. Total protein of the supernatant was determined by a modification of the Bradford assay [13,16]. Fifty ~zg/cm-lane of the total supernatant protein from different devel-
E18
P1
opmental stages were loaded onto 8% SDS-polyacrylamide gels [7] and examined by immunoblot. The difference in levels of PKC isozymes between CA1, CA3 and dentate gyrus regions of the hippocampus were modest (data not shown); therefore, total hippocampus was used in all experiments for consistency. Immunoblotting was performed as previously described [18]. The nitrocellulose lane of each sample was cut into eight strips, each of which was incubated with an isozyme-specific primary antibody for 1 h, followed by alkaline phosphatase-conjugated anti-rabbit goat IgG (1:2500; Promega) for 1 h. The immunoreactive bands were visualized with B C I P / N B T phosphatase substrate (Kirkegaard and Perry Laboratories). The bands were quantified by densitometry with an XRS 6cx scanner (OmniMedia) using NIH Image software. We used a method that is both quantifiable and allows different experiments to be compared with each other. The immunoblots were in the linear range of detection for each antiserum with respect to protein loaded on the gel. Since, however, the time for devel-
7
14
21
28
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? q-related 6 PKC( PKM( : i Fig. 1. Representative immunoblots showing PKC isozymes during rat hippocampal development from El8 to P28. All postnatal hippocampal samples were from rats of a single litter. E18 hippocampus was from a single rat of a separate litter, analyzed on a separate immunoblot and is shown magnified 3 x . The molecular weights for each isozyme (lower band for doublets) are in kDa: a, 80; /3I, 77; flII, 79; T, 79; e, 89; r/-related, 97; 8, 76; PKC~', 70; PKM~r, 51.
X. Jiang et al. / Deuelopmental Brain Research 78 (1994) 291-295
293
except that the mean values for each experiment were preset to 1.5 instead of zero. Hence, the resulting normalized data set for each experiment has a standard deviation equal to 1 and a mean equal to 1.5. To
opment of the colored substrate may vary among the four experiments, the corresponding optical densities could not be combined. Therefore, we applied the normalization procedure employed in factor analysis,
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Fig. 2. Time-courses of postnatal PKC isozyme expression in rat hippocampus. Data from four separate experiments were normalized as described in text. Y-axis scalings are in arbitrary units. Mean values for each day examined, standard errors and fitted regression lines are shown for each isozyme. The slopes of the regression lines for each isozyme were: a, 0.0872*; /3I, 0.0897*; flII, 0.0955*; y, 0.1069"; e, 0.0557*; v-related, 0.0900*; 6, 0.0065; PKC#, -0.0485*; PKM~', -0.0105. (* denotes values significantly different from 0, P = 0.01; the regression line for PKCsr has a significant negative slope.)
294
x. Jiang et al. / Developmental Brain Research 78 (1994) 291-295
determine whether the isozymes changed significantly during development, we applied the simple linear regression model to the time course of each isozyme. The antisera detected specific PKC isozymes as single molecular species or doublets [14], with three exceptions: (1) as noted above, antiserum to the carboxyterminus of P K C r / d e t e c t e d primarily the high molecular weight PKC and only low levels of B in hippocampus; the latter was not measured; (2) PKC6 may be present as a minor P K M form in hippocampus, but preliminary evidence suggests that, in contrast to ~', proteolysis during homogenization may contribute to a portion of P K M 6 (unpublished observations.); (3) the multiple forms of ~', previously described [14]. (PKC0, which is predominantly expressed in muscle [12], was found only at low levels in hippocampus with antiserum provided by Dr. Shin-ichi Osada, Yokohama University [data not shown.] Antiserum to PKCA is not currently available.) Earlier reports examining the Ca2+-dependent PKC isozymes in extracts of total brain [3,19] or in cerebellum [4] found relatively low levels of expression of these isoforms at birth and a steady rise postnatally, with P K C y expression beginning later than the other Ca2+-dependent isozymes. Our results in the hippocampus are consistent with these findings. However, when we analyze the full complement of isozymes in hippocampus, we find a markedly different developmental profile for three forms of PKC: PKC6, PKC~" and PKM~" (Figs. 1,2). Regression analysis (Fig. 2) indicates that the patterns of developmental expression of the isozymes can be divided into two groups: the first group ( a , / 3 I , / 3 I I , y, E and the B-related PKC) is expressed at low levels at birth but increases significantly during postnatal development; the second group ( P K C ( , PKM~', and P K C 6 ) is expressed at birth and does not increase significantly through postnatal development. P K C sr is detectable as early as El8, the earliest time point measured. (PKC~" may be decreasing with time postnatally, but the significance of the negative slope is highly influenced by the values for the first postnatal day.) The association of PKC~" and PKC6 is further substantiated by the observation, as noted above, that P K C a may also have a PKM form. These two patterns of expression suggest discrete roles for individual PKC isozyrnes during development. Isozymes that are present prenatally may be required for developmental processes that occur primarily prior to birth, such as neuronal proliferation and migration. This notion is supported by the observation of the importance of the ~" isozyme during the regulation of maturation in X e n o p u s oocytes [1,2] and mitosis in mouse fibroblasts [1]. The appearance of the other isozymes postnatally correlates with the rapid outgrowth of neuronal processes. The similarity of the developmental profiles of this group suggests a coordi-
nate regulation. It is curious that PKMff is not increased during this period of postnatal synapse formation, considering its potential role in activity-dependent synaptic plasticity. However, changes in PKM~" that may be accrued with experience are likely to be subtle, and specific to subsets of neurons. Stable levels during postnatal development may be particularly relevant for a constitutively active enzyme such as PKMff; its phosphotransferase activity is presumably controlled by its concentration in the cell, not by the levels of second messengers (Ca 2 + or diacylglycerol), which regulate the typical PKC's. It has been hypothesized that the molecular mechanisms that regulate synaptic efficacy during learning and memory may also control neuronal development [5,15]. We find an increasing expression of typical isoforms during the period of synapse formation, in contrast to the stable levels of the atypical ~" isoform, which may participate in LTP maintenance. These results suggest that the plasticity of synapses during development and learning may utilize related but distinct signal transduction pathways. [1] Berra, E., Diaz-Meco, M.T., Dominguez, I., Municio, M.M., Sanz, L., Lozano, J., Chapkin, R.S. and Moscat, J., Protein kinase C ~ isoform is critical for mitogenic signal transduction, Cell, 74 (1993) 555-563. [2] Dominguez, I., Diaz-Meco, M.T., Municio, M.M., Berra, E., de Herreros, A.G., Cornet, M.E., Sanz, L. and Moscat, J., Evidence for a role of protein kinase C ( subspecies in maturation of Xenopus laevis oocytes, Mol. Cell. Biol., 12 (1992) 3776-3783. [3] Hashimoto, T., Ase, K., Sawamura, S., Kikkawa, U., Saito, N., Tanaka, C. and Nishizuka, Y., Postnatal development of a brain-specific subspecies of protein kinase C in rat, J. Neurosci., 8 (1988) 1678-1683. [4] Huang, F.L., Young, W.S., Yoshida, Y. and Huang, K.-P., Developmental expression of protein kinase C isozymes in rat cerebellum, Dev. Brain Res., 52 (1990) 121-130. [5] Kandel, E.R. and O'DelI, T.J., Are adult learning mechanisms also used for development?, Science, 258 (1992) 243-245. [6] Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S.-i., Tominaga, M., Kuroda, T. and Nishizuka, Y,, Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J. Biol. Chem., 264 (1989) 4088-4092. [7] Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. [8] Nakanishi, H., Brewer, K.A. and Exton, J.H., Activation of the ( isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate, J. Biol. Chem., 268 (1993) 13-16. [9] Nishizuka, Y., Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607-614. [10] Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. and Nishizuka, Y., Protein kinase C ~ subspecies from rat brain: its structure, expression and properties, Proc. Natl. Acad. Sci. USA, 86 (1989) 3099-3103. [11] Osada, S., Mizuno, K., Saido, T.C., Akita, Y., Suzuki, K., Kuroki, T. and Ohno, S., A phorbol ester receptor/protein kinase, nPKCrl, a new member of the protein kinase C family predominantly expressed in lung and skin, J. Biol. Chem., 265 (1990) 22434-22440.
X. Jiang et aL / Det~elopmental Brain Research 78 (1994) 291-295 [12] Osada, S.-i., Mizuno, K., Saido, T.C., Suzuki, K., Kuroki, T. and Ohno, S., A new member of the protein kinase C family, nPKC0, predominantly expressed in skeletal muscle, MoL Cell BioL, 12 (1992) 3930-3938. [13] Read, S.M. and Northcote, D.H., Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein, Anal Biochem., 116 (1981) 53-64. [14] Sacktor, T.C., Osten, P., Valsamis, H., Jiang, X., Naik, M.U. and Sublette, E., Persistent activation of the ~" isoform of protein kinase C in the maintenance of long-term potentiation, Proc. NatL Acad. Sci. USA, 90 (1993) 8342-8346. [15] Schatz, C.J., Impulse activity and the patterning of connections during CNS development, Neuron, 10 (1990) 745-756. [16] Simpson, I.A. and Sonne, O., A simple, rapid and sensitive
295
method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation, Anal Biochem., 119 (1982)424-427. [17] Sublette, E., Naik, M., Jiang, X. and Sacktor, T.C., Evidence for a new isoform of PKC specific to rat brain, Neurosci. Lett., 159 (1993) 175-178. [18] Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 79 (1979) 4350-4354. [19] Yoshida, Y., Huang, F.L., Nakabayashi, H. and Huang, K.-P., Tissue distribution and developmental expression of protein kinase C isozymes, J. BioL Chem., 263 (1988) 9868-9873.
ELSEVIER
Developmental Brain Research 78 (1994) 291-295
Short Communication
Developmental expression of the protein kinase C family in rat hippocampus Xiaolan Jiang a, Meghna U. Naik a, Jan Hrabe b, Todd Charlton Sacktor ~'* " Laboratory of Molecular Neuroscience, Departments of Pharmacology and Neurology, Box 29, SUNY Health Science Center at Brooklyn, Brooklyn, N Y 11203, USA, b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, N Y 10461. USA (Accepted 30 November 1993)
Abstract
Protein kinase C (PKC) is a heterogeneous family of ten or more isoforms which plays an important role in neuronal signal transduction. Isoforms from all subclasses are prominently expressed in the rat hippocampus, as demonstrated by immunoblot with isozyme-specific antisera: Ca2+-dependent (a, /3I, /3II and y), CaZ+-independent (& e and a newly characterized PKC related to 7) and atypical (~'). In addition, the ~" isoform is also found as the free, constitutively active catalytic domain, protein kinase M ~" (PKM~). Two distinct patterns of expression of PKC isozymes in rat hippocampus are found during development from El8 to P28. PKC~', PKM( and PKCa are present at birth and their expression does not increase postnatally. In contrast, the other isoforms are expressed only at low levels at birth and then increase in the first 4 weeks postnatally. These two patterns of expression suggest distinct functions for PKC isozymes during development. Key words: Protein kinase M; Zeta; Synaptic plasticity; Phosphorylation; Long-term potentiation
The PKC family of s e r i n e / t h r e o n i n e protein kinases is thought to regulate both neuronal development and synaptic transmission. Nishizuka [9] has recently classified the isozymes of PKC into three groups: 'conventional', CaZ+-dependent PKC's (a, /3I, /3II, y), 'new', Ca2+-independent PKC's (6, e, rt, 0) and 'atypical' PKC's (~', A). These groups are activated by different signal transduction mechanisms (reviewed in ref. 9). Conventional PKC's possess a region (C2), which binds Ca 2+ in the presence of lipid. The new PKC's lack C2, do not require Ca 2+ for activation and share with the conventional PKC's a diacylglycerol/phorbol ester binding site (C1), containing two cysteine-rich regions. Atypical PKC's contain only a single cysteine-rich region, do not appear to bind phorbol esters [10] and may be activated by products of phosphatidylinositol 3-kinase [8]. In addition to these species, a high-molecular weight PKC [17] has recently been characterized. This PKC, which is expressed in brain [17], is recognized by antiserum to the C4-V5 region of ft. The
* Corresponding author. Fax: (1) (718) 270-2241. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 3 ) E 0 2 1 1 - 3
latter isozyme is expressed predominantly in lung and skin [11], but negligibly in neural tissue [17]. In addition to the heterogeneity produced by multiple genes for PKC, the ~" isozyme is expressed in brain as both PKC~" and PKM~', the free, constitutively active catalytic domain of the isozyme [14]. While PKM may be produced from several PKC isozymes in vitro by proteolytic removal of the amino-terminal regulatory domain [6], the endogenous formation of PKM in hippocampus appears to be largely specific to the ,~ isozyme. PKM~" increases in the maintenance phase of LTP in the CA1 region of the hippocampus [14], suggesting a specific role for this constitutively-active form of PKC in long-term modifications of synaptic efficacy. In order to delineate further the potential functional roles of PKMg" and the individual isoforms of PKC, we examined their expression during rat hippocampal development. PKC isozyme-specific antisera were made as previously described [14] against carboxy-terminal sequences of each of the PKC isoforms: a: PQFVHPILQSAV, /3I: SYTNPEFVINV, /3II: SFVNSEFLKPEVKS, -/: PDARSPTSPVPVPVM, 6: N P K Y E Q F L E , e: Y F G E D L M P , ~: EYINPLLLSAEESV, rt: QTSTKQK-
292
X. Jiang et aL / Decelopmental Brain Research 78 (1994) 291-295
T N K P T Y N E E F C . The rabbit polyclonal antisera were obtained by injection of the peptides conjugated to maleimide-activated bovine serum albumin (Pierce) and emulsified in Titermax (Vaxcel). All antisera were affinity-purified with SulfoLink Coupling Gel (Pierce). The postnatal levels of PKC isozymes were determined by four experiments, each consisting of 13 rats of different ages from a single litter. After anaesthesia with ketamine (20 m g / m l , 0.2-0.3 ml/rat), the hippocampi were dissected immediately after decapitation, frozen and stored in liquid nitrogen. The hippocampi were homogenized by hand (40 strokes at 4°C) in buffer containing Tris-HCl, pH 7.5 (50 raM), E D T A (1 mM), E G T A (1 mM), 2-mercaptoethanol (5 mM), phenylmethylsulfonyl fluoride (0.1 mM), aprotinin (16.7 kallikrein units/rot), benzamidine (5 mM) and leupeptin (0.1 mM) and centrifuged at 4000 g for 15 min. All chemicals, unless specified, were from Sigma. Total protein of the supernatant was determined by a modification of the Bradford assay [13,16]. Fifty ~zg/cm-lane of the total supernatant protein from different devel-
E18
P1
opmental stages were loaded onto 8% SDS-polyacrylamide gels [7] and examined by immunoblot. The difference in levels of PKC isozymes between CA1, CA3 and dentate gyrus regions of the hippocampus were modest (data not shown); therefore, total hippocampus was used in all experiments for consistency. Immunoblotting was performed as previously described [18]. The nitrocellulose lane of each sample was cut into eight strips, each of which was incubated with an isozyme-specific primary antibody for 1 h, followed by alkaline phosphatase-conjugated anti-rabbit goat IgG (1:2500; Promega) for 1 h. The immunoreactive bands were visualized with B C I P / N B T phosphatase substrate (Kirkegaard and Perry Laboratories). The bands were quantified by densitometry with an XRS 6cx scanner (OmniMedia) using NIH Image software. We used a method that is both quantifiable and allows different experiments to be compared with each other. The immunoblots were in the linear range of detection for each antiserum with respect to protein loaded on the gel. Since, however, the time for devel-
7
14
21
28
£t
13I i
? q-related 6 PKC( PKM( : i Fig. 1. Representative immunoblots showing PKC isozymes during rat hippocampal development from El8 to P28. All postnatal hippocampal samples were from rats of a single litter. E18 hippocampus was from a single rat of a separate litter, analyzed on a separate immunoblot and is shown magnified 3 x . The molecular weights for each isozyme (lower band for doublets) are in kDa: a, 80; /3I, 77; flII, 79; T, 79; e, 89; r/-related, 97; 8, 76; PKC~', 70; PKM~r, 51.
X. Jiang et al. / Deuelopmental Brain Research 78 (1994) 291-295
293
except that the mean values for each experiment were preset to 1.5 instead of zero. Hence, the resulting normalized data set for each experiment has a standard deviation equal to 1 and a mean equal to 1.5. To
opment of the colored substrate may vary among the four experiments, the corresponding optical densities could not be combined. Therefore, we applied the normalization procedure employed in factor analysis,
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Fig. 2. Time-courses of postnatal PKC isozyme expression in rat hippocampus. Data from four separate experiments were normalized as described in text. Y-axis scalings are in arbitrary units. Mean values for each day examined, standard errors and fitted regression lines are shown for each isozyme. The slopes of the regression lines for each isozyme were: a, 0.0872*; /3I, 0.0897*; flII, 0.0955*; y, 0.1069"; e, 0.0557*; v-related, 0.0900*; 6, 0.0065; PKC#, -0.0485*; PKM~', -0.0105. (* denotes values significantly different from 0, P = 0.01; the regression line for PKCsr has a significant negative slope.)
294
x. Jiang et al. / Developmental Brain Research 78 (1994) 291-295
determine whether the isozymes changed significantly during development, we applied the simple linear regression model to the time course of each isozyme. The antisera detected specific PKC isozymes as single molecular species or doublets [14], with three exceptions: (1) as noted above, antiserum to the carboxyterminus of P K C r / d e t e c t e d primarily the high molecular weight PKC and only low levels of B in hippocampus; the latter was not measured; (2) PKC6 may be present as a minor P K M form in hippocampus, but preliminary evidence suggests that, in contrast to ~', proteolysis during homogenization may contribute to a portion of P K M 6 (unpublished observations.); (3) the multiple forms of ~', previously described [14]. (PKC0, which is predominantly expressed in muscle [12], was found only at low levels in hippocampus with antiserum provided by Dr. Shin-ichi Osada, Yokohama University [data not shown.] Antiserum to PKCA is not currently available.) Earlier reports examining the Ca2+-dependent PKC isozymes in extracts of total brain [3,19] or in cerebellum [4] found relatively low levels of expression of these isoforms at birth and a steady rise postnatally, with P K C y expression beginning later than the other Ca2+-dependent isozymes. Our results in the hippocampus are consistent with these findings. However, when we analyze the full complement of isozymes in hippocampus, we find a markedly different developmental profile for three forms of PKC: PKC6, PKC~" and PKM~" (Figs. 1,2). Regression analysis (Fig. 2) indicates that the patterns of developmental expression of the isozymes can be divided into two groups: the first group ( a , / 3 I , / 3 I I , y, E and the B-related PKC) is expressed at low levels at birth but increases significantly during postnatal development; the second group ( P K C ( , PKM~', and P K C 6 ) is expressed at birth and does not increase significantly through postnatal development. P K C sr is detectable as early as El8, the earliest time point measured. (PKC~" may be decreasing with time postnatally, but the significance of the negative slope is highly influenced by the values for the first postnatal day.) The association of PKC~" and PKC6 is further substantiated by the observation, as noted above, that P K C a may also have a PKM form. These two patterns of expression suggest discrete roles for individual PKC isozyrnes during development. Isozymes that are present prenatally may be required for developmental processes that occur primarily prior to birth, such as neuronal proliferation and migration. This notion is supported by the observation of the importance of the ~" isozyme during the regulation of maturation in X e n o p u s oocytes [1,2] and mitosis in mouse fibroblasts [1]. The appearance of the other isozymes postnatally correlates with the rapid outgrowth of neuronal processes. The similarity of the developmental profiles of this group suggests a coordi-
nate regulation. It is curious that PKMff is not increased during this period of postnatal synapse formation, considering its potential role in activity-dependent synaptic plasticity. However, changes in PKM~" that may be accrued with experience are likely to be subtle, and specific to subsets of neurons. Stable levels during postnatal development may be particularly relevant for a constitutively active enzyme such as PKMff; its phosphotransferase activity is presumably controlled by its concentration in the cell, not by the levels of second messengers (Ca 2 + or diacylglycerol), which regulate the typical PKC's. It has been hypothesized that the molecular mechanisms that regulate synaptic efficacy during learning and memory may also control neuronal development [5,15]. We find an increasing expression of typical isoforms during the period of synapse formation, in contrast to the stable levels of the atypical ~" isoform, which may participate in LTP maintenance. These results suggest that the plasticity of synapses during development and learning may utilize related but distinct signal transduction pathways. [1] Berra, E., Diaz-Meco, M.T., Dominguez, I., Municio, M.M., Sanz, L., Lozano, J., Chapkin, R.S. and Moscat, J., Protein kinase C ~ isoform is critical for mitogenic signal transduction, Cell, 74 (1993) 555-563. [2] Dominguez, I., Diaz-Meco, M.T., Municio, M.M., Berra, E., de Herreros, A.G., Cornet, M.E., Sanz, L. and Moscat, J., Evidence for a role of protein kinase C ( subspecies in maturation of Xenopus laevis oocytes, Mol. Cell. Biol., 12 (1992) 3776-3783. [3] Hashimoto, T., Ase, K., Sawamura, S., Kikkawa, U., Saito, N., Tanaka, C. and Nishizuka, Y., Postnatal development of a brain-specific subspecies of protein kinase C in rat, J. Neurosci., 8 (1988) 1678-1683. [4] Huang, F.L., Young, W.S., Yoshida, Y. and Huang, K.-P., Developmental expression of protein kinase C isozymes in rat cerebellum, Dev. Brain Res., 52 (1990) 121-130. [5] Kandel, E.R. and O'DelI, T.J., Are adult learning mechanisms also used for development?, Science, 258 (1992) 243-245. [6] Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S.-i., Tominaga, M., Kuroda, T. and Nishizuka, Y,, Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain), J. Biol. Chem., 264 (1989) 4088-4092. [7] Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. [8] Nakanishi, H., Brewer, K.A. and Exton, J.H., Activation of the ( isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate, J. Biol. Chem., 268 (1993) 13-16. [9] Nishizuka, Y., Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607-614. [10] Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. and Nishizuka, Y., Protein kinase C ~ subspecies from rat brain: its structure, expression and properties, Proc. Natl. Acad. Sci. USA, 86 (1989) 3099-3103. [11] Osada, S., Mizuno, K., Saido, T.C., Akita, Y., Suzuki, K., Kuroki, T. and Ohno, S., A phorbol ester receptor/protein kinase, nPKCrl, a new member of the protein kinase C family predominantly expressed in lung and skin, J. Biol. Chem., 265 (1990) 22434-22440.
X. Jiang et aL / Det~elopmental Brain Research 78 (1994) 291-295 [12] Osada, S.-i., Mizuno, K., Saido, T.C., Suzuki, K., Kuroki, T. and Ohno, S., A new member of the protein kinase C family, nPKC0, predominantly expressed in skeletal muscle, MoL Cell BioL, 12 (1992) 3930-3938. [13] Read, S.M. and Northcote, D.H., Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein, Anal Biochem., 116 (1981) 53-64. [14] Sacktor, T.C., Osten, P., Valsamis, H., Jiang, X., Naik, M.U. and Sublette, E., Persistent activation of the ~" isoform of protein kinase C in the maintenance of long-term potentiation, Proc. NatL Acad. Sci. USA, 90 (1993) 8342-8346. [15] Schatz, C.J., Impulse activity and the patterning of connections during CNS development, Neuron, 10 (1990) 745-756. [16] Simpson, I.A. and Sonne, O., A simple, rapid and sensitive
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