- Email: [email protected]
Addition of phosphatidylcholine-phospholipase C induces cellular redistribution and phosphorylation of protein kinase C ξ in C 6 glial cells
ELSEVIER
Neuroscience Letters 219 (1996) 68-70
HEUROSClENCE LETTERS
Addition of phosphatidylcholine-phospholipase C induces cellular redistribution and phosphorylation of protein kinase C in C 6 glial cells Ismael Galve-Roperh, Jose M. Malpartida, Amador Haro, In6s Diaz-Laviada* Departamento de Bioquimica y Biologia Molecular 1, Facultad de Qu[micas, Universidad Complutense, 28040 Madrid, Spain
Received 7 August 1996; revised version received 5 October 1996; accepted 8 October 1996
Abstract
Phosphatidylcholine breakdown has been shown to play a critical role in signal transduction involving generation of a number of second messengers [Exton, J.H., Biochim. Biophys. Acta, 1212 (1994) 26-42]. In the present report we demonstrate by immunofluorescence that short-treatment of C 6 glial cells with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracellular localization of protein kinase C (PKC) ~" from the cytoplasm to a perinuclear region. Western blot analysis also showed a redistribution of PKC g"after incubation of cells with PC-PLC. To test whether these changes were accompanied by an activation of the enzyme, we measured the extent of phosphorylation of PKC ~"by immunoprecipitation from 32p-labelled cells. Short-treatment with PC-PLC resulted in enhanced phosphorylation of the higher Mr PKC ~"in C 6 glial cells. Keywords: Protein kinase C ~'; Phosphatidylcholine-phospholipase C; Glial cells
Protein kinase C (PKC) is one of the major mediators of signals upon external stimulation of cells by hormones, neurotransmitters, and growth factors. PKC has been found in most mammalian tissues, but is particularly abundant in the brain [17]. The mammalian PKC polypeptides identified to date by cDNA cloning have been classified into three groups based on two criteria, namely structural features and regulation by cofactor. The atypical PKCs (~" and t/X isoforms) are dependent on phosphatidylserine, but do not require Ca 2÷ or diacylglycerol for activation [4,13]. In astrocytes and glial cells the presence of PKC c~,/3, % 6, e and ~"isoforms has been reported [5,7,11,14]. Profiles of PKC isoforms in rat astrocytes and C 6 glioma cells are similar, although a higher expression of c~, e, and ~" isoforms in glioma cells has been detected [14]. Recently, it has been demonstrated that the PKC ~" isoform plays an important role in mitogenic signal transduction [ 1], neuronal differentiation [19], and maintenance of long-term potentiation [15]. However, the precise mechanism by which PKC ~"activity is regulated is not fully understood. PKC ~"is not activated by diacylglycerol or phorbol esters [13,18] and in many cell types including glial cells, PKC ~" * Corresponding author. Tel.: +34 1 3944668; fax: +34 1 3944159; e-mail: [email protected]
is neither translocated from cytosol to membrane nor down-regulated in response to acute or chronic treatment with phorbol esters [2]. Activation of PKC ~" may be achieved by ceramide [10]. The second messenger ceramide is generated by the action of sphingomyelinase, which is in turn functionally coupled to phosphatidylhydrolyzing phospholipase C (PC-PLC) [16]. We have recently shown that PC-PLC enhances nerve growth factor synthesis in cultured glial ceils by a mechanism which is partially independent of the classical isoforms of PKC [8]. In the present report we show that addition of PC-PLC induces cellular redistribution and phosphorylation of PKC ~"in C 6 glial cells. To test whether PC-PLC could have an effect on PKC ~" we analyzed the intracellular localization and the extent of phosphorylation of this PKC isoform. Short-term-treatment of C 6 cells with exogenous PC-PLC produced a change in the localization of PKC ~"from cytoplasm to a perinuclear membrane region (Fig. 1A,B). Non-specific labeling was shown after incubation with anti-PKC ~"antibody in the presence of a competing peptide used to rinse the antibody (Fig. 1C). As a further proof of the effect of PC-PLC on the localization of PKC ~', particulate, and soluble fractions of cells were separated by centrifugation, and PKC ~"was detected
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13165-7
I. Galve-Roperh et al. / Neuroscience Letters 219 (1996) 6 8 - 7 0
CONTROL
PC-PLC 1 U / m l
69
PC-PLC 1 U / m l + peptide
A
Fig. 1. Immunocytochemical localization of PKC ~"in C 6 glial cells. Cells were seeded (104 cells/cm 2) in glass coverslips, grown in serum-containing medium, and made quiescent by serum starvation. Then, no addition (A) or 1 U/ml PC-PLC from Bacillus cereus (B,C) were added for 15 min. Immunolocalization was performed as in [3] and coverslips were incubated with an anti-PKC ~"antibody (Life Technologies, Inc.) (A,B) or with the antiPKC ~"antibody in the presence of the competing peptide (C). Identical results were obtained in three independent experiments. by i m m u n o b l o t t i n g . P K C ~"m i g r a t e d as two distinct bands, one c o r r e s p o n d i n g to the ' e m b r y o n i c f o r m ' (75 kDa) and another c o r r e s p o n d i n g to the 'adult f o r m ' (83 kDa). B o t h
Particulate fraction
Soluble fraction
97.4 kDa-
66 kDa/ 4
1
2
3 m
r::3
4 High Mw
forms h a v e b e e n d e t e c t e d in cultured glial cells [14]. In cells shortly treated with P C - P L C , an increase in the total a m o u n t o f P K C ~" b o u n d to the particular fraction versus control was o b s e r v e d (from 1828 + 161 to 2479 + 193 d e n s i t o m e t r i c units). A decrease in total soluble P K C ~" o f an equal extent (from 1470 + 97 to 931 + 102 densitometric units) was also detected (Fig. 2). This observation indicates that w h e n cells are treated with P C - P L C , an association o f P K C ~" to a m e m b r a n e structure occurs. This translocation affected m a i n l y the 83 k D a form, w h i c h m a y indicate the existence of different regulatory properties o f both isoforms. As m a y be inferred f r o m i m m u n o f l u o r e s c e n c e labeling (Fig. 1), this structure seems to be located in a perinuclear region. P r e v i o u s reports s h o w e d in another system the intimate association o f P K C ~" with a perinuclear region o f the cells [9]. As brain P K C ~" activity is significantly dependent on phospholipids [13], the translocation of the e n z y m e from the c y t o p l a s m to perinuclear r e g i o n m a y indicate an activation process. A c t i v a t i o n o f P K C is regulated by phosphorylation, w h i c h reflects the activity state of the kinase [6]. In
~"2 Fig. 2. Identification of PKC ~"by immunoblotting. C 6 cells were either untreated (lanes 1 and 3) or treated for 15 min with 1 U/ml PC-PLC (lanes 2 and 4). Then, particulate and cytosolic fractions were obtained from cell lysates by centrifugation at 50 000 g and resolved by electrophoresis. The markers on the left correspond to the migration position of 97.4 and 66 kDa molecular weight markers. Luminograms obtained with the ECL detection kit were scanned with a Scanjet 4c/T (Hewlett Packard). Densitometric analyses were performed with the computer program Sigma Gel 1.0 (Jandel Scientific). Arbitrary units are related to densitometric units from control cells. Identical results were obtained in three independent experiments.
70
1. Gah,e-Roperh et al. / Neuroscience Letters 219 (1996) 68-70
97.4 kDa-
66 kDa-
1
2
4--
3
e3
--
2-
t~ 1
--
0
-
-
-
55
Fig. 3. Phosphorylation level of PKC ~-. C 6 cells were incubated with 32p-orthophosphate (Amersham) and then either untreated (lane 1) or treated for 15 rain 1 U/ml PC-PLC (lane 2). After immunoprecipitation with an anti-PKC ~" antibody, pellets were resolved by electrophoresis and subjected to autoradiography. Densitometric analysis was performed as described for Western blot. The markers on the left correspond to the migration position of 97.4 and 66 kDa molecular weight markers. Identical results were obtained in three independent experiments.
order to know whether translocation of PKC ~" correlates with an activation of the enzyme, we determined the extent of phosphorylation of PKC ~"by immunoprecipitation from 32P-labeled C 6 cells. Thus, incubation of 32P-labeled cells with PC-PLC led to an enhanced phosphorylation of PKC ~" (Fig. 3). Increased phosphorylation of PKC ~" affected one form, probably corresponding to 83 kDa, which agrees with its observed translocation. A similar effect has been demonstrated in epidermal cells, where phorbol myristate acetate-treated cells showed an increased phosphorylation affecting to higher-Mr PKC ~"isoform [12]. Taken together these results indicate that treatment of C 6 cells with PC-PLC causes a change in the intracellular localization of PKC ~"which becomes associated to a perinuclear region. Redistribution of this kinase was accompanied by an increase in the extent of phosphorylation of the enzyme, confirming the hypothesis that PC-PLC treatment of C 6 glioma cells leads to the translocation and consequent activation of PKC ~"isoform. The authors gratefully acknowledged Dr. M. Guzman for critical reading of the manuscripts and Dr. P. Brachet for providing C6 glial cells. This work was supported by a grant from FIS (94/0300). I. Galve has a fellowship from University Complutense.
[1] Berra, E., Dfaz-Meco, M.T., Domfnguez, 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] Chen, C., Protein kinase C a, 6, e and ~"in C 6 glioma cells. TPA induces translocation and down-regulation of conventional and new PKC isoforms but not atypical PKC ~', FEBS Lett., 332 (1993) 169-173. [3] Diaz-Laviada, L, Larrodera, P., Nieto, J.L., Comet, M.E., DfazMeco, M.T., S~nchez, M.J., Guddal, P.H., Johansen, T., Haro, A. and Moscat, J., Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine, J. Biol. Chem., 266 (1991) 1170-1176. [4] Exton, J.H., Phosphatidylcholine breakdown and signal transduction, Biochim. Biophys. Acta, 1212 (1994) 26-42. [5] Gott, A.L., Mellon, B.S., Paton, A., Groome, N. and Rumsby, M.G., Rat brain glial cells in primary culture and subculture contain the 6, e and ~" subspecies of protein kinase C as well as the conventional subspecies, Neurosci. Lett., 171 (1994) 117-120. [6] Keranen, L.M., Dutil, E.M. and Newton, A., Protein kinase C is regulated in vivo by three functionally distinct phosphorylations, Curt. Biol., 5 (1995) 1394-1403. [7] Kumar, K., Kim, B., Rupp, H. and Madhukar, B., Expression of protein kinase C isozymes in rat glial cell line, NeuroReport, 4 (1993) 431-434. [81 Laviada, I.D., Baudet, C., Galve-Roperh, I., Naveilhan, P. and Brachet, P., Phosphatidylcholine-phospholipase C mediates the induction of nerve growth factor in cultured glial cells, FEBS Lett., 364 (1995) 301-304. [9] Lehrich, R.W. and Forrest, J.N., Protein kinase C ~ is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland, J. Biol. Chem., 269 (1994) 32446-32450. [10] Lozano, J., Berra, E., Municio, M.M., Diaz-Meco, M.T., Dominguez, I., Sanz, L. and Moscat, J., Protein kinase C ~"isoform is critical for KB-dependent promoter activation by sphingomyelinase, J. Biol. Chem., 269 (1994) 19200-19202. [11] Masliah, E., Yoshida, K., Shimohama, S., Gage, F.H. and Saitoh, T., Differential expression of protein kinase C isozymes in rat glial cell cultures, Brain Res., 549 (1991) 106-111. [12] Nishikawa, K., Yamamoto, S., Nagumo, H., Maruyama, K. and Kato, R., The presence of phorbol ester responsive and non-responsive forms of the ~"isozyme of protein kinase C in mouse epidermal cells, Cell. Signal., 7 (1995) 491-504. [13] 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. [14] Roisin, M. and Deschepper, C.F., Identification and cellular localization of protein kinase C isoforms in cultures of rat type-I astrocytes, Brain Res., 701 (1995) 297 300. [15] 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. [16] Schtitze, S., Pottboff, K., Machieidt, T., Berkovic, D., Wiegmann, K. and Kr6nke, M., TNF activates NF-KB by phosphatidylcholinespccific phospholipase C-induced acidic sphingomyelin breakdown, Cell, 71 (1992) 765-776. [17] Tanaka, C. and Nishizuka, Y., The protein kinase C family for neuronal signaling, Annu. Rev. Neurosci., 17 (1994) 551-567. [18] Ways, D.K., Cook, P.P., Webster, C. and Parker, P., Effect of phorbol esters on protein kinase C-~-, J. Biol. Chem., 267 (1992) 4799-4805. [19] Wooten, M.W., Zhou, G., Seibenhener, M.L. and Coleman, E.S., A role for zeta protein kinase C in nerve growth factor-induced differentiation of PC12 cells, Cell Growth Diff., 5 (1994) 395-403.
Neuroscience Letters 219 (1996) 68-70
HEUROSClENCE LETTERS
Addition of phosphatidylcholine-phospholipase C induces cellular redistribution and phosphorylation of protein kinase C in C 6 glial cells Ismael Galve-Roperh, Jose M. Malpartida, Amador Haro, In6s Diaz-Laviada* Departamento de Bioquimica y Biologia Molecular 1, Facultad de Qu[micas, Universidad Complutense, 28040 Madrid, Spain
Received 7 August 1996; revised version received 5 October 1996; accepted 8 October 1996
Abstract
Phosphatidylcholine breakdown has been shown to play a critical role in signal transduction involving generation of a number of second messengers [Exton, J.H., Biochim. Biophys. Acta, 1212 (1994) 26-42]. In the present report we demonstrate by immunofluorescence that short-treatment of C 6 glial cells with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracellular localization of protein kinase C (PKC) ~" from the cytoplasm to a perinuclear region. Western blot analysis also showed a redistribution of PKC g"after incubation of cells with PC-PLC. To test whether these changes were accompanied by an activation of the enzyme, we measured the extent of phosphorylation of PKC ~"by immunoprecipitation from 32p-labelled cells. Short-treatment with PC-PLC resulted in enhanced phosphorylation of the higher Mr PKC ~"in C 6 glial cells. Keywords: Protein kinase C ~'; Phosphatidylcholine-phospholipase C; Glial cells
Protein kinase C (PKC) is one of the major mediators of signals upon external stimulation of cells by hormones, neurotransmitters, and growth factors. PKC has been found in most mammalian tissues, but is particularly abundant in the brain [17]. The mammalian PKC polypeptides identified to date by cDNA cloning have been classified into three groups based on two criteria, namely structural features and regulation by cofactor. The atypical PKCs (~" and t/X isoforms) are dependent on phosphatidylserine, but do not require Ca 2÷ or diacylglycerol for activation [4,13]. In astrocytes and glial cells the presence of PKC c~,/3, % 6, e and ~"isoforms has been reported [5,7,11,14]. Profiles of PKC isoforms in rat astrocytes and C 6 glioma cells are similar, although a higher expression of c~, e, and ~" isoforms in glioma cells has been detected [14]. Recently, it has been demonstrated that the PKC ~" isoform plays an important role in mitogenic signal transduction [ 1], neuronal differentiation [19], and maintenance of long-term potentiation [15]. However, the precise mechanism by which PKC ~"activity is regulated is not fully understood. PKC ~"is not activated by diacylglycerol or phorbol esters [13,18] and in many cell types including glial cells, PKC ~" * Corresponding author. Tel.: +34 1 3944668; fax: +34 1 3944159; e-mail: [email protected]
is neither translocated from cytosol to membrane nor down-regulated in response to acute or chronic treatment with phorbol esters [2]. Activation of PKC ~" may be achieved by ceramide [10]. The second messenger ceramide is generated by the action of sphingomyelinase, which is in turn functionally coupled to phosphatidylhydrolyzing phospholipase C (PC-PLC) [16]. We have recently shown that PC-PLC enhances nerve growth factor synthesis in cultured glial ceils by a mechanism which is partially independent of the classical isoforms of PKC [8]. In the present report we show that addition of PC-PLC induces cellular redistribution and phosphorylation of PKC ~"in C 6 glial cells. To test whether PC-PLC could have an effect on PKC ~" we analyzed the intracellular localization and the extent of phosphorylation of this PKC isoform. Short-term-treatment of C 6 cells with exogenous PC-PLC produced a change in the localization of PKC ~"from cytoplasm to a perinuclear membrane region (Fig. 1A,B). Non-specific labeling was shown after incubation with anti-PKC ~"antibody in the presence of a competing peptide used to rinse the antibody (Fig. 1C). As a further proof of the effect of PC-PLC on the localization of PKC ~', particulate, and soluble fractions of cells were separated by centrifugation, and PKC ~"was detected
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13165-7
I. Galve-Roperh et al. / Neuroscience Letters 219 (1996) 6 8 - 7 0
CONTROL
PC-PLC 1 U / m l
69
PC-PLC 1 U / m l + peptide
A
Fig. 1. Immunocytochemical localization of PKC ~"in C 6 glial cells. Cells were seeded (104 cells/cm 2) in glass coverslips, grown in serum-containing medium, and made quiescent by serum starvation. Then, no addition (A) or 1 U/ml PC-PLC from Bacillus cereus (B,C) were added for 15 min. Immunolocalization was performed as in [3] and coverslips were incubated with an anti-PKC ~"antibody (Life Technologies, Inc.) (A,B) or with the antiPKC ~"antibody in the presence of the competing peptide (C). Identical results were obtained in three independent experiments. by i m m u n o b l o t t i n g . P K C ~"m i g r a t e d as two distinct bands, one c o r r e s p o n d i n g to the ' e m b r y o n i c f o r m ' (75 kDa) and another c o r r e s p o n d i n g to the 'adult f o r m ' (83 kDa). B o t h
Particulate fraction
Soluble fraction
97.4 kDa-
66 kDa/ 4
1
2
3 m
r::3
4 High Mw
forms h a v e b e e n d e t e c t e d in cultured glial cells [14]. In cells shortly treated with P C - P L C , an increase in the total a m o u n t o f P K C ~" b o u n d to the particular fraction versus control was o b s e r v e d (from 1828 + 161 to 2479 + 193 d e n s i t o m e t r i c units). A decrease in total soluble P K C ~" o f an equal extent (from 1470 + 97 to 931 + 102 densitometric units) was also detected (Fig. 2). This observation indicates that w h e n cells are treated with P C - P L C , an association o f P K C ~" to a m e m b r a n e structure occurs. This translocation affected m a i n l y the 83 k D a form, w h i c h m a y indicate the existence of different regulatory properties o f both isoforms. As m a y be inferred f r o m i m m u n o f l u o r e s c e n c e labeling (Fig. 1), this structure seems to be located in a perinuclear region. P r e v i o u s reports s h o w e d in another system the intimate association o f P K C ~" with a perinuclear region o f the cells [9]. As brain P K C ~" activity is significantly dependent on phospholipids [13], the translocation of the e n z y m e from the c y t o p l a s m to perinuclear r e g i o n m a y indicate an activation process. A c t i v a t i o n o f P K C is regulated by phosphorylation, w h i c h reflects the activity state of the kinase [6]. In
~"2 Fig. 2. Identification of PKC ~"by immunoblotting. C 6 cells were either untreated (lanes 1 and 3) or treated for 15 min with 1 U/ml PC-PLC (lanes 2 and 4). Then, particulate and cytosolic fractions were obtained from cell lysates by centrifugation at 50 000 g and resolved by electrophoresis. The markers on the left correspond to the migration position of 97.4 and 66 kDa molecular weight markers. Luminograms obtained with the ECL detection kit were scanned with a Scanjet 4c/T (Hewlett Packard). Densitometric analyses were performed with the computer program Sigma Gel 1.0 (Jandel Scientific). Arbitrary units are related to densitometric units from control cells. Identical results were obtained in three independent experiments.
70
1. Gah,e-Roperh et al. / Neuroscience Letters 219 (1996) 68-70
97.4 kDa-
66 kDa-
1
2
4--
3
e3
--
2-
t~ 1
--
0
-
-
-
55
Fig. 3. Phosphorylation level of PKC ~-. C 6 cells were incubated with 32p-orthophosphate (Amersham) and then either untreated (lane 1) or treated for 15 rain 1 U/ml PC-PLC (lane 2). After immunoprecipitation with an anti-PKC ~" antibody, pellets were resolved by electrophoresis and subjected to autoradiography. Densitometric analysis was performed as described for Western blot. The markers on the left correspond to the migration position of 97.4 and 66 kDa molecular weight markers. Identical results were obtained in three independent experiments.
order to know whether translocation of PKC ~" correlates with an activation of the enzyme, we determined the extent of phosphorylation of PKC ~"by immunoprecipitation from 32P-labeled C 6 cells. Thus, incubation of 32P-labeled cells with PC-PLC led to an enhanced phosphorylation of PKC ~" (Fig. 3). Increased phosphorylation of PKC ~" affected one form, probably corresponding to 83 kDa, which agrees with its observed translocation. A similar effect has been demonstrated in epidermal cells, where phorbol myristate acetate-treated cells showed an increased phosphorylation affecting to higher-Mr PKC ~"isoform [12]. Taken together these results indicate that treatment of C 6 cells with PC-PLC causes a change in the intracellular localization of PKC ~"which becomes associated to a perinuclear region. Redistribution of this kinase was accompanied by an increase in the extent of phosphorylation of the enzyme, confirming the hypothesis that PC-PLC treatment of C 6 glioma cells leads to the translocation and consequent activation of PKC ~"isoform. The authors gratefully acknowledged Dr. M. Guzman for critical reading of the manuscripts and Dr. P. Brachet for providing C6 glial cells. This work was supported by a grant from FIS (94/0300). I. Galve has a fellowship from University Complutense.
[1] Berra, E., Dfaz-Meco, M.T., Domfnguez, 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] Chen, C., Protein kinase C a, 6, e and ~"in C 6 glioma cells. TPA induces translocation and down-regulation of conventional and new PKC isoforms but not atypical PKC ~', FEBS Lett., 332 (1993) 169-173. [3] Diaz-Laviada, L, Larrodera, P., Nieto, J.L., Comet, M.E., DfazMeco, M.T., S~nchez, M.J., Guddal, P.H., Johansen, T., Haro, A. and Moscat, J., Mechanism of inhibition of adenylate cyclase by phospholipase C-catalyzed hydrolysis of phosphatidylcholine, J. Biol. Chem., 266 (1991) 1170-1176. [4] Exton, J.H., Phosphatidylcholine breakdown and signal transduction, Biochim. Biophys. Acta, 1212 (1994) 26-42. [5] Gott, A.L., Mellon, B.S., Paton, A., Groome, N. and Rumsby, M.G., Rat brain glial cells in primary culture and subculture contain the 6, e and ~" subspecies of protein kinase C as well as the conventional subspecies, Neurosci. Lett., 171 (1994) 117-120. [6] Keranen, L.M., Dutil, E.M. and Newton, A., Protein kinase C is regulated in vivo by three functionally distinct phosphorylations, Curt. Biol., 5 (1995) 1394-1403. [7] Kumar, K., Kim, B., Rupp, H. and Madhukar, B., Expression of protein kinase C isozymes in rat glial cell line, NeuroReport, 4 (1993) 431-434. [81 Laviada, I.D., Baudet, C., Galve-Roperh, I., Naveilhan, P. and Brachet, P., Phosphatidylcholine-phospholipase C mediates the induction of nerve growth factor in cultured glial cells, FEBS Lett., 364 (1995) 301-304. [9] Lehrich, R.W. and Forrest, J.N., Protein kinase C ~ is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland, J. Biol. Chem., 269 (1994) 32446-32450. [10] Lozano, J., Berra, E., Municio, M.M., Diaz-Meco, M.T., Dominguez, I., Sanz, L. and Moscat, J., Protein kinase C ~"isoform is critical for KB-dependent promoter activation by sphingomyelinase, J. Biol. Chem., 269 (1994) 19200-19202. [11] Masliah, E., Yoshida, K., Shimohama, S., Gage, F.H. and Saitoh, T., Differential expression of protein kinase C isozymes in rat glial cell cultures, Brain Res., 549 (1991) 106-111. [12] Nishikawa, K., Yamamoto, S., Nagumo, H., Maruyama, K. and Kato, R., The presence of phorbol ester responsive and non-responsive forms of the ~"isozyme of protein kinase C in mouse epidermal cells, Cell. Signal., 7 (1995) 491-504. [13] 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. [14] Roisin, M. and Deschepper, C.F., Identification and cellular localization of protein kinase C isoforms in cultures of rat type-I astrocytes, Brain Res., 701 (1995) 297 300. [15] 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. [16] Schtitze, S., Pottboff, K., Machieidt, T., Berkovic, D., Wiegmann, K. and Kr6nke, M., TNF activates NF-KB by phosphatidylcholinespccific phospholipase C-induced acidic sphingomyelin breakdown, Cell, 71 (1992) 765-776. [17] Tanaka, C. and Nishizuka, Y., The protein kinase C family for neuronal signaling, Annu. Rev. Neurosci., 17 (1994) 551-567. [18] Ways, D.K., Cook, P.P., Webster, C. and Parker, P., Effect of phorbol esters on protein kinase C-~-, J. Biol. Chem., 267 (1992) 4799-4805. [19] Wooten, M.W., Zhou, G., Seibenhener, M.L. and Coleman, E.S., A role for zeta protein kinase C in nerve growth factor-induced differentiation of PC12 cells, Cell Growth Diff., 5 (1994) 395-403.