Protein kinase C isoforms and cell proliferation in neuroblastoma cells (original) (raw)
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Biochemical and Biophysical Research Communications, 1989
By the use of cloned cDNAs for protein kinase C isozymes a , /I I, fi II, y , and those for novel protein kinase C, E and C , the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell tines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for B I/ ,Q II mRNAs in spleen. These expression patterns are in clear contrast to that for fl I/ fl II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC ,9 mRNAs in addition to PKC a. Further, nPKC E mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC& and (: in addition to PKCn. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC a and nPKC E was observed in association with in vitro differentiation.-, 1989 Academic press, Inc. Tumor-promoting phorbol esters and the naturally occurring 1,2-diacylglycerols have been shown to utilize the Ca2+-and phospholipid-dependent protein kinase, protein kinase C (PKC), to mediate signal transduction in a variety of cellular processes such as cell proliferation and differentiated functions (1). Recent molecular cloning experiments in conjunction with the biochemical characterization of mammalian brain PKC have established the presence of four distinct PKC types (a , ,9 I, p 11, and y) which are encoded by three distinct genes, a , p , and y. The four conventional PKC types share closely related sequences and similar biochemical properties including kinase and phorbol ester-binding activities (2-7). More recently, a cDNA related to cDNAs for conventional PKCs was isolated and the encoded protein, nPKC E , has been characterized as a novel phorbol ester receptor/protein kinase distinct from the conventional PKCs (8). The presence of two additional PKC-related cDNAs, which encode proteins more related to nPKC E , have also been identified (nPKC6 and c) (9). * Supported in part by a Grant-in-Aid for Cancer-Bioscience from the Ministry of Education, Science and Culture of Japan.
Biochemical and Biophysical Research Communications, 1989
By the use of cloned cDNAs for protein kinase C isozymes a , /I I, fi II, y , and those for novel protein kinase C, E and C , the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell tines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for B I/ ,Q II mRNAs in spleen. These expression patterns are in clear contrast to that for fl I/ fl II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC ,9 mRNAs in addition to PKC a. Further, nPKC E mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC& and (: in addition to PKCn. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC a and nPKC E was observed in association with in vitro differentiation.-, 1989 Academic press, Inc. Tumor-promoting phorbol esters and the naturally occurring 1,2-diacylglycerols have been shown to utilize the Ca2+-and phospholipid-dependent protein kinase, protein kinase C (PKC), to mediate signal transduction in a variety of cellular processes such as cell proliferation and differentiated functions (1). Recent molecular cloning experiments in conjunction with the biochemical characterization of mammalian brain PKC have established the presence of four distinct PKC types (a , ,9 I, p 11, and y) which are encoded by three distinct genes, a , p , and y. The four conventional PKC types share closely related sequences and similar biochemical properties including kinase and phorbol ester-binding activities (2-7). More recently, a cDNA related to cDNAs for conventional PKCs was isolated and the encoded protein, nPKC E , has been characterized as a novel phorbol ester receptor/protein kinase distinct from the conventional PKCs (8). The presence of two additional PKC-related cDNAs, which encode proteins more related to nPKC E , have also been identified (nPKC6 and c) (9). * Supported in part by a Grant-in-Aid for Cancer-Bioscience from the Ministry of Education, Science and Culture of Japan.
Brain Research, 1992
We have studied the effect of protein kinase C inhibitors l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and caiphostin C on the cycle of Neuro-2a cells. Both compounds inhibited cell proliferation and DNA synthesis. Transition from G2 to M phase was not altered by these compounds. Calphostin C blocked the cells in Go/G 1, while H7 did not at any specific point in the cell cycle. We a~o show ~hat the antiproliferative effect induced by both inhibitors is reversible.
Experimental Cell Research, 1996
eration and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induc-To establish whether protein kinase C was involved tion and persistence of the differentiated neuronal in the nuclear events underlying cell differentiation phenotype of PC12 cells. ᭧ 1996 Academic Press, Inc. and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mito-INTRODUCTION genic stimulus. Western blot analysis with isoformspecific antibodies, performed on whole cell homoge-Protein kinase C (PKC) was originally described as nates, cytoplasms, and purified nuclei, showed that a serine/threonine protein phosphotransferase modu-PKC isotypes a, bI, bII, d, e, h, and z were expressed lated by calcium and lipid cofactors, including phosphain PC12 cells and that all of them, except for bI, were tidylserine (PS), other anionic phospholipids, and diacfound at the nuclear level, variably modulated deylglycerol (DG) . It has been recently found that pending on the cell treatment. Compared to serum-PKC represents a large gene family of isoenzymes, difstimulated cells, in which an early (1 day) and marked fering remarkably in their structure and expression in rise of protein kinase C activity was followed by a plavarious tissues, in their mode of activation, cofactor teau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coinci-requirement, and substrate specificity . Therefore, dent with the onset and maintenance of the differenti-PKC regulation appears rather complex and is still inated phenotype. Western blot analysis of nuclei completely understood. The diversity of PKC-mediated isolated from fully differentiated cells demonstrated cell responses is further increased by the evidence that an increase of protein kinase C a, paralleled by enonly PKC a, bI, bII, and g out of the several isoforms hanced phosphotransferase activity along with the identified so far are Ca 2/ dependent and that the nerve growth factor treatment, and complete loss of endogenous activator DG can derive from turnover of the d isotype. In contrast, in nuclei of proliferating either inositol lipids or phosphatidylcholine . In ad-PC12 cells, after an early but modest increase at 1 day dition, PKC autophosphorylation seems an essential of mitogenic stimulation, protein kinase C activity step for enzyme activation [6-8], while the proteolytic reached a plateau. Isotype-specific analysis indicated cleavage of PKC by calpains, generating the constitua concomitant increase of protein kinase C bII, d, and tively active fragment PKM [9, 10], can also participate z and the appearance of protein kinase C e and h at in long-term PKC activation. the nuclear level. Considering the relative intensity
Protein kinase C isoenzymes in human neuroblasts involvement of PKCε in cell differentiation
FEBS Letters, 1993
Although neuronal cells are a major target of phorbol ester action, the activity of the various protein kinase C (PKC) isoenzymes have not been studied in detail in human neuroblasts. Differentiation of the LAN-5 human neuroblastoma cell line by interferon-gamma (IFN-gamma) is accompanied by a twofold increase in PKC activity. Since PKC is a multigene family, we investigated which isoforms were expressed in control and differentiated cells, and which of these isoenzymes is involved in neuronal differentiation. We found that: (1) PKC activity is higher in differentiated than in undifferentiated cells; (2) RT-PCR analysis showed the expression of mRNA for PKC alpha, -gamma, -delta, -epsilon and -zeta and the absence of mRNA for beta in untreated LAN-5 cells; (3) Western blot evaluation with PKC isoform-specific antibodies showed the same pattern of PKC expression in non-differentiated cells; (4) Expression of PKC epsilon mRNA was significantly enhanced by IFN-gamma-induced differentiation, while the other isoforms were not affected; (5) Differentiation of LAN-5 cells with IFN-gamma or retinoic acid induced overexpression of the PKC epsilon protein, while inhibition of cell proliferation by fetal calf serum starvation was without effect. These findings suggest that expression of PKC epsilon isoform is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state.
Journal of Cellular Physiology, 1989
The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPAj. A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2 + mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of
FEBS Letters, 1994
Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-SY cells with 2-0-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-SY PKCa by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKCa. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an 'unregulated' kinase, since its persistence is apparently strictly regulated by proteolysis.
Human Neuroblastoma Cell Differentiation Requires Protein Kinase C-θ
Biochemical and Biophysical Research Communications, 2000
Neuroblastoma LAN-5 cells exposed to retinoic acid cease to multiply and extend neurite outgrowths acquiring a neuronal phenotype. We now report that protein kinase C-(PKC-) isozyme is involved in this differentiation process due to the following findings: (i) PKC-is expressed by LAN-5 cells as a nuclear and perinuclear protein; (ii) cell stimulation with retinoic acid promotes in a large increase in the expression level of the kinase and its intracellular redistribution; and (iii) a PKCantisense oligonucleotide reduces at the same time the expression level of the kinase and the cell response to retinoic acid. Altogether these data are consistent with a specific role played by PKCin the differentiation program of neuronal cells.
Regulation of neuronal differentiation by the α and ϵ isoforms of protein kinase C
Neuroscience Research Communications, 1996
Previous studies utilizing cell-permeant inhibitors and intracellular delivery of anti-PKC antibodies and active enzyme have indicated that inhibition of protein kinase C (PKC) promotes the initial stage(s) of neurite elaboration, while PKC activation inhibits and reverses this phenomenon. In the present study we treated SH-SY-SY human neuroblastoma cells with antisense oligonucleotides directed against unique sequences of the a, 0, and E PKC isoforms for a length of time (5 days) sufficient to reduce imunocytochemical levels of their specific target isoforms. We observed that treatment for 5 days with antisense oligonucleotides directed against PKCa and PKCE, but not PKCp, induced a marked increase in the percentage of cells elaborating neurites. By contrast, treatment with the corresponding sense-oriented oligonucleotides for each isoform had no detectable effect. These data confirm the participation of PKC in regulation of the initial stage(s) of neuronal differentiation, and further imply specific roles for the PKCa and E isoforms in this regulation.