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REVIEW
Protein Kinase C Involvement in Apoptosis M I G U E L L U C A S * and V I C T O R S , ~ N C H E Z - M A R G A L E T
Departamento de Bioquimica Mbdica y Biologia Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda. S~chez Pizju~n 4, 41009 Sevilla, Spain [Tel./Fax: 345.455. 73.52]
(Received 20 October 1994)
Abstract--l. Conflicting observations on the involvement of PKC in apoptosis point to a great variability depending on cell type, agent or condition causing apoptosis, phase of the cell cycle and intracellular signaling pathway.
2. Inhibition by PKC of store-operated calcium entry mechanisms, which are sensitive to the oncoprotein bcl-2, should block the activation of calcium-dependent enzymes triggering the apoptotic cell death.
3. Activation of phosphatases by ceramide and inhibition of PKC by sphingosine seem to mediate the sphingomyelin pathway to apoptosis.
4. A putative target protein appears to be p34 cdc2 which is regulated by a network of kinases and phosphatases. The uncoupling of timing for p34 cat2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that shares some features with apoptosis.
The death of cells in normal tissue turnover is called apoptosis or programmed cell death (Kerr et al., 1972). Unlike necrotic cell death, the process requires that target cell be active in its own death since it depends on the integrity of the cell and the biosyn- thesis of RNA and proteins (Schwartz et al., 1990). However, the last requirement is not a general characteristic as in the case of TNF<t- and CTL- mediated cell death. Morphological and molecular events include chromatin condensation, formation of the so-called apoptotic bodies, shrinkage, fragmenta- tion of DNA into oligonucleosome-sized fragments and, at a later state, progressive cell degradation, swelling and membrane rupture (Wyllie et al., 1980; Wyllie, 1988). Apoptosis occurs during fundamental biological processes such as embryo morphogenesis, the development of immune tolerance, ageing and tissue degeneration as well as cell proliferation and tumorigenesis (For reviews see: McConkey et aL,
1990; Fesus e ta l . , 1991; Golstein et al., 1991; Raft, 1992; Collins and Lrpez-Rivas, 1993; Green and Scott 1994; Wright et al., 1994).
*To whom all correspondence should be addressed.
Agents or conditions inducing apoptosis show variable degree of dependency on different pathways with the main feature relying on: (a) the induction of c-myc, p53 or bcl-2 transcripts; (b) the cell cycle phase; (c) the biosynthesis of proteins; (d) the acti- vation of kinases; and (e) the hydrolysis of sphin- gomyelin. These apparently polymorphic pathways could be related to its intracellular control which may vary significantly as a function of cell type, the state of the cell and the apoptosis-inducing agent (Golstein et al., 1991). Oncogenes and tumor suppressor genes appears to be clearly involved: in fact, p53-dependent and independent pathways have been described (Lowe et al., 1993; Clarke et al., 1993) as well as altered expression of oncogenes c-fos and c-myc (Buttyan et al., 1988), whereas a protein encoded by the oncogene bcl-2 has been shown to block pro- grammed cell death (Hockenberry et al., 1990). In- deed, apoptotic cell death induced by c-myc is inhibited by bcl-2, indicating a novel mechanism for oncogene interaction of potential interest in carcino- genesis (Bisonnette et al., 1992; Fanidi et al., 1992). Recently, the v-raf kinase has been described to trigger a pathway, alternative to bcl-2, which sup-
881
882 Miguel Lucas and Victor Sfinchez-Margalet
presses apoptosis and promotes cell cycle progression and cell survival (Cleveland et al., 1994).
A final common step should recruit different path- ways to apoptosis and lead to the enzymatic degra- dation of the nuclear membrane, the fragmentation of DNA and the formation of apoptotic bodies. The main candidate for the final step seems to be calcium since a number of calcium-mediated events are crucial in the apoptotic process (Arends et al., 1990; Fesus et al., 1991; Sarin et al., 1993). These events include activation of enzymes, such as endonuclease, protease, transglutaminase and phospholipase, and changes in ionic and water fluxes through plasma membrane leading to condensation of cytosol. More- over, DNA fragmentation and apoptosis have been related to influx of calcium (McConkey et al., 1989a), ATP-dependent Ca 2+ uptake in nuclei (Nicotera et aL, 1989) and to increases in intranuclear free Ca :+ concentration (Bellomo et al., 1992); nonetheless, calcium-independent pathways have been proposed (Lennon et al., 1992; Iseki et al., 1993; Baffy et al.,
1993). An alternative point of view is the consider- ation of calcium and other events, such as the acti- vation of kinases, as both regulatory signals of the pathways to apoptosis and effectors of the final reactions causing apoptosis.
The role of PKC in the induction of apoptosis has been complicated by conflicting reports. For instance, the observation that activation of PKC by exposure to PMA, either alone or in combination with calcium- ionophore, induces apoptosis in lymphoid cells (Mercep et al., 1989) and that PKC inhibitors prevent glucocorticoid-induced apoptosis of thymocytes (Ojeda et al., 1990) suggest that PKC activation promotes apoptosis. On the other hand, the ability of PMA to oppose steroid-induced apoptosis in thymic lymphocytes (McConkey et al., 1989b), to prevent the death of T-lymphocytes deprived of interleukin-2 (Nieto and Lrpez-Rivas, 1989; Rodriguez-Tarducy and Lrpez-Rivas, 1989), radiation-induced apoptosis in vi tro (Tomei et al., 1988) as well as serum depri- vation-induced apoptosis of mature lymphocytes (Lucas et al., 1991) support an antagonistic effect of PKC in the apoptotic process (see also Table 1 that summarizes more recent data on the involvement of PKC in apoptotic cell death). It is conceivable that conflicting observations regarding the apparent role of PKC in the regulation of apoptosis reflect cell type-specific responses to triggering agents (Jarvis et al., 1994a). The present article deals with the implication of PKC in programmed cell death and in cell survival pointing to possible mechanisms and PKC-targets during the apoptotic process. The role of other kinases, such as tyrosine-kinases (Otani et al., 1993) or the double-stranded RNA-activated
kinase recently described to induce apoptosis (Lee and Esteban, 1994) is out of the scope of this review.
STORE-OPERATED CALCIUM ENTRY, BCL-2 AND APOPTOSIS
bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death (Hockenberry et al., 1990) by mechanisms which cannot be related to metabolic process such as ATP depletion since bcl-2 blocks apoptosis, induced by either serum depri- vation or staurosporine, even in cells lacking mito- chondrial DNA. Moreover bel-2 protein is known to be associated with the nuclear envelope and the endoplasmic reticulum as well as the mitochondrial membrane (Jacobson et al., 1993). An interesting observation was the inhibition by bcl-2 oncoprotein of the apoptosis induced by withdrawal of inter- leukins that was clearly associated with the reparti- tioning of intracellular calcium (Baffy et al., 1993). These observations have been reinforced by the experiments with thapsigargin, an inhibitor of the
1 2 3 4
Fig. 1. Internucleosomal breakdown of DNA. Peripheral blood lymphocytes (PBL) were incubated for 72hr as follows: medium lacking fetal bovine serum (lanes 1 and 2) and supplemented with 50riM PMA (lane I); complete medium, including 10% heat-inactivated fetal bovine serum and 1/~M staurosporine (lane 4). DNA was extracted and labelled with [~32p]-dCTP and the Klenow fragment of DNA polimerase. Lane 3 was loaded with a 100 bp molecu-
lar size marker.
Protein kinase C involvement in apoptosis
Table I.
883
Dnta supporting that PKC activation Mocks apopto~lis DNA fragmentation is associated with down regulation of PKC in promyclocytic leukemia cells (Jarvis et aL, 1994b, c) PKC inhibitors enhance apoptosis in mouse natural killer cells and cytotoxic T lymphocytes (Migliorati et al., 1994) The activation of PKC promotes cell survival of mature lymphocytes prone to apoptosis (Lucas et al., 1994) and protects from radiation-induced apoptony (Radford, 1994) Cyclosporine A proteCts B cells against calcium-dependent apoptosis, by blockade of the phosphoprotein phosphatase calcineurin, but not against the apoptosis triggered by the PKC inhibitor chelerytrine (Bonnefoyberard et al., 1994) Proper combination of calcium ionophore and protein kinase activator (PMA) inhibits corticosterone-induced apoptosis in lymphocytes (Iseki et al., 1993) Apoptosis, occurring at a high rate among B cells in germinal centers can be arrested by protein kinase C-activating phorbol esters (Knox et aL, 1993) Inhibition, as well as down regulation, of PKC cause apoptosis in freshly isolated rat bepatocytes (S/mchez et al., 1992) The inhibition of PKC by staurosporine triggers apoptosis of insulin-secreting RIN m5F cells without raising cytosolic free calcium (Shnchez-Margalet et al., 1993) Inhibitors of PKC block the prolongation of neutrophil survival induced by granulocyte colony-stimulating factor (G-CSF) and induce DNA fragmentation at concentrations that fail to alter the priming effect of G-CSF (Adachi et al., 1993) Translocation of PKC from the cytosol mediates phosphatidyl inositol-dependent pathway of rescue germinal center B cells from apoptosis (Knox and Gordon, 1994) Basic fibroblast growth factor (bFGF) and phorbol esters protect endothelial cells against radiation-induced apoptosis. The mechanism of action of bFGF involves activation of tyrosine-kinase which in turns causes the translocation of the alpha isotype of cytoplasmic PKC into the membrane (Haimovitzfriedman et al., 1994a) PKC inhibitors induce apoptosis of malignant glioma cells (Couldwell et aL, 1994) Selective PKC inhibitors block IL-2-mediated proliferation of routine T cells and cause apoptosis (G6mez et al., 1994) PKC activation blocks both radiation-induced sphingomyelin hydrolysis and apoptosis of aortic endothelial cells (Haimovitzfriedman et al., 1994b) Haggerty and Monroe (1994) describe a mutant of a B lymphocyte line sensitive to apoptosis caused by signaling components that lie downstream of PKC
Data supporting that PKC activation promotes apoptosis Fragmentation of DNA induced by tyrosine kinase-inhibitors in mouse thymocytes is enhanced by phorbol esters capable of activating PKC (Azuma et al., 1993) Serine-threonine phosphatase inhibitors synergistically augment TNF-induced apoptosis in several TNF-sensitive tumor cell lines including histiocytic lymphoma, mammary carcinoma, and prostatic tumor cells (Wright et al., 1993) Phorbol ester induces apoptosis in promyelocytic leukemia cells (Macfarlane and O'Dnnnell, 1993; Macfarlane and Manzel, 1994) Phorbol ester enhances mitoxantrone-induced internucleosomal DNA fragmentation in human myeloid leukemia cells, whereas PKC inhibitors have no effects (Bhalla et al., 1994) Retinoic acid and activators of PKC enhance apoptosis of neuronal cells induced by serum deprivation down regulation of PKC reduces the ability of retinoic acid to induce apoptosis (Mailhos et al., 1994) Radiation-induced apoptosis of mouse thymocytes is prevented by a PKC inhibitor and. is potentiated by the PKC activator phorbol ester (Shaposhnikova et al., 1994) Phorbol esters leads to growth arrest an apoptosis of a continuously proliferating B lymphocyte line (Haggerty and Monroe, 1994)
calcium pumping ATPase of the endoplasmic reticu- lum that causes persistent depletion of intracellular calcium stores and produces apoptosis of hepatoma cell lines (Kaneko and Tsukamoto, 1994). This ap- parent paradox (the association of calcium depletion and apoptosis) can be explained taking into account
t he so-ca l led " c a p a c i t a t i v e " ( s t o r e -ope ra t ed ) m o d e l
o f c a l c ium en t ry t h a t is r egu l a t ed by the degree
o f dep l e t i on o f the e n d o p l a s m i c r e t i c u l u m c a l c i u m
pool . In te res t ing ly , this s t o r e - o p e r a t e d c a l c i u m en t ry
m e c h a n i s m is inh ib i t ed by s t i m u l a t i o n o f p ro t e in
k ina se C ( M o n t e r o e l a l . , 1993). T h e inh ib i t i on o f
Fig. 2. Thapsigargin (Tg), by inhibiting Ca2+-pumping ATPase in endoplasmic reticulum (er), depletes this intracellular calcium pool which in turn triggers the entry of calcium. The oncoprotein bcl-2 blocks the effect o f thapsigargin in the Ca2+-pumping ATPase. The activation of PKC inhibits the entry of
calcium by the store operated "capacitative" mechanism (Montero e t al . , 1993).
884 Miguel Lucas and Victor Shnchez-Margalet
Sphingomyel in
Ceramide Sphingosine
Phosphatase Kinase
APOPTOSIS
Fig. 3. Scheme of the putative mechanism of the sphin- gomyelinase pathway to apoptosis. The hydrolysis of sphin- gomyelin produces ceramide and sphingosine which, by activating phosphatase and inhibiting kinase respectively, regulate the activity of target proteins (T.P.) hypothetical
effectors of apoptosis.
calcium entry should block the activation of calcium- dependent enzymes triggering the apoptotic cell death. In fact Lam e t al. (1994) have recently explained the role of bcl-2 in the repression of apoptosis by regulating endoplasmic reticulum- associated calcium fluxes. The induction of apoptosis by thapsigargin is blocked by bcl-2 and may be explained assuming that the oncoprotein, by inhibit- ing calcium leaking from the endoplasmic reticulum hinder the thapsigargin-induced store-operated cal- cium entry and therefore prevents the permanent increase of cytosolic free calcium that causes apopto- sis. This could also be a general mechanism of the abrogation of apoptosis by phorbol esters since it inhibits the store-operated calcium entry into the cytosol (Montero et al. , 1993). Nonetheless, a direct effect of PKC on bcl-2 has been described indicating that hematopoietic growth factors may inhibit apoptosis by a molecular mechanism involv- ing activation and phosphorylation of bcl-2 (May e t al. , 1993).
PKC AND THE SPHINGOMYELIN PATHWAY TO APOPTOSIS
Sphingolipid breakdown products have anti-prolif- erative and tumor-suppressor properties (Hannun and Linardic, 1993) and the hydrolysis of sphin- gomyelin and ceramide generation have been impli- cated in a signal transduction pathway that mediates the effects of tumor necrosis factor alpha, TNF~. Moreover, it has been described that ceramide (Obeid e t al. , 1993) as well as the activation of the sphin- gomyelin hydrolysis (Jarvis et al. , 1994a) induce programmed cell death, a process that is inhibitible by the protein kinase C activators phorbol-myristate
acetate and synthetic diglycerides, suggesting oppo- site roles for digyiceride- and' ceramide-mediated signals in the regulation of apoptosis. Therefore, ceramide is a possible mediator of apoptosis in response to a number of agents, including interferon and hypoxia, that causes sphingomyelin hydrolysis. Besides, a ceramide-activated protein phosphatase could mediate the effects of ceramide (Dobrowsky and Hannum, 1992). On the other hand, sphingosine, a breakdown product of sphingolipids, is well known for its pharmacological inhibition of PKC. The co- incidence of both complementary events may argue in favor of a PKC mediated pathway by inhibiting the phosphorlyation and activating the phosphatase of target proteins.
Very recently, it has been described that ionizing radiation acts on cellular membranes of bovine and aortic endothelial cells to generate ceramide and initiate apoptosis, suggesting an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell killing. The authors (Haimovitzfriedman e t al., 1994a, b) indicate that PKC activation blocked both radiation-induced sph- ingomyelin hydrolysis and apoptosis. Cell type seems important since radiation-induced apoptosis of mouse thymocytes is prevented by PKC inhibitors
I Synthosis I I Mitosis I P P
J f Y T Y T
?l ?
Y T
Fig. 4. The entry into the mitosis phase requires the activation of p34 ~2 kinase following dephosphorylation and association with cyclins. The uncoupling of these events, by p34 ¢d¢2 activation before DNA replication is completed, causes "mitosis catastrophe", a process quite similar to apoptosis. The triggering of apoptosis by the uncoupling of completion of DNA synthesis with events leading to nuclear membrane disintegration and apoptotic bodies formation is an interesting hypothesis with a number of interrogations
(see also Oberharmer et al., 1994).
Protein kinase C involvement in apoptosis 885
whereas it is potentiated by activation of PKC (Sha- posnikova et al., 1994).
APOPTOSIS VERSUS MITOSIS
The uncoupling of timing for p34 ~c2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that appears results from mitosis during DNA replication (Nurse, 1990; Heald et aL, 1993). P34 ~c2 is a highly regulated serine-threonine kinase that controls cell entry into mitosis. The regulation of p34 cd~2 is known to involve a network of kinases and phosphatases, that may respond to the state of DNA replication, as well as the formation of complexes with cyclins (Nurse,
1990). Entry into M-phase is determined by acti- vation of p34 ¢dc2 which requires p34 °de dephosphory-
lation of phosphotyrosine and phosphothreonine and association with cyclins. The active form of the kinase leads to the phosphorylation of key substrates: H1- hitone, p60src, iamins, centrosomal proteins and other proteins which need to be displaced from chromatin to allow chromosome condensation. The complex of p34~C2/cyclins initiates the dissolution of nuclear membrane and promote chromatin conden- sation, events that take place in both mitosis and apoptosis (Meikrantz et al., 1994). It has been re- cently proposed that premature p34 ~dc2 activation
may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus. This has been deduced from experiments with fragmentin and with staurosporine that induces dephosphorylation of p34 ~dc2 and apoptosis in lymphoma and mammary carcinoma cell lines (Shi et al., 1994). Staurosporine is a potent PKC inhibitor with a broad apoptotic activity in cell lines from different origins (Bertrand et al., 1994). This hypoth- esis may be questioned since Oberharmer et al. (1994) have recently shown that chromatin condensation during apoptosis seems to be due to a rapid proteol- ysis of nuclear matrix proteins which does not involve the p34 ~¢2 kinase; in contrast to mitosis, dephospho- rylation and activation of p34 ~dc2 does not occur in apoptotic cells.
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