Protein kinases and phosphatases
DURING THE PAST DECADE, phospholipids have been shown to be precursors of second messengers for cell signalling, and the importance of protein kinase C (PKC) activation coupled to phosphatidylinositol (PtdIns) hydrolysis is well appreciatedL It is now clear that phosphatidylcholine (PC) hydrolysis also contributes to the generation of diacylglycerol (DAG) in the relatively later phases of cellular responses, and a possible role of phospholipase D in the activation of PKC has been proposed 2,3. In a similar way, phospholipase A2 (PLA2) has also been shown to be activated by most of the signals which induce Ptdlns hydrolysis 4. The reaction products of PC hydrolysis by PLA2, cis- unsaturated fatty acids and lysophosphatidylcholine (lysoPC), are both enhancer molecules of PKC activation 5,6. It is plausible that various metabolites of signal-induced degradation of membrane phospholipids may take part in sustained PKC activation, which is a prerequisite for long-term physiological responses such as cell proliferation and differentiation. This review will briefly describe the intimate interactions among various phospholipases and calcium (Ca2+) mobilization. Most results available to date appear to favour PKC playing a role in this phospholipid degradation cascade.
TIBS 17 - OCTOBER1992
Protein kinase C, calcium and phospholipid degradation
In most cells, calcium signals are transient, while the resulting physiological responses often persist longer. The sustained activation of protein kinase C has been postulated to be essential for maintaining such cellular responses. It is becoming clear that an elaborate network involving protein kinase C, calcium and degradation of membrane phospholipids may generate several molecules that are necessary for sustaining the activation of protein kinase C itself. Multiple members of the protein kinase C family show distinct responses to calcium and the phospholipid degradation products, suggesting their unique functions in cell signalling.
tremely rapid 7. On the other hand, tumour-promoting phorbol esters that mimic DAG for PKC activation are metabolically stable, and thus the cellular responses caused by phorbol esters differ somewhat from those caused by a membrane-permeant DAG8. Recent
experiments with repeated additions of a membrane-permeant DAG have confirmed that the sustained activation of PKC is a prerequisite essential for causing long-term physiological responses such as cell proliferation and differentiation 7.
PC
Sustained elevation of DAG and PKC activation
When receptors are stimulated, DAG is initially produced as a result of Ptdlns hydrolysis, particularly phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2]. This DAG production is normally transient, and temporally corresponds to the formation of inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], which is frequently followed by a more sustained elevation of DAG. Extensive analysis of fatty acid compositions has indicated that this second phase of DAG formation is most likely due to the hydrolysis of PC in various stimulated cells 2'3. Phosphatidylethanolamine (PE) appears to be a minor source of DAG. Tracer experiments indicate that the metabolic turnover rate of DAG is exY. Asaoka is at the Biosignal Research Center, Kobe University, Kobe 657, Japan. S. Nakamura, K. Yoshidaand Y. Nishizuka are at the Departmentof Biochemistry, Kobe University School of Medicine, Kobe 650, Japan. 414
myo-inositol -.~ ~ - ~ Choline I PC-PLC
PA
fins
Ptdlns-PLCI P choline - ~
k. p i DAG
Figure 1 Three potential pathways for the generation of DAG from PC. PC, phosphatidylcholine; P-choline, phosphocholine; Pi, inorganic phosphate; DAG, diacyiglycerol; Ptdlns, phosphatidylinositot; PA, phosphatidic acid; PLD, phospho~ipaseD; PC-PLC,PC-reactive phospholipase C; Ptdlns-PLC, phosphatidylinositol-specific phospholipase C; PAP, phosphatidic acid phosphohydrolase. © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
TIBS 17 - OCTOBER 1992
Obviously, the DAG level in membranes depends on the balance between its formation and degradation. The biochemical mechanism to maintain the DAG level sufficient to sustain PKC activation is discussed below.
Protein kinases and phosphatases
C a 2+
SIGNAL
Ptdlns(4, 5)P 2
PC
I PhospholipaseC I
I PhospholipaseA21
L,
I
PhospholipaseD
0
I
Generation of DAG from PC %,* Several mechanisms may DAG Ins(l, 4, 5)P 3 DAG FFA LysoPC be responsible for the signal-induced formation of DAG from PC (Fig. 1). PLCs previously identified in mammalian tissues are Ca2+ specific to PtdIns, but the existence of PLC that is reactive with PC has been Sustained PKC activation Transient PKC activation proposed to occur in several tissues 2,3. However, the relationship between this type of PLC and the signalLate responses Early responses induced generation of DAG e.g. Proliferation e.g. Secretion remains unclear. It has been Differentiation Release reaction suggested that PC-reactive PLC requires protein-tyrosine phosphorylation for its Figure 2 activation 9. Signal-induced degradation of membrane phospholipids and cellular responses. Ptdlns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; Ins(1,4,5)P 3, inositol 1,4,5-trisphosphate; It is more likely that PC DAG, diacylglycerol; FFA, cis-unsaturated fatty acid; LysoPC, lysophosphatidyicboline; PKC, protein is hydrolysed by phosphokinase C. Dashed lines and the crosses inside the circles indicate the positive feedback effect of PKC lipase D (PLD) in a signalon the activities of phospholipases. dependent manner, resulting in the formation of phosphatidic acid, which is then con- been shown to stimulate PLD as well Unsaturated fatty acid and lysoPC PLA2 hydrolyses phospholipids to verted to DAG by the removal of its as base-exchange reactions H. More rephosphate 2,3. Mammalian PLD is as- cently, evidence obtained from the liberate free fatty acids and lysophossociated with primarily particulate frac- authors' laboratory suggests that the pholipids. It is abundant in mammalian tions, presumably membranes, and its choline moiety of PC may be exchanged tissues, and it has been proposed that kinetic properties vary greatly from with free inositol to produce PtdIns, this enzyme undergoes receptortissue to tissue, suggesting its extensive and this exchange reaction is stimu- mediated activation 4. The signals that heterogeneity. The enzyme reacts pref- lated by a phorbol ester or a membrane- provoke PtdIns hydrolysis frequently erentially with PC and also PE. Note permeant DAG (S. Nakamura et al., release arachidonic acid. It has recently that PLD partially purified from rat unpublished). It may therefore be poss- been found that cis-unsaturated fatty brain and liver membranes shows a ible that stimulation of the cell results acids and lysoPC, which are produced considerable activity at the submicro- in the conversion of PC into Ptdlns by from PC by PLA2, can both enhance submolar range of Ca2÷ concentrations ~°'H. an exchange reaction catalysed by PLD; sequent cellular responses, most likely PLD from mammalian tissues also cata- the resulting PtdIns may subsequently by interacting with the PKC pathway lyses the trans-phosphatidylation reac- be hydrolysed by PLC to produce DAG (Fig. 2) 5,6. For PKC activation, DAG was pretion (base-exchange reaction), but the and inositol phosphate. The concenrelationship between PLD (hydrolytic tration of free myo-inositol in the cell is viously shown to increase the apparent reaction) and base-exchange enzymes known to be in the region of one milli- affinity of PKC for Ca2+, thereby rendermolar. remains unclear. ing the enzyme active at micromolar Although none of these mechanisms Ca2+concentrations 1. It has subsequently In various intact cells, phorbol esters have been shown to stimulate PC break- has yet been firmly established on a been found that cis-unsaturated fatty down to produce choline and phospho- molecular basis, it is plausible that l~he acids, including oleic, linoleic, linolenic, choline ~2. In addition, PLD has been sustained levels of DAG may come from arachidonic and docosahexaenoic acids, shown to be activated by a phorbol PC. Also, PKC activated initially by greatly enhance PKC activation13; that ester or a membrane-permeant DAG, PtdIns hydrolysis may play a role in is, in the presence of these fatty acids sometimes synergistically with a Ca2÷ enhancing PC hydrolysis and providing and DAG, PKC exhibits near-full activity ionophore 2,3. In experiments with mem- DAG through the action of PLD and/or at submicromolar Ca2+ concentrations. brane fractions and permeabilized cells, PLC, which in turn sustains its own acti- Kinetic analysis that uses intact human phorbol esters and GTP analogues have vation. platelets as a model system has
1
I
415
Protein kinases and phosphatases Table I. PKC subspecies in mammalian tissues Group
Subspecies
Apparentmolecular mass (kDa)
Activatorsb
Tissue expression
cPKC
c~
76799
Ca2+, DAG, PS, FFA,LysoPC Universal
~1
76 790
Ca2+, DAG, PS, FFA, LysoPC Sometissues
[311
76933
Ca 2+, DAG, PS, FFA,LysoPC Manytissues
y
78366
Ca 2+, DAG, PS, FFA, LysoPC Brainonly
TIBS 17 - OCTOBER 1 9 9 2
alone is inactive. Mong with physiological and pathological agonists including antigens, this type of PLA 2 may take part in the propagation of inflammatory responses. Heterogeneity of PKC and mode of activation There are more than one species of
PKC molecule, and multiple discrete subspecies have been defined. These nPKC 5 77 517 DAG, PS Universal show distinct enzymological charac83 474 DAG, PS, FFA Brain and others teristics and differential tissue exq (L) a 77 972 ? Lung, skin, heart pression with specific intracellular localization 1. Further, molecular cloning 0a 81571 ? Skeletal muscle (mainly) techniques and biochemical analyses have uncovered additional subspecies 67 740 PS, FFA Universal aPKC of the enzyme, and their enzymological properties have been clarified in detail. Xa 67 200 ? Ovary, testis and others There are presently ten identified subspecies of PKC in mammalian tissues aThedetailed enzymologicalpropertiesof the q (L)-, 0-, and X-subspecieshave not yet been clarified (K. (Table I). The first group of four classiAkimoto et al., unpublished). cal or conventional PKC (cPKC) subbThe activators for each subspecies are determined with calf thymus H1 histone and bovine myelin species (o¢, ]~I, [5II and 7) emerged from basic protein as model phosphate accepters. the initial screening 1, while the second Abbreviations: DAG, diacylglycerol;PS, phosphatidylserine; FFA, c/s-unsaturatedfatty acid; LysoPC, consists of four new PKC (nPKC) sublysophosphatidylcholine. species [8, ~, rl (L) and 0] 1,18-2°.The third and most recently identified group contains two atypical PKC (aPKC) subspecies (4 and ~)21. revealed that these fatty acids act as PLA~activation The members of the PKC family so Arachidonic acid-selective and nonenhancer molecules and that the coexistence of a membrane-permeant selective cytosolic PLA2 molecules are far examined are dependent on phosDAG is always needed for the platelet both present within cells, but only the phatidylserine (PS), but show clearly activation. Measurement of the intracel- former is well characterized. PLA2 is different requirements of Ca2* and phoslular Ca2* level using the fluorescent known to be activated by long-acting pholipid metabolites for their activation. dye fura-2 indicates that platelets signals such as some growth factors, cPKC enzymes show characteristics respond well to a membrane-permeant suggesting that the activity of cytosolic that are typical of the previously deDAG without an appreciable increase in PLA2 may be regulated by some second scribed Ca2--activated, phospholipidthe Ca2*concentration in the presence of messengers TM.Recent studies with intact dependent protein kinase. These encis-unsaturated fatty acids 5. Presumably cell systems as well as with cell mem- zymes are activated by Ca2+ and DAG, PKC activated by PtdIns hydrolysis in branes suggest that PKC plays a role in and this activation is further enhanced stimulated cells may intensify its enzy- the signal-induced activation of PLA2, by c/s-unsaturated fatty acids and lysoPC, matic activity even after the Ca 2÷concen- since phorbol esters and membrane- as described above. On the other hand, tration returns to the basal level if DAG permeant DAG provoke arachidonic nPKC enzymes are insensitive to Ca 2", and cis-unsaturated fatty acids both acid release, sometimes in synergy with although they respond well to DAG and physiological agonists 15. In neutrophils, phorbol esters 22'23. The E-subspecies become available. LysoPC, the other product of PC it appears that phorbol esters activate may be activated further by cis-unsatuhydrolysis by PLA2, shows a membrane- both arachidonic acid-selective and rated fatty acid. The & and E-sublyric activity, and is toxic to intact cells. non-selective PLA2, thereby increasing species, which are the best-characHowever, when lysoPC is added to the the intracellular levels of various terized enzymes in this group so far, cell together with a membrane-per- cis-unsaturated fatty acids ~6. Plausible exist in phosphorylated forms in native meant DAG or a phorbol ester, the long- evidence available to date strongly sug- tissues, and show doublet bands upon term physiological responses are en- gests that the signal-induced PLA2 acti- electrophoresis 22,23. This is not simply hanced in particular. For example, vation may also be involved directly in due to their autophosphorylation. In contrast, aPKC enzymes have not yet lysoPC alone does not activate human cell signalling. Low molecular weight arachidonic been fully characterized, but the 4- subresting T-lymphocytes. However, when both a membrane-permeant DAG and acid-non-selective PLA2 molecules (extra- species does not respond to Ca2~, DAG ionomycin are present, the lympho- cellular secretory PLA2) are known to or phorbol esters 2~,24. The enzyme recytes are activated 6, indicating that this be secreted by many cell types into quires PS, and is activated by cis-unsatulysophospholipid interacts with the extracellular spaces, especially those at rated fatty acids. The signal to activate PKC pathway. Biochemical analysis inflammatory sites 17. This type of en- aPKC enzymes remains unknown. shows that low concentrations of zyme, when added directly to the cell, lysoPC indeed intensify the DAG-depen- can greatly enhance the signal-induced Implications and perspectives Despite extensive studies, our knowldent PKC activation over a wide range cellular responses, such as T-lymphocyte activation. Note that the PLA2 edge of specific functions of the indiof Ca2+concentrations.
416
TIBS 17 - OCTOBER 1992
Protein kinases and phosphatases
vidual PKC subspecies is still limited. It is clear, however, that the members of the PKC family respond differently to various combinations of Ca2+, DAG and other phospholipid degradation products, thus producing distinct activation patterns with respect to the extent and duration of response and perhaps to their intracellular localization. Biochemical and immunocytochemical studies have shown that the PKC subspecies have different distributions in particular cell types within limited intracellular localization. Presumably each subspecies of the PKC family is present in the right intracellular compartment of the cell at the right time, in association with its specific target substrate proteins. Such spatio-temporal aspects of phospholipid degradation and the activation of PKC subspecies with the cell will inevitably be explored. It is tempting to suggest that the (z-subspecies plays a crucial role in cell growth and differentiation, since it is expressed universally in all cell types so far examined, and responds well to the various phospholipid degradation products discussed above. However, the members of nPKC, particularly the 5-subspecies, may also play a role in the control of nuclear events. This enzyme may persist in its activation because it is not sensitive to Ca2÷. nPKC enzymes may be integrated directly or indirectly in a protein kinase cascade, which is initiated by the activation of some growth factor receptors, eventually lead-
ing to the regulation of the cell cycle. Recent experiments have revealed that treatment with phorbol esters of CHO cells that overexpress the 5-subspecies prevents their cell division possibly at the G2/M phase 2s. In the membrane, however, crucial roles have been assigned to PKC in receptor down-regulation, ion channel modulation, and various steps of the crosstalk of cell signalling pathways. Again, the distinct mode of activation, Ca2+sensitivity, apparent patterns of tissue expression and intracellular localization of the multiple subspecies imply their unique functions in cell signalling.
J
~~ ~
Conclusion A stimulation of the cell-surface receptor elicits a series of degradation cascades of membrane phospholipids, and many of the degradation products may directly contribute to the control of cellular responses through activation of multiple subspecies of the PKC family. Elucidation of the biochemical mechanism of the interactions among various phospholipases to cause such a phospholipid degradation cascade is vital for our understanding of cellular regulation.
Acknowledgements Work from the authors' laboratory was supported in part by research grants from the Special Research Fund of Ministry of Education, Science and Culture, Japan.
References 1 Nishizuka, Y. (1988) Nature 334, 661-665 2 Exton, J. H. (1990) J. Biol. Chem. 265, 1-4 3 Billah, M. M. and Anthes, J. C. (1990) Biochem. J. 269, 281-291 4 Axelrod, J., Butch, R. M. and Jelsema, C. L. (1988) Trends Neurosci. 11, 117-123 5 Yoshida, K., Asaoka, Y. and Nishizuka,Y. (1992) Proc. Natl Acad. Sci. USA 89, 6443-6446 6 Asaoka, Y. et al. (1992) Proc. Natl Acad. Sci. USA 89, 6447-6451 7 Asaoka,Y., Oka, M., Yoshida,K. and Nishizuka,Y. (1991) Proc. Natl Acad. Sci. USA 88, 8681-8685 8 Yamamoto, S., Gotoh, H., Aizu, E. and Kato, R. (1985) J. Biol. Chem. 260, 14230-14234 9 Choudhury, G. G., Sylvia, V. L. and Sakaguchi, A. Y. (1991) J. Biol. Chem. 266, 23147-23151 10 Chalifa, V., M6hn, H. and Liscovitch, M. (1990) J. Biol. Chem. 265, 17512-17519 11 Siddiqui, R. A. and Extort, J. H. (1992) J. Biol. Chem. 267, 5755-5761 12 Pelech, S. L. and Vance, D. E. (1989) Trends Biochem. Sci. 14, 28-30 13 Shinomura, T. et al. (1991) Proc. Natl Acad. Sci. USA 88, 5149-5153 14 Bonventre, J. V., Gronich, J. H. and Nemenoff, R. A. (1990) J. Biol. Chem. 265, 4934-4938 15 Rehfeldt, W., Hass, R. and Goppelt-Struebe, M. (1991) Biochem. J. 276, 631-636 16 Conquer, J. and Mahadevappa, V. G. (1991) J. Lipid Mediators 3, 113-123 17 Kudo, I. et al. (1989) Dermatologica 179, 72-76 18 Liyanage, M., Frith, D., Livneh, E. and Stabel, S. (1992) Biochem. J. 283, 781-787 19 Osada, S. et al. (1990) J. Biol. Chem. 265, 22434-22440 20 Osada, S. et al. (1992) MoI. Ceil. Biol. 12, 3930-3938 21 Ono, Y. et al. (1989) Proc. Natl Acad. Sci. USA 86, 3099-3103 22 Ogita, K. et al. (1992) Proc. Natl Acad. Sci. USA 89, 1592-1596 23 Koide, H., Ogita, K., Kikkawa,U. and Nishizuka,Y. (1992) Proc. Natl Acad. Sci. USA 89, 1149-1153 24 Nakanishi, H. and Exton, J. H. J. Biol. Chem. (in press) 25 Watanabe, T. et al. Proc. Natl Acad. Sci. USA (in press)
STIMULUS
receptor
{f'~k'l~ ]
proteinkinaseC
phospholipaseC
~
iuositolP3~
substrate
"
RESP~ONSE 417
DURING THE PAST DECADE, phospholipids have been shown to be precursors of second messengers for cell signalling, and the importance of protein kinase C (PKC) activation coupled to phosphatidylinositol (PtdIns) hydrolysis is well appreciatedL It is now clear that phosphatidylcholine (PC) hydrolysis also contributes to the generation of diacylglycerol (DAG) in the relatively later phases of cellular responses, and a possible role of phospholipase D in the activation of PKC has been proposed 2,3. In a similar way, phospholipase A2 (PLA2) has also been shown to be activated by most of the signals which induce Ptdlns hydrolysis 4. The reaction products of PC hydrolysis by PLA2, cis- unsaturated fatty acids and lysophosphatidylcholine (lysoPC), are both enhancer molecules of PKC activation 5,6. It is plausible that various metabolites of signal-induced degradation of membrane phospholipids may take part in sustained PKC activation, which is a prerequisite for long-term physiological responses such as cell proliferation and differentiation. This review will briefly describe the intimate interactions among various phospholipases and calcium (Ca2+) mobilization. Most results available to date appear to favour PKC playing a role in this phospholipid degradation cascade.
TIBS 17 - OCTOBER1992
Protein kinase C, calcium and phospholipid degradation
In most cells, calcium signals are transient, while the resulting physiological responses often persist longer. The sustained activation of protein kinase C has been postulated to be essential for maintaining such cellular responses. It is becoming clear that an elaborate network involving protein kinase C, calcium and degradation of membrane phospholipids may generate several molecules that are necessary for sustaining the activation of protein kinase C itself. Multiple members of the protein kinase C family show distinct responses to calcium and the phospholipid degradation products, suggesting their unique functions in cell signalling.
tremely rapid 7. On the other hand, tumour-promoting phorbol esters that mimic DAG for PKC activation are metabolically stable, and thus the cellular responses caused by phorbol esters differ somewhat from those caused by a membrane-permeant DAG8. Recent
experiments with repeated additions of a membrane-permeant DAG have confirmed that the sustained activation of PKC is a prerequisite essential for causing long-term physiological responses such as cell proliferation and differentiation 7.
PC
Sustained elevation of DAG and PKC activation
When receptors are stimulated, DAG is initially produced as a result of Ptdlns hydrolysis, particularly phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2]. This DAG production is normally transient, and temporally corresponds to the formation of inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], which is frequently followed by a more sustained elevation of DAG. Extensive analysis of fatty acid compositions has indicated that this second phase of DAG formation is most likely due to the hydrolysis of PC in various stimulated cells 2'3. Phosphatidylethanolamine (PE) appears to be a minor source of DAG. Tracer experiments indicate that the metabolic turnover rate of DAG is exY. Asaoka is at the Biosignal Research Center, Kobe University, Kobe 657, Japan. S. Nakamura, K. Yoshidaand Y. Nishizuka are at the Departmentof Biochemistry, Kobe University School of Medicine, Kobe 650, Japan. 414
myo-inositol -.~ ~ - ~ Choline I PC-PLC
PA
fins
Ptdlns-PLCI P choline - ~
k. p i DAG
Figure 1 Three potential pathways for the generation of DAG from PC. PC, phosphatidylcholine; P-choline, phosphocholine; Pi, inorganic phosphate; DAG, diacyiglycerol; Ptdlns, phosphatidylinositot; PA, phosphatidic acid; PLD, phospho~ipaseD; PC-PLC,PC-reactive phospholipase C; Ptdlns-PLC, phosphatidylinositol-specific phospholipase C; PAP, phosphatidic acid phosphohydrolase. © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
TIBS 17 - OCTOBER 1992
Obviously, the DAG level in membranes depends on the balance between its formation and degradation. The biochemical mechanism to maintain the DAG level sufficient to sustain PKC activation is discussed below.
Protein kinases and phosphatases
C a 2+
SIGNAL
Ptdlns(4, 5)P 2
PC
I PhospholipaseC I
I PhospholipaseA21
L,
I
PhospholipaseD
0
I
Generation of DAG from PC %,* Several mechanisms may DAG Ins(l, 4, 5)P 3 DAG FFA LysoPC be responsible for the signal-induced formation of DAG from PC (Fig. 1). PLCs previously identified in mammalian tissues are Ca2+ specific to PtdIns, but the existence of PLC that is reactive with PC has been Sustained PKC activation Transient PKC activation proposed to occur in several tissues 2,3. However, the relationship between this type of PLC and the signalLate responses Early responses induced generation of DAG e.g. Proliferation e.g. Secretion remains unclear. It has been Differentiation Release reaction suggested that PC-reactive PLC requires protein-tyrosine phosphorylation for its Figure 2 activation 9. Signal-induced degradation of membrane phospholipids and cellular responses. Ptdlns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; Ins(1,4,5)P 3, inositol 1,4,5-trisphosphate; It is more likely that PC DAG, diacylglycerol; FFA, cis-unsaturated fatty acid; LysoPC, lysophosphatidyicboline; PKC, protein is hydrolysed by phosphokinase C. Dashed lines and the crosses inside the circles indicate the positive feedback effect of PKC lipase D (PLD) in a signalon the activities of phospholipases. dependent manner, resulting in the formation of phosphatidic acid, which is then con- been shown to stimulate PLD as well Unsaturated fatty acid and lysoPC PLA2 hydrolyses phospholipids to verted to DAG by the removal of its as base-exchange reactions H. More rephosphate 2,3. Mammalian PLD is as- cently, evidence obtained from the liberate free fatty acids and lysophossociated with primarily particulate frac- authors' laboratory suggests that the pholipids. It is abundant in mammalian tions, presumably membranes, and its choline moiety of PC may be exchanged tissues, and it has been proposed that kinetic properties vary greatly from with free inositol to produce PtdIns, this enzyme undergoes receptortissue to tissue, suggesting its extensive and this exchange reaction is stimu- mediated activation 4. The signals that heterogeneity. The enzyme reacts pref- lated by a phorbol ester or a membrane- provoke PtdIns hydrolysis frequently erentially with PC and also PE. Note permeant DAG (S. Nakamura et al., release arachidonic acid. It has recently that PLD partially purified from rat unpublished). It may therefore be poss- been found that cis-unsaturated fatty brain and liver membranes shows a ible that stimulation of the cell results acids and lysoPC, which are produced considerable activity at the submicro- in the conversion of PC into Ptdlns by from PC by PLA2, can both enhance submolar range of Ca2÷ concentrations ~°'H. an exchange reaction catalysed by PLD; sequent cellular responses, most likely PLD from mammalian tissues also cata- the resulting PtdIns may subsequently by interacting with the PKC pathway lyses the trans-phosphatidylation reac- be hydrolysed by PLC to produce DAG (Fig. 2) 5,6. For PKC activation, DAG was pretion (base-exchange reaction), but the and inositol phosphate. The concenrelationship between PLD (hydrolytic tration of free myo-inositol in the cell is viously shown to increase the apparent reaction) and base-exchange enzymes known to be in the region of one milli- affinity of PKC for Ca2+, thereby rendermolar. remains unclear. ing the enzyme active at micromolar Although none of these mechanisms Ca2+concentrations 1. It has subsequently In various intact cells, phorbol esters have been shown to stimulate PC break- has yet been firmly established on a been found that cis-unsaturated fatty down to produce choline and phospho- molecular basis, it is plausible that l~he acids, including oleic, linoleic, linolenic, choline ~2. In addition, PLD has been sustained levels of DAG may come from arachidonic and docosahexaenoic acids, shown to be activated by a phorbol PC. Also, PKC activated initially by greatly enhance PKC activation13; that ester or a membrane-permeant DAG, PtdIns hydrolysis may play a role in is, in the presence of these fatty acids sometimes synergistically with a Ca2÷ enhancing PC hydrolysis and providing and DAG, PKC exhibits near-full activity ionophore 2,3. In experiments with mem- DAG through the action of PLD and/or at submicromolar Ca2+ concentrations. brane fractions and permeabilized cells, PLC, which in turn sustains its own acti- Kinetic analysis that uses intact human phorbol esters and GTP analogues have vation. platelets as a model system has
1
I
415
Protein kinases and phosphatases Table I. PKC subspecies in mammalian tissues Group
Subspecies
Apparentmolecular mass (kDa)
Activatorsb
Tissue expression
cPKC
c~
76799
Ca2+, DAG, PS, FFA,LysoPC Universal
~1
76 790
Ca2+, DAG, PS, FFA, LysoPC Sometissues
[311
76933
Ca 2+, DAG, PS, FFA,LysoPC Manytissues
y
78366
Ca 2+, DAG, PS, FFA, LysoPC Brainonly
TIBS 17 - OCTOBER 1 9 9 2
alone is inactive. Mong with physiological and pathological agonists including antigens, this type of PLA 2 may take part in the propagation of inflammatory responses. Heterogeneity of PKC and mode of activation There are more than one species of
PKC molecule, and multiple discrete subspecies have been defined. These nPKC 5 77 517 DAG, PS Universal show distinct enzymological charac83 474 DAG, PS, FFA Brain and others teristics and differential tissue exq (L) a 77 972 ? Lung, skin, heart pression with specific intracellular localization 1. Further, molecular cloning 0a 81571 ? Skeletal muscle (mainly) techniques and biochemical analyses have uncovered additional subspecies 67 740 PS, FFA Universal aPKC of the enzyme, and their enzymological properties have been clarified in detail. Xa 67 200 ? Ovary, testis and others There are presently ten identified subspecies of PKC in mammalian tissues aThedetailed enzymologicalpropertiesof the q (L)-, 0-, and X-subspecieshave not yet been clarified (K. (Table I). The first group of four classiAkimoto et al., unpublished). cal or conventional PKC (cPKC) subbThe activators for each subspecies are determined with calf thymus H1 histone and bovine myelin species (o¢, ]~I, [5II and 7) emerged from basic protein as model phosphate accepters. the initial screening 1, while the second Abbreviations: DAG, diacylglycerol;PS, phosphatidylserine; FFA, c/s-unsaturatedfatty acid; LysoPC, consists of four new PKC (nPKC) sublysophosphatidylcholine. species [8, ~, rl (L) and 0] 1,18-2°.The third and most recently identified group contains two atypical PKC (aPKC) subspecies (4 and ~)21. revealed that these fatty acids act as PLA~activation The members of the PKC family so Arachidonic acid-selective and nonenhancer molecules and that the coexistence of a membrane-permeant selective cytosolic PLA2 molecules are far examined are dependent on phosDAG is always needed for the platelet both present within cells, but only the phatidylserine (PS), but show clearly activation. Measurement of the intracel- former is well characterized. PLA2 is different requirements of Ca2* and phoslular Ca2* level using the fluorescent known to be activated by long-acting pholipid metabolites for their activation. dye fura-2 indicates that platelets signals such as some growth factors, cPKC enzymes show characteristics respond well to a membrane-permeant suggesting that the activity of cytosolic that are typical of the previously deDAG without an appreciable increase in PLA2 may be regulated by some second scribed Ca2--activated, phospholipidthe Ca2*concentration in the presence of messengers TM.Recent studies with intact dependent protein kinase. These encis-unsaturated fatty acids 5. Presumably cell systems as well as with cell mem- zymes are activated by Ca2+ and DAG, PKC activated by PtdIns hydrolysis in branes suggest that PKC plays a role in and this activation is further enhanced stimulated cells may intensify its enzy- the signal-induced activation of PLA2, by c/s-unsaturated fatty acids and lysoPC, matic activity even after the Ca 2÷concen- since phorbol esters and membrane- as described above. On the other hand, tration returns to the basal level if DAG permeant DAG provoke arachidonic nPKC enzymes are insensitive to Ca 2", and cis-unsaturated fatty acids both acid release, sometimes in synergy with although they respond well to DAG and physiological agonists 15. In neutrophils, phorbol esters 22'23. The E-subspecies become available. LysoPC, the other product of PC it appears that phorbol esters activate may be activated further by cis-unsatuhydrolysis by PLA2, shows a membrane- both arachidonic acid-selective and rated fatty acid. The & and E-sublyric activity, and is toxic to intact cells. non-selective PLA2, thereby increasing species, which are the best-characHowever, when lysoPC is added to the the intracellular levels of various terized enzymes in this group so far, cell together with a membrane-per- cis-unsaturated fatty acids ~6. Plausible exist in phosphorylated forms in native meant DAG or a phorbol ester, the long- evidence available to date strongly sug- tissues, and show doublet bands upon term physiological responses are en- gests that the signal-induced PLA2 acti- electrophoresis 22,23. This is not simply hanced in particular. For example, vation may also be involved directly in due to their autophosphorylation. In contrast, aPKC enzymes have not yet lysoPC alone does not activate human cell signalling. Low molecular weight arachidonic been fully characterized, but the 4- subresting T-lymphocytes. However, when both a membrane-permeant DAG and acid-non-selective PLA2 molecules (extra- species does not respond to Ca2~, DAG ionomycin are present, the lympho- cellular secretory PLA2) are known to or phorbol esters 2~,24. The enzyme recytes are activated 6, indicating that this be secreted by many cell types into quires PS, and is activated by cis-unsatulysophospholipid interacts with the extracellular spaces, especially those at rated fatty acids. The signal to activate PKC pathway. Biochemical analysis inflammatory sites 17. This type of en- aPKC enzymes remains unknown. shows that low concentrations of zyme, when added directly to the cell, lysoPC indeed intensify the DAG-depen- can greatly enhance the signal-induced Implications and perspectives Despite extensive studies, our knowldent PKC activation over a wide range cellular responses, such as T-lymphocyte activation. Note that the PLA2 edge of specific functions of the indiof Ca2+concentrations.
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Protein kinases and phosphatases
vidual PKC subspecies is still limited. It is clear, however, that the members of the PKC family respond differently to various combinations of Ca2+, DAG and other phospholipid degradation products, thus producing distinct activation patterns with respect to the extent and duration of response and perhaps to their intracellular localization. Biochemical and immunocytochemical studies have shown that the PKC subspecies have different distributions in particular cell types within limited intracellular localization. Presumably each subspecies of the PKC family is present in the right intracellular compartment of the cell at the right time, in association with its specific target substrate proteins. Such spatio-temporal aspects of phospholipid degradation and the activation of PKC subspecies with the cell will inevitably be explored. It is tempting to suggest that the (z-subspecies plays a crucial role in cell growth and differentiation, since it is expressed universally in all cell types so far examined, and responds well to the various phospholipid degradation products discussed above. However, the members of nPKC, particularly the 5-subspecies, may also play a role in the control of nuclear events. This enzyme may persist in its activation because it is not sensitive to Ca2÷. nPKC enzymes may be integrated directly or indirectly in a protein kinase cascade, which is initiated by the activation of some growth factor receptors, eventually lead-
ing to the regulation of the cell cycle. Recent experiments have revealed that treatment with phorbol esters of CHO cells that overexpress the 5-subspecies prevents their cell division possibly at the G2/M phase 2s. In the membrane, however, crucial roles have been assigned to PKC in receptor down-regulation, ion channel modulation, and various steps of the crosstalk of cell signalling pathways. Again, the distinct mode of activation, Ca2+sensitivity, apparent patterns of tissue expression and intracellular localization of the multiple subspecies imply their unique functions in cell signalling.
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~~ ~
Conclusion A stimulation of the cell-surface receptor elicits a series of degradation cascades of membrane phospholipids, and many of the degradation products may directly contribute to the control of cellular responses through activation of multiple subspecies of the PKC family. Elucidation of the biochemical mechanism of the interactions among various phospholipases to cause such a phospholipid degradation cascade is vital for our understanding of cellular regulation.
Acknowledgements Work from the authors' laboratory was supported in part by research grants from the Special Research Fund of Ministry of Education, Science and Culture, Japan.
References 1 Nishizuka, Y. (1988) Nature 334, 661-665 2 Exton, J. H. (1990) J. Biol. Chem. 265, 1-4 3 Billah, M. M. and Anthes, J. C. (1990) Biochem. J. 269, 281-291 4 Axelrod, J., Butch, R. M. and Jelsema, C. L. (1988) Trends Neurosci. 11, 117-123 5 Yoshida, K., Asaoka, Y. and Nishizuka,Y. (1992) Proc. Natl Acad. Sci. USA 89, 6443-6446 6 Asaoka, Y. et al. (1992) Proc. Natl Acad. Sci. USA 89, 6447-6451 7 Asaoka,Y., Oka, M., Yoshida,K. and Nishizuka,Y. (1991) Proc. Natl Acad. Sci. USA 88, 8681-8685 8 Yamamoto, S., Gotoh, H., Aizu, E. and Kato, R. (1985) J. Biol. Chem. 260, 14230-14234 9 Choudhury, G. G., Sylvia, V. L. and Sakaguchi, A. Y. (1991) J. Biol. Chem. 266, 23147-23151 10 Chalifa, V., M6hn, H. and Liscovitch, M. (1990) J. Biol. Chem. 265, 17512-17519 11 Siddiqui, R. A. and Extort, J. H. (1992) J. Biol. Chem. 267, 5755-5761 12 Pelech, S. L. and Vance, D. E. (1989) Trends Biochem. Sci. 14, 28-30 13 Shinomura, T. et al. (1991) Proc. Natl Acad. Sci. USA 88, 5149-5153 14 Bonventre, J. V., Gronich, J. H. and Nemenoff, R. A. (1990) J. Biol. Chem. 265, 4934-4938 15 Rehfeldt, W., Hass, R. and Goppelt-Struebe, M. (1991) Biochem. J. 276, 631-636 16 Conquer, J. and Mahadevappa, V. G. (1991) J. Lipid Mediators 3, 113-123 17 Kudo, I. et al. (1989) Dermatologica 179, 72-76 18 Liyanage, M., Frith, D., Livneh, E. and Stabel, S. (1992) Biochem. J. 283, 781-787 19 Osada, S. et al. (1990) J. Biol. Chem. 265, 22434-22440 20 Osada, S. et al. (1992) MoI. Ceil. Biol. 12, 3930-3938 21 Ono, Y. et al. (1989) Proc. Natl Acad. Sci. USA 86, 3099-3103 22 Ogita, K. et al. (1992) Proc. Natl Acad. Sci. USA 89, 1592-1596 23 Koide, H., Ogita, K., Kikkawa,U. and Nishizuka,Y. (1992) Proc. Natl Acad. Sci. USA 89, 1149-1153 24 Nakanishi, H. and Exton, J. H. J. Biol. Chem. (in press) 25 Watanabe, T. et al. Proc. Natl Acad. Sci. USA (in press)
STIMULUS
receptor
{f'~k'l~ ]
proteinkinaseC
phospholipaseC
~
iuositolP3~
substrate
"
RESP~ONSE 417
