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Phosphoinositide 3-kinase and the regulation of cell growth
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ELSEVIER
BB Biochi~ic~a et Biophysica
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Biochimicaet BiophysicaActa 1288(1996)M1 l-M16
Mini Review
Phosphoinositide 3-kinase and the regulation of cell growth Christopher L. Carpenter a, Lewis C. Cantley b,* a Dicision of Signal Transduction, Departmentof Medicine, Beth Israel Hospital HaruardMedical School, Boston, MA 02115, USA b Departmentof Cell Biology, Harvard Medical School, Boston, MA 02115, USA
Received 15 May 1996;accepted 17 May 1996
1. Introduction More than 10 years ago, a phosphatidylinositol (Ptdlns) kinase activity was found associated with the v-src and v-ros oncoproteins [1,2]. Subsequent work revealed that this enzyme phosphorylates the D-3 position of the inositol ring, a reaction not previously known to exist [3]. This work led to the discovery of new signaling pathways involving the phosphoinositide (PI) products of this enzyme, Ptdlns-3-P, Ptdlns-3,4-P 2 and Ptdlns-3,4,5-P 3. Interest in the PI 3-kinases was stimulated by evidence that this enzyme is important for transformation by v-src, polyoma middle T antigen and v-ros and mitogenic signaling by the platelet-derived growth factor (PDGF) receptor [4]. More intense study of the role of P1 3-kinase in cell transformation and mitogenic signaling has led to conflicting data, which likely reflects the redundancy of signals for growth and utilization of different pathways in different cells. The issue of PI 3-kinase in the control of cell growth is further confounded by its role in preventing apoptosis. The recent discoveries of PI 3-kinase family members that are regulated by heterotrimeric G-proteins and by other pathways has greatly expanded the number of signaling systems in which PI 3-kinase seems to be involved. These discoveries also raise the possibility that the role of PI 3-kinase in cell growth in some systems could be quite indirect. To remain concise we have concentrated on pathways in which PI 3-kinase is thought to directly influence cell growth.
2. Background PI 3-kinase was originally purified as a heterodimer of an 85 kDa regulatory subunit and 110 kDa catalytic subunit [5,6]. eDNA cloning of the subunits has revealed five forms of the regulatory subunit [7-12] and five forms of * Corresponding author. Fax: + 1 (617) 2783033; e-mail: [email protected]
catalytic subunits [13-17] to date. The p85 subunit contains, in the amino terminal half, an SH3 domain, a region homologous to the GTPase activating protein (GAP) domain of the breakpoint cluster region (BCR) protein and two proline rich regions that can bind SH3 domains. The carboxy-terminal half has two SH2 domains and a region for binding to p 110. Three recently-described forms of the regulatory subunit have only the carboxy-terminal SH2 domains and the p110-binding domain [10-12]. The catalytic p110 subunit is homologous to protein kinases and, itself, has protein serine/threonine kinase activity, as well as phosphoinositide kinase activity [ 18,19]. The cDNA cloning of p110 a lead to the identification of the yeast gene product, vps34p, as a PtdIns 3-kinase [20]. This gene had been shown to be essential for vesicle trafficking from the golgi to the vacuole. This discovery lead to experiments demonstrating a role for PI 3-kinases in vesicle trafficking in higher organisms [21,22]. The p110 subunits differ in their substrate specificity and regulation. The p85/p110 type PI 3-kinases are regulated by protein-tyrosine kinases and phosphorylate PtdIns, Ptdlns-4-P or PtdIns-4,5-P 2 at the D-3 position (they are named phosphoinositide 3-kinases to indicate their broad in vitro substrate specificity). These enzymes mediate acute increases in PtdIns-3,4,5-P 3 in response to stimulation of receptor-type protein-tyrosine kinases. The p110 3' type enzyme is activated by both the c~ and 13/3' subunits of heterotrimeric G proteins, and this enzyme has similar substrate specificity to the p85/p110 type enzymes [ 16]. In contrast, the yeast vps34p and a mammalian form of vps34p are distinct from the p85/p110-type PI 3-kinase in that they phosphorylate only PtdIns. These enzymes appear to be regulated by an associated protein-serine kinase, vpsl5p. Finally, a recently-described C2 domain-containing catalytic subunit phosphorylates primarily Ptdlns and PtdIns-4-P [17]. This enzyme probably provides an independent pathway for regulated production of PtdIns-3,4-P2. Thus, in vivo levels of PtdIns-3-P, Ptdlns-3,4-P 2 and PtdIns-3,4,5-P 3 can be independently controlled by distinct
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enzymes, suggesting that these lipids each have unique functions in the cell (see below). cDNA cloning has revealed two additional subgroups of proteins with homology to the catalytic domain of PI 3-kinases. The enzymes in the first group are quite homologous to the PI 3-kinases and include human, yeast, and dictyostelium discoideum Ptdlns 4-kinases. The second group is more distant from the PI 3-kinases and Ptdlns 4-kinases and includes the ataxia-telangectasia gene product ( A T M ) , DNA-PK, RAFT/FRAP/TOR, MEC1/RAD3, TEL1, and Mei-41 [23]. Interestingly, all of the proteins in this group, except the R A F T / F R A P / TOR proteins, are implicated in sensing DNA damage. None of them has been shown to have endogenous phosphoinositide kinase activity (although FRAP/RAFT and TOR have been shown to have an associated Ptdlns 4kinase activity [24,25]. DNA-PK phosphorylates proteins and peptides on serine and threonine residues and FRAP/RAFT autophosphorylates on serine [26,27]. Although these proteins are more homologous to the lipid kinases than to conventional protein kinases, it is likely that all of these enzymes function in the cell as protein kinases rather than as lipid kinases.
3. PI 3-kinases and growth regulation Studies of mutants of the polyoma virus middle T antigen that were defective in transforming ability first identified PI 3-kinase as necessary for transformation by middle T [28,29]. Subsequent experiments involving mutations of v-src and the PDGF receptor correlated the stimulation of PI 3-kinase activity with transformation and mitogenesis, respectively [4]. The necessity of PI 3-kinase for PDGF-induced mitogenesis appears to be cell-type dependent, and PI 3-kinase does not seem to be required for mitogenesis by the related CSF-1 receptor. These data emphasize that in some systems, PI 3-kinase is neither necessary nor sufficient for cell growth. It is likely that the signals generated by PI 3-kinase are redundant with other signaling pathways and that PI 3-kinase becomes critical for growth only when these other pathways are missing. Rather than review these data in detail, we will focus on the mechanisms by which PI 3-kinase might regulate growth in cells where its activity appears to be necessary. A net increase in the cell population can be accomplished either by stimulating cells to enter and progress through the cell cycle or by preventing apoptosis. There is evidence for the involvement of PI 3-kinase in both mechanisms.
4. Apoptosis Yao and Cooper found that the wild-type PDGF receptor in the presence of PDGF, could rescue PC-12 cells
from apoptosis and that mutants of the PDGF receptor that failed to bind PI 3-kinase failed to rescue [30]. Scheid et al. found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked the ability of IL-4, IL-3 and steel factor to prevent apoptosis [31]. In contrast, G-M CSF and IL-5 prevented apoptosis in the presence of wortmannin. Minshall et al. found that wortmannin blocked the ability of IGF-1, but not IL-3 to prevent apoptosis in hematopoetic precursors [32]. While the latter two studies should be interpreted cautiously because they rely solely on inhibitors, it appears likely that PI 3-kinase is necessary to prevent apoptosis in some pathways but not in others.
5. Cell cycle progression Several lines of evidence suggest that PI 3-kinase is involved in G o to S progression. Roche et al. injected antibodies raised against p l l 0 into cells as PI 3-kinase inhibitors and determined the effect on growth factorstimulated BrdU incorporation [33]. They found that the antibodies to PI 3-kinase inhibited BrdU incorporation stimulated by PDGF and EGF, but not by CSF-I, LPA or bombesin. The antibodies were inhibitory, if injected up to six hours following growth factor addition. Jhun et al. found that injection of the SH2 domains of the p85 subunit of PI 3-kinase or antibodies against p85 could block insulin dependent DNA synthesis in rat fibroblasts (Jhun et al., 1994). Karnitz et al. found that wortmannin treatment of CTTL-2 cells led to an accumulation of cells at G 0 / G ~ [34]. Wortmannin suppressed thymidine incorporation in these cells, but did not completely block it. These findings all suggest a role for PI 3-kinase in progression from G Oto S, but indicate that there are pathways for entering S that do not require the activation of PI 3-kinase. The observation that injection of anti-PI 3-kinase antibodies several hours after growth factor addition (long after activation of MAP kinase and induction of fos has peaked) can still block DNA synthesis suggests that persistent activation of this enzyme is important for entry into S phase.
6. How does PI 3-kinase affect cell growth? PI 3-kinases could affect cell growth in three ways (1) through the production of phosphoinositides (2) through phosphorylation of proteins on serine via the endogenous protein kinase activity, or (3) by acting as an adaptor, not requiring enzymatic activity. The third possibility is raised because the p85/p110 type PI 3-kinase has been shown to bind to a wide variety of molecules in vivo (e.g. proteintyrosine kinases, Grb2, ras, tubulin) and it could potentially act to assemble signaling complexes that do not require the PI 3-kinase activity. However, it appears unlikely that the catalytic activity of PI 3-kinase is irrelevant, since small molecule inhibitors of the catalytic site
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(wortmannin and LY294002) have, in several cases, been shown to have similar effects on cell growth as dominant negative forms of PI 3-kinase and as mutations in receptors that eliminate PI 3-kinase binding. The question as to whether it is the ability of PI 3-kinase to phosphorylate lipids or proteins that is critical for cell growth is more difficult to answer. Wortmannin inhibits both the protein kinase activity and the lipid kinase activity of this enzyme. However, thus far, the only good protein substrate for PI 3-kinase is the p85 subunit and phosphorylation of this subunit (which results in inhibition of lipid kinase activity) is orders of magnitude slower than phosphorylation of Ptdlns [18,19]. In some cells, p85 has been shown to be pbosphorylated at the same site in vivo (serine-608) as is phosphorylated in vitro indicating that the serine kinase activity of p110 is not an in vitro artifact [18,19]. There is also evidence that p110 can phosphorylate IRS-I in vitro, however there is no evidence to date that this occurs in vivo [35]. In contrast, there is clear evidence that PI 3-kinase is responsible for growth-factor dependent increases in Ptdlns-3,4-P2 and Ptdlns-3,4,5-P3 in vivo, and it is likely that these two lipids act as second messengers.
7. Protein kinases as downstream targets of PI 3-kinase
Recent studies have identified several protein kinases as potential downstream targets of PI 3-kinase. The calciumindependent protein kinase C isoforms, PKCS, PKC~ and PKC'q (especially PKC~) were shown to be activated in vitro by Ptdlns-3,4-P 2 and to a lesser extent by Ptdlns3,4,5-P 3 [36]. The finding that Ptdlns-3,4-P 2 was much more potent than the isomer Ptdlns-4,5-P2 argues that this effect may be relevant in vivo. Since these PKC isoforms can also be activated by diacylglycerol, they could be downstream responses to multiple signaling pathways. There was also a report that brain-derived PKC~ is activated by Ptdlns-3,4,5-P 3 [37]. This activation was not observed using recombinant PKC~ [36], raising the possibility that contaminating PKCe in the preparation was responsible for the observed effects. Evidence that a PKC is activated in vivo by a PI 3-kinase product was first provided by the observation that, in thrombin-stimulated platelets, sustained phosphorylation of pleckstrin (the major PKC substrate in platelets) is blocked by PI 3-kinase inhibitors [38,39]. Addition of Ptdlns-3,4-P 2 or Ptdlns-3,4,5-P 3 to permeabilized platelets circumvented the inhibition of pleckstrin phosphorylation by wortmannin. The best evidence to date that a product of PI 3-kinase is activating PKCe in vivo is provided by Moriya et al. [40]. Wild-type PDGF receptors caused the translocation of PKCe to the membrane and mutants that failed to bind PLC-~ or PI 3-kinase did not. Adding back either the PI 3-kinase binding site or the PLC-'y binding site to the
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PDGF receptor restored the PDGF-dependent translocation of PKCe. Wortmannin blocked the PDGF-dependent translocation of PKC~ in the mutants containing the PI 3-kinase binding site, but failed to block translocation in the mutants that bind only PLC-',/ [40]. These data are in agreement with the in vitro data of Toker et al [36] and indicate that both PI 3-kinase and PLC-',/ participate in the in vivo translocation of PKCe in response to PDGF. Further evidence that a PKC is activated by products of PI 3-kinase is provided by Akimoto et al. [41]. They have shown, using both dominant active and dominant negative PI 3-kinase constructs, that the activation of a phorbol ester response element in cells overexpressing PKC k is dependent on PI 3-kinase. The combination of in vitro and in vivo data suggests that the lipid products of PI 3-kinase activate the novel and the atypical protein kinase C isoforms. How activation of these PKC isoforms might be involved in preventing apoptosis or promoting cell division is not yet known. Another protein-serine/threonine kinase that is likely to be directly regulated by the lipid products of PI 3-kinase is the proto-oncoprotein, Akt. Akt was originally isolated as a retrovirus-encoded oncogene [42]. Two groups have provided convincing evidence that growth factor-dependent activation of cellular Akt is dependent upon activation of PI 3-kinase [43,44]. Akt contains a pleckstrin homology (PH) domain at the amino-terminus whose presence is critical for activation. Since other PH domains have been shown to bind phosphoinositides, the PH domain of Akt is a likely site for regulation via the lipid products of P1 3-kinase. Evidence that Akt is directly regulated by PI 3-kinase products was provided by the ability of this enzyme to be activated in vitro by a mixture of phosphoinositides that had been incubated with PI 3-kinase and ATP [44]. Several laboratories are currently investigating the ability of phosphoinositides to bind to the Akt PH domain and to activate purified Akt. Activation of Akt may explain some of the downstream responses to PI 3-kinase. Burgering et al. found that an activated form of Akt led to stimulation of another protein kinase, p70-$6 kinase [43]. P70-$6 kinase is activated by most growth factors and cytokines and is responsible for phosphorylating the ribosomal $6 subunit. Previous studies have shown that PI 3-kinase, protein kinase C and FRAP/RAFT all have a role in the activation of $6 kinase [45-47]. The PI 3-kinase dependence could be explained either by its ability to activate a PKC family member or by its ability to activate Akt, or both. Attempts to demonstrate direct activation of p70-$6 kinase by the protein kinase endogenous to PI 3-kinase or by the lipid products of PI 3-kinase have failed. P70-$6 kinase, by phosphorylating ribosomes is thought to regulate translation of a subgroup of messages. However, the role of p70-$6 kinase in cell cycle progression is not yet clear. In some cell types and in response to some stimuli, PI 3-kinase activation is required for stimulation of mitogen
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activated protein (MAP) kinase [34,48-50]. Different resuits have been obtained from different laboratories, but the PI 3-kinase dependence is probably either at the level of raf activation or MEK activation. Isoforms of protein kinase C that are activated by PI 3-kinase are candidates for mediating activation of this pathway. Activation of MAP kinases could affect both apoptosis and promote cell division since the relative levels of MAP kinases and J N K / p 3 8 kinase activities seem to affect whether a cell undergoes apoptosis [51]. An additional mechanism by which the lipid products of PI 3-kinase may be involved in stimulating protein kinase pathways is via interaction with src-homology 2 (SH2) domains. Rameh et al. [52] showed that Ptdlns-3,4,5-P 3 binds to several SH2 domains in vitro and that this binding is in competition with phosphotyrosine-protein binding. Evidence was also provided that this occurs in vivo since the SH2-mediated interaction between the insulin receptor and PI 3-kinase could be modulated by changing the level of Ptdlns-3,4,5-P 3 in the cell. These results suggest that Ptdlns-3,4,5-P 3 may act to recruit SH2-containing proteins to the membrane independent of the need for tyrosine phosphorylation. In this way, PI 3-kinase could contribute to protein kinase signaling cascades by assembling proteins at the membrane. Wortmannin, at a concentration of 250 nM, stimulates JNK kinase activity [53]. This effect could be due to inhibition of other PI 3-kinase family members such as DNA-dependent protein kinase. If the effect is due to PI 3-kinase inhibition, this finding implies a constitutive function of PI 3-kinase to suppress JNK kinase activity. In combination with the MAP kinase results and the role of relative MAP kinase and J N K / p 3 8 activities in determining apoptosis, these data suggest PI 3-kinase could be at a central point in apoptosis. Absence of PI 3-kinase activity could simultaneously decrease MAP kinase activity and increase JNK kinase activity. Stimulation of PI 3-kinase would have the opposite effect. No data are available that suggest how this regulation might be mediated.
8. Small G proteins and PI 3-kinase PI 3-kinase binds to the GTP form of ras in vitro and this occurs via a domain of the p110 subunit of PI 3-kinase [54]. A dominant-negative form of ras decreases the level of lipid products of PI 3-kinase in NGF-stimulated PC-12 cells, suggesting that PI 3-kinase is downstream of ras [54]. Conversely, expression of a constitutively active mutant of PI 3-kinase leads to increased GTP bound ras, suggesting that PI 3-kinase is upstream of ras [55]. These data have not been reconciled. It is possible that PI 3kinase, in some cells, assists in the activation of ras (e.g. the lipid products may help to bring SOS to the membrane) and is itself further activated by GTP-ras. Whichever model proves to be correct, the presence of PI 3-kinase in
the ras pathway places it in a position to promote cell division and perhaps avert apoptosis. If PI 3-kinase is upstream of ras, this could explain the necessity of PI 3-kinase for activation of the MAP kinase pathway in some cells. However, the available data suggest that PI 3-kinase-dependent activation of MAP kinase is at a step downstream of ras (see above). The realization that ras not only activates raf but also activates PI 3-kinase further complicates what was previously thought to be a linear signaling pathway. The relative contributions of PI 3-kinase and raf in ras-dependent signaling is an area of intense research by several laboratories. For reasons discussed below, it seems likely that PI 3-kinase is playing a role in ras-dependent morphological changes and anchorage-independent cell growth. PI 3-kinase interacts with rac and Cdc42 in a complex way. In vitro, PI 3-kinase binds to the GTP-bound forms of rac and Cdc42, and this occurs via the BCR-homology region of p85 [56]. Although this domain resembles GAPs for r h o / r a c / C d c 4 2 family members, p85 fails to stimulate GTP hydrolysis on any of these proteins. PI 3-kinase has also been shown to bind rac in vivo in response to stimulation of cells with PDGF [57]. These results suggest that PI 3-kinase is downstream of rac a n d / o r Cdc42. However, there is much stronger evidence that PI 3-kinase is upstream of rac. Inhibition of PI 3-kinase blocks membrane ruffling [58] and this can be circumvented by introduction of dominant-positive rac [59]. Also, inhibition of PI 3-kinase prevented PDGF-dependent increases in GTPloading of rac [60]. Whether these interactions might be related to cell growth is not known, but the ability of rac and Cdc42 to activate the J N K / p 3 8 pathways suggests a possible link [61-63].
9. Vesicle trafficking Work from Silvia Corvera's laboratory has established the necessity of PI 3-kinase for endocytic trafficking of the PDGF receptor [64,65]. Both wortmannin-treated cells and cells expressing PDGF receptor mutants that do not activate PI 3-kinase fail to endocytose the PDGF receptor. It may be that transport to an intracellular location is important in signaling either cell division or preventing apoptosis. By preventing transport of the activated PDGF receptor further signaling would be impeded.
I0. Conclusions The role of PI 3-kinase in the control of cell growth is quite complex and, like many signal transduction pathways, the results differ depending on the cell type and stimulus. The pathways appear to be overlapping and a clearer understanding of the role of PI 3-kinase in vivo may need to await knockout studies. Even these studies are
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likely to be complicated by the existence of multiple isoforms of PI 3-kinase. Although the picture is complex, several themes emerge: (1) PI 3-kinase has a role in preventing apoptosis and in promoting cell division in some systems. (2) The lipid products of PI 3-kinase are likely to directly regulate the novel and atypical protein kinases C and Akt, leading to activation of protein kinase cascades that include p70-$6 kinase. (3) PI 3-kinase is likely to indirectly affect protein kinase cascades and actin rearrangement via regulation of small GTP-binding proteins. (4) Finally, PI 3-kinase is a downstream response to ras and may be important for many of the ras-dependent cellular responses.
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[50] Urich, M., el Shemerly, M.Y., Besser, D., Nagamine, Y. and Ballmer-Hofer, K. (1995) J. Biol. Chem. 270, 29286-29292. [51] Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. and Greenberg, M.E. (1995) Science 270, 1326-1331. [52] Rameh, L.E., Chen, C.S. and Cantley, L.C. (1995) Cell 83, 821-830. [53] Kharbanda, S., Saleem, A., Shafman, T., Emoto, Y., Taneja, N., Rubin, E., Weichselbaum, R., Woodgett, J., Avruch, J., Kyriakis, J., et al. (1995) J. Biol. Chem. 270, 18871-18874. [54] Rodriguez, V.P., Warne, P.H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M.J., Waterfield, M.D. and Downward, J. (1994) Nature 370, 527-532. [55] Hu, P., Margolis, B., Skolnik, E., Lammers, R., Ullrich, A. and Schlessinger, J. (1992) Mol. Cell. Biol. 12, 981-990. [56] Zheng, Y., Bagrodia, S. and Cerione, R.A. (1994) J. Biol. Chem. 269, 18727-18730. [57] Tolias, K.F., Cantley, L.C. and Carpenter, C.L. (1995) J. Biol. Chem. 270, 17656-17659.
[58] Wennstrom, S., Siegbahn, A., Yokote, K., Arvidsson, A.K., Heldin, C.H., Mori, S. and Claesson, W.L. (1994) Oncogene 9, 651-660. [59] Nobes, C.D., Hawkins, P., Stephens, L. and Hall, A. (1995) J. Cell. Sci. 108, 225-233. [60] Hawkins, P.T., Eguinoa, A., Qiu, R.-G., Stokoe, D., Cooke, F.T., Waiters, R., Wennstrom, S., Claesson-Welsh, L., Evans, T., Symons, M. and Stephens, L. (1995) Curr. Biol. 5, 393-403. [61] Minden, A., Lin, A., Claret, F.X., Abo, A. and Karin, M. (1995) Cell 81, 1147-1157. [62] Hill, C.S., Wynne, J. and Treisman, R. (1995) Cell 81, 1159-1170. [63] Coso, O.A., Chiariello, M., Yu, J.C., Teramoto, H., Crespo, P., Xu, N., Miki, T. and Gutkind, J.S. (1995) Cell 81, 1137-1146. [64] Joly, M., Kazlauskas, A. and Corvera, S. (1995) J Biol Chem 270, 13225-13230. [65] Joly, M., Kazlauskas, A., Fay, F.S. and Corvera, S. (1994) Science 263, 684-687.
ELSEVIER
BB Biochi~ic~a et Biophysica
A~ta
Biochimicaet BiophysicaActa 1288(1996)M1 l-M16
Mini Review
Phosphoinositide 3-kinase and the regulation of cell growth Christopher L. Carpenter a, Lewis C. Cantley b,* a Dicision of Signal Transduction, Departmentof Medicine, Beth Israel Hospital HaruardMedical School, Boston, MA 02115, USA b Departmentof Cell Biology, Harvard Medical School, Boston, MA 02115, USA
Received 15 May 1996;accepted 17 May 1996
1. Introduction More than 10 years ago, a phosphatidylinositol (Ptdlns) kinase activity was found associated with the v-src and v-ros oncoproteins [1,2]. Subsequent work revealed that this enzyme phosphorylates the D-3 position of the inositol ring, a reaction not previously known to exist [3]. This work led to the discovery of new signaling pathways involving the phosphoinositide (PI) products of this enzyme, Ptdlns-3-P, Ptdlns-3,4-P 2 and Ptdlns-3,4,5-P 3. Interest in the PI 3-kinases was stimulated by evidence that this enzyme is important for transformation by v-src, polyoma middle T antigen and v-ros and mitogenic signaling by the platelet-derived growth factor (PDGF) receptor [4]. More intense study of the role of P1 3-kinase in cell transformation and mitogenic signaling has led to conflicting data, which likely reflects the redundancy of signals for growth and utilization of different pathways in different cells. The issue of PI 3-kinase in the control of cell growth is further confounded by its role in preventing apoptosis. The recent discoveries of PI 3-kinase family members that are regulated by heterotrimeric G-proteins and by other pathways has greatly expanded the number of signaling systems in which PI 3-kinase seems to be involved. These discoveries also raise the possibility that the role of PI 3-kinase in cell growth in some systems could be quite indirect. To remain concise we have concentrated on pathways in which PI 3-kinase is thought to directly influence cell growth.
2. Background PI 3-kinase was originally purified as a heterodimer of an 85 kDa regulatory subunit and 110 kDa catalytic subunit [5,6]. eDNA cloning of the subunits has revealed five forms of the regulatory subunit [7-12] and five forms of * Corresponding author. Fax: + 1 (617) 2783033; e-mail: [email protected]
catalytic subunits [13-17] to date. The p85 subunit contains, in the amino terminal half, an SH3 domain, a region homologous to the GTPase activating protein (GAP) domain of the breakpoint cluster region (BCR) protein and two proline rich regions that can bind SH3 domains. The carboxy-terminal half has two SH2 domains and a region for binding to p 110. Three recently-described forms of the regulatory subunit have only the carboxy-terminal SH2 domains and the p110-binding domain [10-12]. The catalytic p110 subunit is homologous to protein kinases and, itself, has protein serine/threonine kinase activity, as well as phosphoinositide kinase activity [ 18,19]. The cDNA cloning of p110 a lead to the identification of the yeast gene product, vps34p, as a PtdIns 3-kinase [20]. This gene had been shown to be essential for vesicle trafficking from the golgi to the vacuole. This discovery lead to experiments demonstrating a role for PI 3-kinases in vesicle trafficking in higher organisms [21,22]. The p110 subunits differ in their substrate specificity and regulation. The p85/p110 type PI 3-kinases are regulated by protein-tyrosine kinases and phosphorylate PtdIns, Ptdlns-4-P or PtdIns-4,5-P 2 at the D-3 position (they are named phosphoinositide 3-kinases to indicate their broad in vitro substrate specificity). These enzymes mediate acute increases in PtdIns-3,4,5-P 3 in response to stimulation of receptor-type protein-tyrosine kinases. The p110 3' type enzyme is activated by both the c~ and 13/3' subunits of heterotrimeric G proteins, and this enzyme has similar substrate specificity to the p85/p110 type enzymes [ 16]. In contrast, the yeast vps34p and a mammalian form of vps34p are distinct from the p85/p110-type PI 3-kinase in that they phosphorylate only PtdIns. These enzymes appear to be regulated by an associated protein-serine kinase, vpsl5p. Finally, a recently-described C2 domain-containing catalytic subunit phosphorylates primarily Ptdlns and PtdIns-4-P [17]. This enzyme probably provides an independent pathway for regulated production of PtdIns-3,4-P2. Thus, in vivo levels of PtdIns-3-P, Ptdlns-3,4-P 2 and PtdIns-3,4,5-P 3 can be independently controlled by distinct
0304-419X/96/$15.00 Copyright © 1996 ElsevierScienceB.V. All rights reserved. PII S0304-419X(96)00018-2
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enzymes, suggesting that these lipids each have unique functions in the cell (see below). cDNA cloning has revealed two additional subgroups of proteins with homology to the catalytic domain of PI 3-kinases. The enzymes in the first group are quite homologous to the PI 3-kinases and include human, yeast, and dictyostelium discoideum Ptdlns 4-kinases. The second group is more distant from the PI 3-kinases and Ptdlns 4-kinases and includes the ataxia-telangectasia gene product ( A T M ) , DNA-PK, RAFT/FRAP/TOR, MEC1/RAD3, TEL1, and Mei-41 [23]. Interestingly, all of the proteins in this group, except the R A F T / F R A P / TOR proteins, are implicated in sensing DNA damage. None of them has been shown to have endogenous phosphoinositide kinase activity (although FRAP/RAFT and TOR have been shown to have an associated Ptdlns 4kinase activity [24,25]. DNA-PK phosphorylates proteins and peptides on serine and threonine residues and FRAP/RAFT autophosphorylates on serine [26,27]. Although these proteins are more homologous to the lipid kinases than to conventional protein kinases, it is likely that all of these enzymes function in the cell as protein kinases rather than as lipid kinases.
3. PI 3-kinases and growth regulation Studies of mutants of the polyoma virus middle T antigen that were defective in transforming ability first identified PI 3-kinase as necessary for transformation by middle T [28,29]. Subsequent experiments involving mutations of v-src and the PDGF receptor correlated the stimulation of PI 3-kinase activity with transformation and mitogenesis, respectively [4]. The necessity of PI 3-kinase for PDGF-induced mitogenesis appears to be cell-type dependent, and PI 3-kinase does not seem to be required for mitogenesis by the related CSF-1 receptor. These data emphasize that in some systems, PI 3-kinase is neither necessary nor sufficient for cell growth. It is likely that the signals generated by PI 3-kinase are redundant with other signaling pathways and that PI 3-kinase becomes critical for growth only when these other pathways are missing. Rather than review these data in detail, we will focus on the mechanisms by which PI 3-kinase might regulate growth in cells where its activity appears to be necessary. A net increase in the cell population can be accomplished either by stimulating cells to enter and progress through the cell cycle or by preventing apoptosis. There is evidence for the involvement of PI 3-kinase in both mechanisms.
4. Apoptosis Yao and Cooper found that the wild-type PDGF receptor in the presence of PDGF, could rescue PC-12 cells
from apoptosis and that mutants of the PDGF receptor that failed to bind PI 3-kinase failed to rescue [30]. Scheid et al. found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked the ability of IL-4, IL-3 and steel factor to prevent apoptosis [31]. In contrast, G-M CSF and IL-5 prevented apoptosis in the presence of wortmannin. Minshall et al. found that wortmannin blocked the ability of IGF-1, but not IL-3 to prevent apoptosis in hematopoetic precursors [32]. While the latter two studies should be interpreted cautiously because they rely solely on inhibitors, it appears likely that PI 3-kinase is necessary to prevent apoptosis in some pathways but not in others.
5. Cell cycle progression Several lines of evidence suggest that PI 3-kinase is involved in G o to S progression. Roche et al. injected antibodies raised against p l l 0 into cells as PI 3-kinase inhibitors and determined the effect on growth factorstimulated BrdU incorporation [33]. They found that the antibodies to PI 3-kinase inhibited BrdU incorporation stimulated by PDGF and EGF, but not by CSF-I, LPA or bombesin. The antibodies were inhibitory, if injected up to six hours following growth factor addition. Jhun et al. found that injection of the SH2 domains of the p85 subunit of PI 3-kinase or antibodies against p85 could block insulin dependent DNA synthesis in rat fibroblasts (Jhun et al., 1994). Karnitz et al. found that wortmannin treatment of CTTL-2 cells led to an accumulation of cells at G 0 / G ~ [34]. Wortmannin suppressed thymidine incorporation in these cells, but did not completely block it. These findings all suggest a role for PI 3-kinase in progression from G Oto S, but indicate that there are pathways for entering S that do not require the activation of PI 3-kinase. The observation that injection of anti-PI 3-kinase antibodies several hours after growth factor addition (long after activation of MAP kinase and induction of fos has peaked) can still block DNA synthesis suggests that persistent activation of this enzyme is important for entry into S phase.
6. How does PI 3-kinase affect cell growth? PI 3-kinases could affect cell growth in three ways (1) through the production of phosphoinositides (2) through phosphorylation of proteins on serine via the endogenous protein kinase activity, or (3) by acting as an adaptor, not requiring enzymatic activity. The third possibility is raised because the p85/p110 type PI 3-kinase has been shown to bind to a wide variety of molecules in vivo (e.g. proteintyrosine kinases, Grb2, ras, tubulin) and it could potentially act to assemble signaling complexes that do not require the PI 3-kinase activity. However, it appears unlikely that the catalytic activity of PI 3-kinase is irrelevant, since small molecule inhibitors of the catalytic site
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(wortmannin and LY294002) have, in several cases, been shown to have similar effects on cell growth as dominant negative forms of PI 3-kinase and as mutations in receptors that eliminate PI 3-kinase binding. The question as to whether it is the ability of PI 3-kinase to phosphorylate lipids or proteins that is critical for cell growth is more difficult to answer. Wortmannin inhibits both the protein kinase activity and the lipid kinase activity of this enzyme. However, thus far, the only good protein substrate for PI 3-kinase is the p85 subunit and phosphorylation of this subunit (which results in inhibition of lipid kinase activity) is orders of magnitude slower than phosphorylation of Ptdlns [18,19]. In some cells, p85 has been shown to be pbosphorylated at the same site in vivo (serine-608) as is phosphorylated in vitro indicating that the serine kinase activity of p110 is not an in vitro artifact [18,19]. There is also evidence that p110 can phosphorylate IRS-I in vitro, however there is no evidence to date that this occurs in vivo [35]. In contrast, there is clear evidence that PI 3-kinase is responsible for growth-factor dependent increases in Ptdlns-3,4-P2 and Ptdlns-3,4,5-P3 in vivo, and it is likely that these two lipids act as second messengers.
7. Protein kinases as downstream targets of PI 3-kinase
Recent studies have identified several protein kinases as potential downstream targets of PI 3-kinase. The calciumindependent protein kinase C isoforms, PKCS, PKC~ and PKC'q (especially PKC~) were shown to be activated in vitro by Ptdlns-3,4-P 2 and to a lesser extent by Ptdlns3,4,5-P 3 [36]. The finding that Ptdlns-3,4-P 2 was much more potent than the isomer Ptdlns-4,5-P2 argues that this effect may be relevant in vivo. Since these PKC isoforms can also be activated by diacylglycerol, they could be downstream responses to multiple signaling pathways. There was also a report that brain-derived PKC~ is activated by Ptdlns-3,4,5-P 3 [37]. This activation was not observed using recombinant PKC~ [36], raising the possibility that contaminating PKCe in the preparation was responsible for the observed effects. Evidence that a PKC is activated in vivo by a PI 3-kinase product was first provided by the observation that, in thrombin-stimulated platelets, sustained phosphorylation of pleckstrin (the major PKC substrate in platelets) is blocked by PI 3-kinase inhibitors [38,39]. Addition of Ptdlns-3,4-P 2 or Ptdlns-3,4,5-P 3 to permeabilized platelets circumvented the inhibition of pleckstrin phosphorylation by wortmannin. The best evidence to date that a product of PI 3-kinase is activating PKCe in vivo is provided by Moriya et al. [40]. Wild-type PDGF receptors caused the translocation of PKCe to the membrane and mutants that failed to bind PLC-~ or PI 3-kinase did not. Adding back either the PI 3-kinase binding site or the PLC-'y binding site to the
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PDGF receptor restored the PDGF-dependent translocation of PKCe. Wortmannin blocked the PDGF-dependent translocation of PKC~ in the mutants containing the PI 3-kinase binding site, but failed to block translocation in the mutants that bind only PLC-',/ [40]. These data are in agreement with the in vitro data of Toker et al [36] and indicate that both PI 3-kinase and PLC-',/ participate in the in vivo translocation of PKCe in response to PDGF. Further evidence that a PKC is activated by products of PI 3-kinase is provided by Akimoto et al. [41]. They have shown, using both dominant active and dominant negative PI 3-kinase constructs, that the activation of a phorbol ester response element in cells overexpressing PKC k is dependent on PI 3-kinase. The combination of in vitro and in vivo data suggests that the lipid products of PI 3-kinase activate the novel and the atypical protein kinase C isoforms. How activation of these PKC isoforms might be involved in preventing apoptosis or promoting cell division is not yet known. Another protein-serine/threonine kinase that is likely to be directly regulated by the lipid products of PI 3-kinase is the proto-oncoprotein, Akt. Akt was originally isolated as a retrovirus-encoded oncogene [42]. Two groups have provided convincing evidence that growth factor-dependent activation of cellular Akt is dependent upon activation of PI 3-kinase [43,44]. Akt contains a pleckstrin homology (PH) domain at the amino-terminus whose presence is critical for activation. Since other PH domains have been shown to bind phosphoinositides, the PH domain of Akt is a likely site for regulation via the lipid products of P1 3-kinase. Evidence that Akt is directly regulated by PI 3-kinase products was provided by the ability of this enzyme to be activated in vitro by a mixture of phosphoinositides that had been incubated with PI 3-kinase and ATP [44]. Several laboratories are currently investigating the ability of phosphoinositides to bind to the Akt PH domain and to activate purified Akt. Activation of Akt may explain some of the downstream responses to PI 3-kinase. Burgering et al. found that an activated form of Akt led to stimulation of another protein kinase, p70-$6 kinase [43]. P70-$6 kinase is activated by most growth factors and cytokines and is responsible for phosphorylating the ribosomal $6 subunit. Previous studies have shown that PI 3-kinase, protein kinase C and FRAP/RAFT all have a role in the activation of $6 kinase [45-47]. The PI 3-kinase dependence could be explained either by its ability to activate a PKC family member or by its ability to activate Akt, or both. Attempts to demonstrate direct activation of p70-$6 kinase by the protein kinase endogenous to PI 3-kinase or by the lipid products of PI 3-kinase have failed. P70-$6 kinase, by phosphorylating ribosomes is thought to regulate translation of a subgroup of messages. However, the role of p70-$6 kinase in cell cycle progression is not yet clear. In some cell types and in response to some stimuli, PI 3-kinase activation is required for stimulation of mitogen
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activated protein (MAP) kinase [34,48-50]. Different resuits have been obtained from different laboratories, but the PI 3-kinase dependence is probably either at the level of raf activation or MEK activation. Isoforms of protein kinase C that are activated by PI 3-kinase are candidates for mediating activation of this pathway. Activation of MAP kinases could affect both apoptosis and promote cell division since the relative levels of MAP kinases and J N K / p 3 8 kinase activities seem to affect whether a cell undergoes apoptosis [51]. An additional mechanism by which the lipid products of PI 3-kinase may be involved in stimulating protein kinase pathways is via interaction with src-homology 2 (SH2) domains. Rameh et al. [52] showed that Ptdlns-3,4,5-P 3 binds to several SH2 domains in vitro and that this binding is in competition with phosphotyrosine-protein binding. Evidence was also provided that this occurs in vivo since the SH2-mediated interaction between the insulin receptor and PI 3-kinase could be modulated by changing the level of Ptdlns-3,4,5-P 3 in the cell. These results suggest that Ptdlns-3,4,5-P 3 may act to recruit SH2-containing proteins to the membrane independent of the need for tyrosine phosphorylation. In this way, PI 3-kinase could contribute to protein kinase signaling cascades by assembling proteins at the membrane. Wortmannin, at a concentration of 250 nM, stimulates JNK kinase activity [53]. This effect could be due to inhibition of other PI 3-kinase family members such as DNA-dependent protein kinase. If the effect is due to PI 3-kinase inhibition, this finding implies a constitutive function of PI 3-kinase to suppress JNK kinase activity. In combination with the MAP kinase results and the role of relative MAP kinase and J N K / p 3 8 activities in determining apoptosis, these data suggest PI 3-kinase could be at a central point in apoptosis. Absence of PI 3-kinase activity could simultaneously decrease MAP kinase activity and increase JNK kinase activity. Stimulation of PI 3-kinase would have the opposite effect. No data are available that suggest how this regulation might be mediated.
8. Small G proteins and PI 3-kinase PI 3-kinase binds to the GTP form of ras in vitro and this occurs via a domain of the p110 subunit of PI 3-kinase [54]. A dominant-negative form of ras decreases the level of lipid products of PI 3-kinase in NGF-stimulated PC-12 cells, suggesting that PI 3-kinase is downstream of ras [54]. Conversely, expression of a constitutively active mutant of PI 3-kinase leads to increased GTP bound ras, suggesting that PI 3-kinase is upstream of ras [55]. These data have not been reconciled. It is possible that PI 3kinase, in some cells, assists in the activation of ras (e.g. the lipid products may help to bring SOS to the membrane) and is itself further activated by GTP-ras. Whichever model proves to be correct, the presence of PI 3-kinase in
the ras pathway places it in a position to promote cell division and perhaps avert apoptosis. If PI 3-kinase is upstream of ras, this could explain the necessity of PI 3-kinase for activation of the MAP kinase pathway in some cells. However, the available data suggest that PI 3-kinase-dependent activation of MAP kinase is at a step downstream of ras (see above). The realization that ras not only activates raf but also activates PI 3-kinase further complicates what was previously thought to be a linear signaling pathway. The relative contributions of PI 3-kinase and raf in ras-dependent signaling is an area of intense research by several laboratories. For reasons discussed below, it seems likely that PI 3-kinase is playing a role in ras-dependent morphological changes and anchorage-independent cell growth. PI 3-kinase interacts with rac and Cdc42 in a complex way. In vitro, PI 3-kinase binds to the GTP-bound forms of rac and Cdc42, and this occurs via the BCR-homology region of p85 [56]. Although this domain resembles GAPs for r h o / r a c / C d c 4 2 family members, p85 fails to stimulate GTP hydrolysis on any of these proteins. PI 3-kinase has also been shown to bind rac in vivo in response to stimulation of cells with PDGF [57]. These results suggest that PI 3-kinase is downstream of rac a n d / o r Cdc42. However, there is much stronger evidence that PI 3-kinase is upstream of rac. Inhibition of PI 3-kinase blocks membrane ruffling [58] and this can be circumvented by introduction of dominant-positive rac [59]. Also, inhibition of PI 3-kinase prevented PDGF-dependent increases in GTPloading of rac [60]. Whether these interactions might be related to cell growth is not known, but the ability of rac and Cdc42 to activate the J N K / p 3 8 pathways suggests a possible link [61-63].
9. Vesicle trafficking Work from Silvia Corvera's laboratory has established the necessity of PI 3-kinase for endocytic trafficking of the PDGF receptor [64,65]. Both wortmannin-treated cells and cells expressing PDGF receptor mutants that do not activate PI 3-kinase fail to endocytose the PDGF receptor. It may be that transport to an intracellular location is important in signaling either cell division or preventing apoptosis. By preventing transport of the activated PDGF receptor further signaling would be impeded.
I0. Conclusions The role of PI 3-kinase in the control of cell growth is quite complex and, like many signal transduction pathways, the results differ depending on the cell type and stimulus. The pathways appear to be overlapping and a clearer understanding of the role of PI 3-kinase in vivo may need to await knockout studies. Even these studies are
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likely to be complicated by the existence of multiple isoforms of PI 3-kinase. Although the picture is complex, several themes emerge: (1) PI 3-kinase has a role in preventing apoptosis and in promoting cell division in some systems. (2) The lipid products of PI 3-kinase are likely to directly regulate the novel and atypical protein kinases C and Akt, leading to activation of protein kinase cascades that include p70-$6 kinase. (3) PI 3-kinase is likely to indirectly affect protein kinase cascades and actin rearrangement via regulation of small GTP-binding proteins. (4) Finally, PI 3-kinase is a downstream response to ras and may be important for many of the ras-dependent cellular responses.
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