- Email: [email protected]
p70 S6 kinase: an enigma with variations
REVIEWS
TIBS 21 - MAY 1996 1314-1321 24 Reinhard, M. et al. (1995) EMBO J. 14, 25 26 27 28 29 30
1583-1589 Matuoka, K., Shibasaki, F., Shibata, M. and Takenawa, T. (1993) EMBO J. 12, 3467-3473 Symons, M. (1995) Curr. Opin. Biotech. 6, 668-674 Qiu, R-G. et al. (1995) Nature 374, 457-459 Khosravi-Far, R. et al. (1995) Mol. Cell. Biol. 15, 6443-6453 Prendergast, G. C. et al. (1995) Oncogene 10, 2289-2296 Qiu, R-G. et al. (1996) Proc. Natl Acad. Sci. USA 92, 11781-11785
31 Joneson, T., White, M. A., Wigler, M. H. and Bar-Sagi, D. (1996) Science 271, 810-812 32 Simon, M. N. et al. (1995) Nature 376,
25951-25954
Christopher G. Proud The $6 ribosomal protein is phosphorylated by p70 $6 kinase (p7OS6k). Although the cellular role of $6 phosphorylation is still not fully clear, studies on p70 s6k and its activation have revealed the existence of a novel signalling pathway, clues to the mechanism of action of certain immunosuppressants and insights into the control of gene expression at the levels of transcription and translation.
C. G. Proud is at the Department of Biosciences, University of Kent at Canterbury, Canterbury, UK CT2 7NJ, 9 1996,ElsevierScienceLtd
5931-5938 40 Watanabe, G. et al. (1996) Science 271,
645-648
702-705 33 Zhao, Z. S., Leung, T., Manser, E. and Lim, L. (1995) Mol. Cell. Biol. 15, 5246-5257 34 Coso, O. A. et al. (1995) Cell 81, 1137-1146 35 Minden, A. et al. (1995) Cell 81, 1147-1157 36 Olson, M. F., Ashworth, A. and Hall, A. (1995) Science 269, 1270-1272 37 Hill, C. S., Wynne, J. and Treisman, R. (1995) Cell 81, 1159-1170 38 Malcolm, K. C. et al. (1994) J. Biol. Chem. 269,
p70 $6 kinase: an enigma with variations
THE $6 PROTEIN is a component of the small (40S) subunit of eukaryotic ribosomes. It has been known since the 1970s that $6 undergoes phosphorylation and that this reaction is enhanced by agents that stimulate protein synthesis, such as hormones (e.g. insulin) and growth factors. Up to five Ser residues in the carboxyl terminus of $6 become phosphorylated ~ (Ser235, 236, 240, 244 and 247). Given that $6 phosphorylation occurs in a wide range of cell types in response to a variety of stimuli, a burning question, which was tackled with vigour in the 1980s, was what is the identity of the kinase(s) responsible for the phosphorylation of $6 in vivo? Two candidates eventually emerged. They could be distinguished by various criteria including their apparent molecular masses, which gave rise to the current nomenclature: p70 s6k (apparent M = 70 kDa) and p90 "k. It now appears that p70 s6k is
39 Nonaka, H. et al. (1995) EMBO J. 14,
the major physiological $6 kinase in mammalian cellsL Evidence in favour of this includes the observations that p70 sBk phosphorylates 40S subunits more efficiently than p90 rsk (Refs 3, 4) and, most convincingly, that rapamycin (which, as described in detail below, blocks activation of p70 sBk, but not that of p90 rsk) prevents phosphorylation of $6 in response to various stimuli in intact cells 5,6. Having found the probable kinase, the focus of attention has shifted to the key question of how this enzyme is regulated physiologically. Structure of S6 kinase Three research groups almost simultaneously reported the initial isolation of cDNA clones for p70 s6k from rat 7.s and rabbit 9. The enzyme has a single catalytic domain related most closely to the more amino terminal of the two catalytic domains found in p90 ~sk and to second-messenger-regulated kinases. It is encoded by at least two distinct transcripts differing at their 5' ends 1~ and encoding polypeptides with different amino termini (Fig. 1; predicted masses
41 Amano, M. et al. (1996) Science 271,
648-650 42 Leung, T., Manser, E., Tan, L. and Lim, L. (1995) J. Biol. Chem. 270, 29051-29054 43 Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830 44 Burbelo, P. D., Drechsel, D. and Hall, A. (1995) J. Biol. Chem. 270, 29071-29074 45 Peppelenbosch, M. P. et al. (1995) Cell 81, 849-856
56.2 and 59.2kDa). The longer protein contains an extension of 23 amino acids including a hexa-Arg sequence typical of nuclear localization signals: both forms are synthesized in vivo. The larger species has an apparent molecular mass of 85kDa and is hence called p85sGk.From here on, unless otherwise specified, the term p70 s6k will be used to include the larger form too, and the numbering of residues will be based on p70 s6k. (To convert to p85 s6k numbering, add 23.) Data from Thomas' group show that the p85 isoform is targeted to the nucleus by its amino-terminal sequence 12 (although a modest amount of the shorter form might also be present in the nucleus13). This nuclear form might play a key role in the regulation of cell growth as indicated by the observation that antibodies specific for this species block cell growth when injected into the nucleus (but not when introduced into the cytoplasm) and that this block is overcome by co-injection of the shorter form into the nucleus~L p70 s6k contains a sequence that resembles the region of $6 containing the phosphorylation sites. This sequence lies immediately carboxy-terminal to the catalytic domain of the kinase (Fig. 1) and it was initially suggested that it might act as an autoregulatory domain by occluding the active site of the kinaseS.~4: it contains four Ser residues that are phosphorylated as the kinase is activated. However, the situation is more complex than this, as deletion of this sequence does not generate an active enzyme and phosphorylation of sites elsewhere in the enzyme is also associated with activation (see below). How is p70 s6kactivated? A key observation made during the isolation of p70 s6k was that it rapidly lost activity unless I~-glycerophosphate or other phosphatase inhibitors were
PII: S0968-0004(96) 10016-5
181
REVIEWS pathway activating p70 s6~ was distinct from that leadI I p70s6k II ing to activation of MAP kinase (and thus p90rsk)19. .~. 9 I I p85 s6k Avruch's group 2~used a peptide based on the autoreguMRRRRRRD ~'N',. Potential autoregulatory latory region of p70s6k to Acidic region sequence identify possible upstream kinases. They found several 'set 1' (b) kinases that could phosrThr229 Thr389 Ser404~ phorylate the peptide, including members of the MAP I I P70s6k kinase family and a form of II cdc2. However, these kinases s e r 4 1 1 J / ~~ - Ser424 also phosphorylated recomSer418 Thr421 J L binant p70s6k in vitro at sites 'set 2' that correspond to some of those labelled in vivo, but Figure 1 they were unable to activate Structure of members of the p70 s6k family and model p70 s6k, indicating a requirefor their activation. (a) The structures of p70 s6k and ment for additional kinases. p85 s6k are shown schematically. The magenta bar shows the amino-terminal extension present in the When the four residues at longer form, which contains the Arg-rich, potential the carboxyl terminus are nuclear-targeting signal. Also shown are the catalytic mutated to Asp or Glu (to domain (green), the proposed autoregulatory region mimic phosphoserine or (blue) and an acidic region (red) near the amino terphosphothreonine, respecminus with which it has been proposed to interact. tively), the kinase shows This interaction is suggested to close off the active higher basal activity than site, rendering the kinase inactive8. (b) The positions of the phosphorylation sites identified in p70 ssk and the wild-type enzyme 21. Howp85 s6k are indicated. Thr229, Thr389 and Ser404 beever, it can still be activated long to the group referred to in this article as further by serum, again show'set 1': phosphorylation of these sites accompanies ing that additional mechaactivation of the kinase and is blocked by rapamycin. nisms, involving the phosThe four sites indicated towards the carboxyl termiphorylation of other sites, nus of p70 s6k and p85 s6k are also phosphorylated are involved in switching on when the kinase is activated, but not all are sensitive to rapamycin ('set 2'). p70s6k (Ref. 22). However, as the activity of the mutant, with all four Ser residues conincluded in the buffers. This suggested verted to Ala, is greatly reduced, these that p70s6k was itself activated by residues are important for the activation phosphorylation. In addition, treatment or activity of p70s6k~ef. 21; see below). with protein phosphatase-2A ~P-2A) led to inactivation, suggesting that Ser/ Rapamycin Thr phosphorylation was importanfl 5,~6. Rapamycin inhibits T-cell activation It has since been shown that acti- by blocking progression through the GI vation of p70s6k is accompanied by phase of the cell cycle (for reviews, see phosphorylation of seven Ser/Thr resi- Refs 23, 24), and selectively prevents dues including the four referred to activation of p70 sGk (Refs 5, 6, 24-26), above n,~s Gig. 1). Thus, the identifi- i.e. it does not block the activation of cation of kinases that activate p70s6k kinases in the MAP kinase cascade. seems a crucial step in elucidating the Neutralizing antibodies against p70s6k signalling cascade leading to the stimu- also block the ability of serum to induce lation of p70s6k. It is interesting to note the shift of quiescent cells through G1 that each of the four carboxy-terminal into S phase [2,27,providing evidence that phosphorylation sites (Ser411, Ser418, pT0s6kis indeed a key component underThr421 and Ser424) is followed by a Pro lying the ability of rapamycin to block residue. Ser/Thr-Pro motifs are tar- G1 progression. However, it should be geted by a range of kinases including noted that: (1) for reasons that are not mitogen-activated protein (MAP) kinase yet clear, rapamycin has less profound and cyclin-dependent kinases (CDKs) effects on growth and on protein synthesis than does microinjection of antisuch as cdc2. A further important observation made p70s6k antibodies; and (2) rapamycin by Thomas' group came in 1991 when also blocks activation of CDKs (see Ref. they provided data indicating that the 28 and lit. cit. therein).
(a)
182
Catalytic domain
TIBS 2 1 -
MAY 1996
Rapamycin causes dephospho~lation of p70s6kat sites other than those in the carboxy-terminaJ domain 22. This fits with the data above, which indicate that other phosphorylation events were required for pT0s6kactivation 22. Additional phosphorylation sites have now been identified as Thr229, Thr389 and Ser404 [here termed 'set 1' ~efs 21, 29; Fig. 1)]. Detailed analysis revealed that the four most carboxy-terminal sites ('set 2') are partially phosphorylated in quiescent cells and that they and set 1 (which are not basally phosphorylated) undergo rapid phosphorylation in response to stimuli that activate p70 s6k (Ref. 21). Rapamycin blocks phosphorylation of set 1 and also Ser411 of set 2. These sites also undergo dephosphorylation in response to wortmannin or the methylxanthine SQ20006. Wortmannin is reported to be a selective inhibitor of phosphoinositide 3-kinases ~I 3-kinases), while methylxanthines are well-known inhibitors of cyclic AMP-phosphodiesterase, but the action of SQ20006 seems not to be related to elevated levels of cAMP. Instead, it might block a protein kinase upstream of p70s6kand act in a manner similar to that of rapamycin rather than that of wortmannin (based on the fact that SQ20006 and rapamycin both block the activation of p70s6k by phorbol esters whereas wortmannin does not2~). The kinases that phosphorylate any of the sites in p70 s6khave yet to be identified. Thr229, Thr389 and Ser404 all lie in similar sequence contexts, suggesting that a common kinase could be responsible for phosphorylating each of them. This context is rich in aromatic residues and, thus, differs markedly from that around the set 2 sites. Detailed analysis of the correlation between changes in the phosphorylation of individual sites in p70s6~and its activity suggests that Thr389 and Ser404 are particularly important (see below). Each of these residues has now been mutated to either Ala or Asp/Glu. The Thr389~a mutant is catalytically inactive, suggesting a key role for this residue, a notion that is strongly supported by the observation that the Thr389Glu mutant is largely insensitive to rapamycin 29. This evidence has been interpreted as indicating that Thr389 is a key target residue for the rapamycin-sensitive input, which leads to kinase activation.
Truncation mutants An alternative approach to investigating the regulation of p70 s6kis the use
TIBS 21 - MAY 1996
of truncation mutants of the enzyme. (These studies actually used the longer form, but to save further confusion, here I will use the numbering of the 70 kDa form). Removal of the carboxyl terminus (amino acids 399-502, containing the autoregulatory domain and the set 2 sites) generates a mutant (ACT104), which is still strongly activated by serum 3~ This activation is sensitive to wortmannin, but only partially blocked by rapamycin, indicating that these inhibitors block activation in different ways. Consistent with the data discussed above, activation of ACT104 is accompanied by phosphorylation and both rapamycin and wortmannin cause dephosphorylation of the same set of sites in this mutant, again showing that phosphorylation events outside the carboxyl terminus are important for activation. However, as this mutant does not contain Ser404, this residue is clearly not essential for activation by serum. Additional insights were obtained using a mutant in which the acidic region adjacent to the amino terminus was deleted 3~ This mutant exhibited sharply reduced activity, but was still activated by serum and sensitive to wortmannin. However, it was entirely unaffected by rapamycin, again showing that two separate inputs are involved. The effects of rapamycin require amino acids 6-23, but this does not depend on phosphorylation within this region. Instead, phosphorylation at site(s) between residues 23 and 398, which includes Thr229 (see below) and Thr389, is required for activation. Consistent with this, the double truncation mutant, lacking both termini, can be activated by serum, is resistant to rapamycin, but is inhibited by wortmannin 3~ While data obtained from the relatively drastic truncation mutants must be treated with caution, as they differ substantially from the intact protein, it must be pointed out that these data do not agree with the idea that Thr389 plays the key role in the rapamycinsensitive activation of p70 s6k. For example, the amino-terminal truncation and the double truncation both contain this site, but are not sensitive to rapamycin, while the ACT104 mutant (which also contains Thr389) is only partially sensitive to this drug 3~
The target of rapamycin is homologousto PI 3-kinase Despite the identification of the target of rapamycin in yeast (target of rapamycin, TOR31) and in mammalian cells
REVIEWS (mTOR, also called RAFT or FRAp32), there is little information on the mechanism of action of this drug. It should be noted that rapamycin does not itself interact with mTOR; rather, rapamycin binds to a protein termed FKBP12 Ras PI 3-kinase Others? (FK506-binding protein 12, where FK506 is another immunosuppressant) and the rapamycin-FKBP12 complex PKB mTOR actually interacts with mTOR Rapamycin (Fig. 2). It is interesting to Unknown kinases FKBP12 note that TOR shows homology with PI 3-kinases, whereas the mammalian protein has Activation of p70 s6k been reported to possess phosphoinositide 4-kinase acFigure 2 tivity33, although this remains Mechanism of activation of p70 s6k. Ras does not play controversial. a role in p70 s6k activation. However, most (but not Recent data demonstrate all) of the available evidence suggests that phosthat mTOR is indeed a rapaphoinositide 3-kinase (PI 3-kinase) is important, and mycin-sensitive regulator of that it might act through protein kinase B (PKB). Wortmannin blocks activation of p70 s6k presumably p70 s6k in vivo 28 and that by inhibiting PI 3-kinase. The target of rapamycin in mTOR undergoes autophosmammalian cells (mTOR) confers rapamycin sensitivphorylation, which is blocked ity to the activation of p70 s6k, but lies on a pathway by rapamycin. This suggests that provides an input into p70 s6k activation that is that the kinase activity of separate from that provided by the PI 3-kinase. mTOR is essential for its Rapamycin does not bind directly to mTOR, but instead forms a complex with FKBP12, which in turn binds to function in the activation of and blocks mTOR. Activation of p70 s6k is associated p70 sBk, an idea that is conwith phosphorylation at multiple sites, probably catafirmed by 'kinase-dead' mulysed by several distinct protein kinases. It is not entants of TOR that cannot tirely clear whether PKB acts upstream of roTOR or function to activate p70 s6k parallel to it (the variant shown here). The obser(Ref. 28). By contrast, autovation that rapamycin and wortmannin affect differphosphorylation of mTOR is ent inputs into p70 s6k is more consistent with this situation, assuming that wortmannin is inhibiting the not blocked by wortmannin, PI 3-kinase upstream of PKB. Not shown is the actiagain showing that this comvation of p70 s6k by phorbol esters, which is insensipound acts differently from tive to wortmannin and could bypass PKB (see text). rapamycin and might not be merely acting to inhibit the links and suggested possible roles for putative kinase activity of mTOR. Deletion mutations of mTOR reveal others, while also providing some apthat the kinase domain alone is not parently conflicting data. Also, it is not enough for activation of p70 s6k and that clear which steps lie above, and which amino-terminal sequences are also re- act in parallel to, the step blocked by quired 28. Perhaps this region interacts rapamycin. with p70 s6k, although there is no direct Neither Ras nor Raf plays a role in the evidence for this. So far, mTOR has not activation of p70 s6k.The platelet-derived been shown to phosphorylate p70 s6k.As growth factor (PDGF) receptor contains rapamycin decreases the phosphoryl- a so-called 'kinase insert', which contains ation of p70 s6k, an additional com- Tyr residues that might act as docking ponent, most likely a protein kinase sites for SH2-containing proteins such or phosphatase, must lie in between as P1 3-kinase when phosphorylated 34. mTOR and p70 s6k(Fig. 2). In cells synthesizing PDGF receptors from which the kinase insert domain Upstream signallingevents has been deleted, Ras is still activated What links the rapamycin-sensitive normally, but p70 s6k is not 35. These and step to cell surface receptors such as other data (e.g. use of dominant-negathose with intrinsic tyrosine kinase ac- tive mutants of Ras) indicate that Ras is tivity? The short answer is that we do not involved in the activation of p70 s6k. not know, although recent work has Similarly, dominant-negative mutants of apparently rule(] out some potential Raf (the kinase immediately downstream
183
REVIEWS
TIBS 2 1 -
MAY 1996
by Chung e t al. 36 had provided several pieces of evidence supporting a role for PI 3-kinase in pT0s6k actiR a p a m y c i n - - t roTOR vation. In addition to finding that the Tyr740/751 mutant could not activate p70ssk,they Other also showed that two differp70 s6k . ~ kinases ent inhibitors of PI 3-kinase ' (wortmannin and LY294002) blocked activation of p70s6k p27Kip1 .,~,.~ by insulin as well as PDGF l CREM $6 4E-BP1 ~ef. 37). The explanation CDKs 1 for these discrepancies remains unclear. A more informative way of employing mutants of the PDGF receptor might be to carry out 'add-back' experiments: in this strategy, all Figure 3 five T~ phosphorylation The role of p70 s6k in cellular regulation, where it directly phosphorylates both the cyclic-AMP-responsivesites in the PDGF receptor element modulator (CREM), a transcription factor inare mutated to Phe, and one volved in the control of expression of specific genes, or more of the Tyrs are then and ribosomal protein $6, whose role in controlling 'added back' so that one can translation remains unclear. Rapamycin interferes with examine the roles of individthe control of the cyclin-dependent kinase (CDK) p27 Kip1 ual residues against a backand the elF-4E-binding protein (4E-BP1), implying links to p70 s6k, but it is not known if p70 s6k is itself inground in which the other volved in their regulation, or whether the relevant sigphosphotyrosines have been nalling pathways merely share common features with eliminated. those controlling p70 s6k (indicated by broken lines). Chung et al. 36 provided the Abbreviation: rnTOR, mammalian target of rapamycin. first evidence that wortmannin and rapamycin inhibit of Ras in the MAP kinase cascade) p70s6k activation in different ways, as also fail to block activation of p70s6k activation by phorbol esters is sensitive (Ref. 35). to rapamycin, but not to wortmannin, suggesting that protein kinase C ~KC)Role of PI 3-kinase in the activation of p70m mediated activation bypasses PI 3-kiMing e t al. 35 individually mutated the nase. two Tyrs of the PDGF receptor, which Recent work has provided further are thought to act as docking sites for strong evidence for a role for PI 3PI 3-kinase. Mutation of Tyr740 causes kinase in activating p70s6k. Weng e t al. 3~ complete abolition of activation of PI showed that synthesis of a constitu3-kihase, but leaves activation of p70s6k tively active PI 3-kinase causes actiintact, thus apparently ruling out vation of either p70s6k or the ACT104 involvement of PI 3-kinase. As mutation mutant. These data show that PI 3of the Tyr751 residue blocked both, it kinase acts at sites outside set 2, and seems likely that another SH2-domain Thr229 was subsequently identified as protein is the key here, but its identity a key residue for the control of p70s6k, is unknown. its phosphorylation increasing or deThe findings of Ming e t al. 3s came as a creasing in response to PI 3-kinase overconsiderable surprise, as earlier work production or wortmannin treatment.
This site also undergoes phosphorylation in response to serum, and this is blocked by wortmannin (see above2'). Incidentally, Thr229 is phosphorylated even in a 'kinase-dead' p70s6k mutant, showing that it is not a target for autophosphorylation and suggesting that the Thr229 kinase could be a key player in the control of p70s6k. The hunt for this kinase is on. It will provide muchneeded further information on the events involved in the upstream control of p70s6k. Additional evidence for a link to PI 3-kinase comes from the demonstration that protein kinase B (PKB, the cellular homologue of the transforming kinase v-Akt) lies downstream of PI 3-kinase but upstream of p70s6k ~ef. 38). Intriguingly, Franke e t a l ) 9 have shown that PKB can be activated in vitro by phosphatidylinositol 3-phosphate, perhaps by binding to the amino-terminal pleckstrin-homolo~ domain of PKB, thus providing a clear potential link to PI 3-kinase. Activation of PKB by PDGF or insulin is accompanied by its phosphorylation and is sensitive to treatments that block or prevent activation of PI 3-kinase (e.g. wortmannin)39,4~ Burgering and Coffer38 conclude that PKB is either upstream of, or acts in parallel to, the rapamycin-sensitive step involved in the activation of p70S6k: the latter would be consistent with the data, discussed above, which indicate that wortmannin and rapamycin block p70s6k activation by inhibiting different inputs (Fig. 2). Mutants of PKB can block activation of the wild-type ldnase4~ This provides the possibility of using such dominant-negatives to assess the requirement for PKB for activation of p70s6k,although this has not yet been done. Information on the requirement for Ras in the activation of PKB is conflicting3s,39. Incidentally, phorbol esters activate PKB only very weakly38,4~ This suggests that they feed into p70s6k activation by an alternative route and could explain why wortmannin does not block the activation of p70s6k by these agents36.
Table I. Selected examples of rapamycin-sensitive signalling pathways
Other substrates and other roles of p70 ~ p70s6k is implicated in the regulation of cellular processes other than $6 phosphorylation (Fig. 3; and Table I) by data showing that rapamycin interferes with the control of the process in question. For reasons of space, and because none of these examples is yet fully characterized, they will not be discussed in detail. Briefly, rapamycin
Hormones, growth factors
/
/
\
\
Target
Physiological role
Comments
Ref.
4E-BP1a
Control of translation initiation
Not a direct substrate for p70s6k
41
CREMb
Regulation of transcription
Physiological substrate for p70s6k
13
Regulatory role of p70s6k unclear
48
p34cdc2and p33cdk2 Cell-cycle control (G1/S transition) aelF-4E-bindingprotein. bCyclic-AMP-responsive-elementmodulator.
REVIEWS
TIBS 2 1 - M A Y 1 9 9 6
blocks changes in the phosphorylation/activity of another protein involved in the control of mRNA translation. This is the initiation factor elF-4E-binding protein 4E-BP1 (Refs 41, 42). Insulin (and other stimuli) lead to changes in the phosphorylation of 4E-BP1, which is linked to activation of translation, and this is blocked by rapamycin, suggesting that the p70 s6k pathway or a related one is involved. The initiation factor elF-4E appears to play a crucial role in the regulation of cell growth and the control of the translation of specific mRNAs with highly structured 5' untranslated regions (for review, see Ref. 43). In this case, p70 s6k is not directly involved itself (i.e. it does not phosphorylate 4E-BP1)41. The above findings might help explain the observation that rapamycin blocks the activation of protein synthesis by angiotensin II in vascular smooth muscle cells 44. Rapamycin also blocks the translational upregulation by serum of mRNAs that possess a polypyrimidine tract at their extreme 5' ends (next to the cap) 45,46.Initially this was interpreted in terms of a role for phosphorylation of $6, which lies close to the mRNA in the ribosome. However, as rapamycin also influences the phosphorylation of 4E-BP1 (Refs 41, 42), which in turn regulates the cap-binding protein elF-4E, other mechanisms are clearly possible. In fact, elF-4E is implicated in the control of the translation of certain members of the class of mRNAs47. It remains unclear why microinjection of antibodies to p70 s6k causes more generalized and profound inhibition of translation than would be expected from these specific types of regulation of mRNA translation 27. The finding that p70 s6k phosphorylates one isoform of the transcription factor cyclic-AMP-responsive element modulator (CREM"0 at Ser117, the site in CREM phosphorylated in response to serum in vivo, also implicates it in the control of transcription. Furthermore, rapamycin blocks both CREM phosphorylation and transactivation in response to serum ~3. Thus, p70 s~k might be important in transcriptional activation by mitogens.
Concludingremarks There is clearly still much to do to tie up the many loose ends in this story. These include the identification of p70 s6k kinase(s) and their links, presumably by mTOR, to cell surface receptors.
The role of PI 3-kinase in p70 s6k 23 Kunz, J. and Hall, M. N. (1993) Trends Biochem. Sci. 18, 334-338 activation still remains enigmatic. The 24 Terada, N. et al. (1993) J. Biol. Chem. 268, basis of the G1 block induced by 12062-12068 rapamycin also remains unexplained: 25 Kuo, C. J. et al. (1992) Nature 358, 70-73 rapamycin-induced growth arrest oc- 26 CaNo, V., Crews, C. M., Vik, T. A. and Bierer, B. E. (1992) Proc. Natl Acad. Sci. USA curs very late in G1 and is associated 89, 7571-7575 with inhibition of the cell-cycle-control 27 Lane, H. A., Fernandez,A., Lamb, N. J. C. and Thomas, G. (1993) Nature 363, 170-172 kinases p34 r and p33 cdk2(Ref. 48), but 28 Brown, E. J. et al. (1995) Nature 377, whether p70 s6k is directly involved is 441-446 not known. Rapamycin has been shown 29 Pearson, R. B. et al. (1995) EMBO J. 14, to block the inactivation of the CDK in5279-5287 hibitor p27 Kip', which normally occurs 30 Weng, Q. R eta/. (1995) Proc. Natl Acad. Sci. USA 92, 5744-5748 in response to interleukin 2 in T cells 49. 31 Kunz, J. et al. (1993) Cell 73, 585-596 The upstream control of 4E-BP1 needs 32 Sabatini, D. M. et aL (1994) Cell 78, 35-43 to be addressed to establish whether 33 Sabatini, D. M. et al. (1995) J. Biol. Chem. 270, 20875-20878 p70 s6k itself is involved, or whether 34 Pawson, T. (1995) Nature 373, 573-580 there is bifurcation (or trifurcation) in 35 Ming, X. F. et al. (1994) Nature 371, 426-429 rapamycin-sensitive signalling pathways. 36 Chung, J. eta/. (1994) Nature 370, 71-75 37 Cheatham, B. et al. (1994) Mol. Cell. Biol. 14,
Acknowledgements l wish to thank G. Thomas and J. Avruch for providing information on the regulation of p70 s6k before publication and G. Welsh for his careful reading of this article.
References 1 Bandi, H. R. et al. (1993) J. Biol. Chem. 268, 4530-4533 2 Kozma, S. C. and Thomas, G. (1994) Cancer Biol. 5, 406-412 3 Lavoinne, A. et al. (1991) Eur. J. Biochem. 199, 721-728 4 Flotow, H. and Thomas, G. (1992) J. Biol. Chem. 267, 3074-3078 5 Chung, J., Kuo, C. J., Crabtree, G. R. and Blenis, J. (1992) Ceil 69, 1227-1236 6 Price, D. J. et al. (1992) Science 257, 973-977 7 Kozma, S. C. et al. (1990) Proc. Natl Acad. Sci. USA 87, 7365-7369 8 Banerjee, P. et al. (1990) Proc. Natl Acad. Sci. USA 87, 8550-8554 9 Harmann, B. and Kilimann, M. W. (1990) FEBS Lett. 273, 248-252 10 Grove, J. R. et al. (1991) Mol. Cell. Biol. 11, 5541-5550 11 Reinhard, C., Thomas, G. and Kozma, S. C. (1992) Proc. Natl Acad. Sci. USA 89, 4052-4056 12 Reinhard, C., Fernandez,A., Lamb, N. J. C. and Thomas, G. (1994) EMBO J. 13, 1557-1565 13 de Groot, R. P., Ballou, L. M. and SassoneCorsi, P. (1994) Cell 79, 81-91 14 Weng, Q-P. et al. (1995) Mol. Cell. Biol. 15, 2333-2340 15 Ballou, L. M., Siegmann, M. and Thomas, G. (1988) Proc. Natl Acad. Sci. USA 85, 7154-7158 16 Ballou, L. M., Jeno, P. and Thomas, G. (1988) J. Biol. Chem. 263, 1188-1194 17 Kozma, S. C. et al. (1993) J. Biol. Chem. 268, 7134-7138 18 Ferrari, S. et al. (1992) Proc. Natl Acad. Sci. USA 89, 7282-7286 19 Ballou, L. M., Luther, H. and Thomas, G. (1991) Nature 349, 348-350 20 Mukhopadhyay, N. K. et al. (1992) J. Biol. Chem. 267, 3325-3335 21 Han, J. W., Pearson, R. B., Dennis, P. B. and Thomas, G. (1995) J. Biol. Chem. 270, 21396-21403 22 Ferrari, S. et al. (1993) J. Biol. Chem. 268, 16091-16094
4902-4911 38 Burgering, B. M. T. and Coffer, P. J. (1995) Nature 376, 599-602 39 Franke, T. F. et al. (1995) Cell 81, 727-736 40 Kohn, A. D., Kovacina, K. S. and Roth, R. A. (1995) EMBO J. 14, 4288-4295 41 Lin, T-A. et al. (1995) J. Biol. Chem. 270,
18531-18538 42 Graves, L. M. eta/. (1995) Proc. Natl Acad. Sci. USA 92, 7222-7226 43 Sonenberg, N. (1994) Biochimie 76, 839-846 44 Giasson, E. and Meloche, S. (1995) J. Biol. Chem. 270, 5225-5231 45 Jefferies, H. B. J., Reinhard, G., Kozma, S. C. and Thomas, G. (1994) Proc. NatlAcad. Sci. USA 91, 4441-4445 46 Terada, N. et al. (1994) Proc. Natl 4cad. Sci. USA 91, 11477-11481 47 Bommer, U. A. et al. (1994) Cell. Mol. Biol. Res. 40, 633-641 48 Morice, W. G. et al. (1993) J. Biol. Chem. 268, 22737-22745 49 Nourse, J. et al. (1994) Nature 372, 570-573
Computer Corner Have you come across any applications (freeware or commercially available), CDs, servers or tips recently that might be of interest to other biochemists and molecular biologists? If so, why not let us know so that we can review them in
Computer Comer? Please contact: Jo McEntyre Tel: +44 1 2 2 3 3 1 5 9 6 1 Fax: +44 1 2 2 3 4 6 4 4 3 0 Email: [email protected] 185
TIBS 21 - MAY 1996 1314-1321 24 Reinhard, M. et al. (1995) EMBO J. 14, 25 26 27 28 29 30
1583-1589 Matuoka, K., Shibasaki, F., Shibata, M. and Takenawa, T. (1993) EMBO J. 12, 3467-3473 Symons, M. (1995) Curr. Opin. Biotech. 6, 668-674 Qiu, R-G. et al. (1995) Nature 374, 457-459 Khosravi-Far, R. et al. (1995) Mol. Cell. Biol. 15, 6443-6453 Prendergast, G. C. et al. (1995) Oncogene 10, 2289-2296 Qiu, R-G. et al. (1996) Proc. Natl Acad. Sci. USA 92, 11781-11785
31 Joneson, T., White, M. A., Wigler, M. H. and Bar-Sagi, D. (1996) Science 271, 810-812 32 Simon, M. N. et al. (1995) Nature 376,
25951-25954
Christopher G. Proud The $6 ribosomal protein is phosphorylated by p70 $6 kinase (p7OS6k). Although the cellular role of $6 phosphorylation is still not fully clear, studies on p70 s6k and its activation have revealed the existence of a novel signalling pathway, clues to the mechanism of action of certain immunosuppressants and insights into the control of gene expression at the levels of transcription and translation.
C. G. Proud is at the Department of Biosciences, University of Kent at Canterbury, Canterbury, UK CT2 7NJ, 9 1996,ElsevierScienceLtd
5931-5938 40 Watanabe, G. et al. (1996) Science 271,
645-648
702-705 33 Zhao, Z. S., Leung, T., Manser, E. and Lim, L. (1995) Mol. Cell. Biol. 15, 5246-5257 34 Coso, O. A. et al. (1995) Cell 81, 1137-1146 35 Minden, A. et al. (1995) Cell 81, 1147-1157 36 Olson, M. F., Ashworth, A. and Hall, A. (1995) Science 269, 1270-1272 37 Hill, C. S., Wynne, J. and Treisman, R. (1995) Cell 81, 1159-1170 38 Malcolm, K. C. et al. (1994) J. Biol. Chem. 269,
p70 $6 kinase: an enigma with variations
THE $6 PROTEIN is a component of the small (40S) subunit of eukaryotic ribosomes. It has been known since the 1970s that $6 undergoes phosphorylation and that this reaction is enhanced by agents that stimulate protein synthesis, such as hormones (e.g. insulin) and growth factors. Up to five Ser residues in the carboxyl terminus of $6 become phosphorylated ~ (Ser235, 236, 240, 244 and 247). Given that $6 phosphorylation occurs in a wide range of cell types in response to a variety of stimuli, a burning question, which was tackled with vigour in the 1980s, was what is the identity of the kinase(s) responsible for the phosphorylation of $6 in vivo? Two candidates eventually emerged. They could be distinguished by various criteria including their apparent molecular masses, which gave rise to the current nomenclature: p70 s6k (apparent M = 70 kDa) and p90 "k. It now appears that p70 s6k is
39 Nonaka, H. et al. (1995) EMBO J. 14,
the major physiological $6 kinase in mammalian cellsL Evidence in favour of this includes the observations that p70 sBk phosphorylates 40S subunits more efficiently than p90 rsk (Refs 3, 4) and, most convincingly, that rapamycin (which, as described in detail below, blocks activation of p70 sBk, but not that of p90 rsk) prevents phosphorylation of $6 in response to various stimuli in intact cells 5,6. Having found the probable kinase, the focus of attention has shifted to the key question of how this enzyme is regulated physiologically. Structure of S6 kinase Three research groups almost simultaneously reported the initial isolation of cDNA clones for p70 s6k from rat 7.s and rabbit 9. The enzyme has a single catalytic domain related most closely to the more amino terminal of the two catalytic domains found in p90 ~sk and to second-messenger-regulated kinases. It is encoded by at least two distinct transcripts differing at their 5' ends 1~ and encoding polypeptides with different amino termini (Fig. 1; predicted masses
41 Amano, M. et al. (1996) Science 271,
648-650 42 Leung, T., Manser, E., Tan, L. and Lim, L. (1995) J. Biol. Chem. 270, 29051-29054 43 Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830 44 Burbelo, P. D., Drechsel, D. and Hall, A. (1995) J. Biol. Chem. 270, 29071-29074 45 Peppelenbosch, M. P. et al. (1995) Cell 81, 849-856
56.2 and 59.2kDa). The longer protein contains an extension of 23 amino acids including a hexa-Arg sequence typical of nuclear localization signals: both forms are synthesized in vivo. The larger species has an apparent molecular mass of 85kDa and is hence called p85sGk.From here on, unless otherwise specified, the term p70 s6k will be used to include the larger form too, and the numbering of residues will be based on p70 s6k. (To convert to p85 s6k numbering, add 23.) Data from Thomas' group show that the p85 isoform is targeted to the nucleus by its amino-terminal sequence 12 (although a modest amount of the shorter form might also be present in the nucleus13). This nuclear form might play a key role in the regulation of cell growth as indicated by the observation that antibodies specific for this species block cell growth when injected into the nucleus (but not when introduced into the cytoplasm) and that this block is overcome by co-injection of the shorter form into the nucleus~L p70 s6k contains a sequence that resembles the region of $6 containing the phosphorylation sites. This sequence lies immediately carboxy-terminal to the catalytic domain of the kinase (Fig. 1) and it was initially suggested that it might act as an autoregulatory domain by occluding the active site of the kinaseS.~4: it contains four Ser residues that are phosphorylated as the kinase is activated. However, the situation is more complex than this, as deletion of this sequence does not generate an active enzyme and phosphorylation of sites elsewhere in the enzyme is also associated with activation (see below). How is p70 s6kactivated? A key observation made during the isolation of p70 s6k was that it rapidly lost activity unless I~-glycerophosphate or other phosphatase inhibitors were
PII: S0968-0004(96) 10016-5
181
REVIEWS pathway activating p70 s6~ was distinct from that leadI I p70s6k II ing to activation of MAP kinase (and thus p90rsk)19. .~. 9 I I p85 s6k Avruch's group 2~used a peptide based on the autoreguMRRRRRRD ~'N',. Potential autoregulatory latory region of p70s6k to Acidic region sequence identify possible upstream kinases. They found several 'set 1' (b) kinases that could phosrThr229 Thr389 Ser404~ phorylate the peptide, including members of the MAP I I P70s6k kinase family and a form of II cdc2. However, these kinases s e r 4 1 1 J / ~~ - Ser424 also phosphorylated recomSer418 Thr421 J L binant p70s6k in vitro at sites 'set 2' that correspond to some of those labelled in vivo, but Figure 1 they were unable to activate Structure of members of the p70 s6k family and model p70 s6k, indicating a requirefor their activation. (a) The structures of p70 s6k and ment for additional kinases. p85 s6k are shown schematically. The magenta bar shows the amino-terminal extension present in the When the four residues at longer form, which contains the Arg-rich, potential the carboxyl terminus are nuclear-targeting signal. Also shown are the catalytic mutated to Asp or Glu (to domain (green), the proposed autoregulatory region mimic phosphoserine or (blue) and an acidic region (red) near the amino terphosphothreonine, respecminus with which it has been proposed to interact. tively), the kinase shows This interaction is suggested to close off the active higher basal activity than site, rendering the kinase inactive8. (b) The positions of the phosphorylation sites identified in p70 ssk and the wild-type enzyme 21. Howp85 s6k are indicated. Thr229, Thr389 and Ser404 beever, it can still be activated long to the group referred to in this article as further by serum, again show'set 1': phosphorylation of these sites accompanies ing that additional mechaactivation of the kinase and is blocked by rapamycin. nisms, involving the phosThe four sites indicated towards the carboxyl termiphorylation of other sites, nus of p70 s6k and p85 s6k are also phosphorylated are involved in switching on when the kinase is activated, but not all are sensitive to rapamycin ('set 2'). p70s6k (Ref. 22). However, as the activity of the mutant, with all four Ser residues conincluded in the buffers. This suggested verted to Ala, is greatly reduced, these that p70s6k was itself activated by residues are important for the activation phosphorylation. In addition, treatment or activity of p70s6k~ef. 21; see below). with protein phosphatase-2A ~P-2A) led to inactivation, suggesting that Ser/ Rapamycin Thr phosphorylation was importanfl 5,~6. Rapamycin inhibits T-cell activation It has since been shown that acti- by blocking progression through the GI vation of p70s6k is accompanied by phase of the cell cycle (for reviews, see phosphorylation of seven Ser/Thr resi- Refs 23, 24), and selectively prevents dues including the four referred to activation of p70 sGk (Refs 5, 6, 24-26), above n,~s Gig. 1). Thus, the identifi- i.e. it does not block the activation of cation of kinases that activate p70s6k kinases in the MAP kinase cascade. seems a crucial step in elucidating the Neutralizing antibodies against p70s6k signalling cascade leading to the stimu- also block the ability of serum to induce lation of p70s6k. It is interesting to note the shift of quiescent cells through G1 that each of the four carboxy-terminal into S phase [2,27,providing evidence that phosphorylation sites (Ser411, Ser418, pT0s6kis indeed a key component underThr421 and Ser424) is followed by a Pro lying the ability of rapamycin to block residue. Ser/Thr-Pro motifs are tar- G1 progression. However, it should be geted by a range of kinases including noted that: (1) for reasons that are not mitogen-activated protein (MAP) kinase yet clear, rapamycin has less profound and cyclin-dependent kinases (CDKs) effects on growth and on protein synthesis than does microinjection of antisuch as cdc2. A further important observation made p70s6k antibodies; and (2) rapamycin by Thomas' group came in 1991 when also blocks activation of CDKs (see Ref. they provided data indicating that the 28 and lit. cit. therein).
(a)
182
Catalytic domain
TIBS 2 1 -
MAY 1996
Rapamycin causes dephospho~lation of p70s6kat sites other than those in the carboxy-terminaJ domain 22. This fits with the data above, which indicate that other phosphorylation events were required for pT0s6kactivation 22. Additional phosphorylation sites have now been identified as Thr229, Thr389 and Ser404 [here termed 'set 1' ~efs 21, 29; Fig. 1)]. Detailed analysis revealed that the four most carboxy-terminal sites ('set 2') are partially phosphorylated in quiescent cells and that they and set 1 (which are not basally phosphorylated) undergo rapid phosphorylation in response to stimuli that activate p70 s6k (Ref. 21). Rapamycin blocks phosphorylation of set 1 and also Ser411 of set 2. These sites also undergo dephosphorylation in response to wortmannin or the methylxanthine SQ20006. Wortmannin is reported to be a selective inhibitor of phosphoinositide 3-kinases ~I 3-kinases), while methylxanthines are well-known inhibitors of cyclic AMP-phosphodiesterase, but the action of SQ20006 seems not to be related to elevated levels of cAMP. Instead, it might block a protein kinase upstream of p70s6kand act in a manner similar to that of rapamycin rather than that of wortmannin (based on the fact that SQ20006 and rapamycin both block the activation of p70s6k by phorbol esters whereas wortmannin does not2~). The kinases that phosphorylate any of the sites in p70 s6khave yet to be identified. Thr229, Thr389 and Ser404 all lie in similar sequence contexts, suggesting that a common kinase could be responsible for phosphorylating each of them. This context is rich in aromatic residues and, thus, differs markedly from that around the set 2 sites. Detailed analysis of the correlation between changes in the phosphorylation of individual sites in p70s6~and its activity suggests that Thr389 and Ser404 are particularly important (see below). Each of these residues has now been mutated to either Ala or Asp/Glu. The Thr389~a mutant is catalytically inactive, suggesting a key role for this residue, a notion that is strongly supported by the observation that the Thr389Glu mutant is largely insensitive to rapamycin 29. This evidence has been interpreted as indicating that Thr389 is a key target residue for the rapamycin-sensitive input, which leads to kinase activation.
Truncation mutants An alternative approach to investigating the regulation of p70 s6kis the use
TIBS 21 - MAY 1996
of truncation mutants of the enzyme. (These studies actually used the longer form, but to save further confusion, here I will use the numbering of the 70 kDa form). Removal of the carboxyl terminus (amino acids 399-502, containing the autoregulatory domain and the set 2 sites) generates a mutant (ACT104), which is still strongly activated by serum 3~ This activation is sensitive to wortmannin, but only partially blocked by rapamycin, indicating that these inhibitors block activation in different ways. Consistent with the data discussed above, activation of ACT104 is accompanied by phosphorylation and both rapamycin and wortmannin cause dephosphorylation of the same set of sites in this mutant, again showing that phosphorylation events outside the carboxyl terminus are important for activation. However, as this mutant does not contain Ser404, this residue is clearly not essential for activation by serum. Additional insights were obtained using a mutant in which the acidic region adjacent to the amino terminus was deleted 3~ This mutant exhibited sharply reduced activity, but was still activated by serum and sensitive to wortmannin. However, it was entirely unaffected by rapamycin, again showing that two separate inputs are involved. The effects of rapamycin require amino acids 6-23, but this does not depend on phosphorylation within this region. Instead, phosphorylation at site(s) between residues 23 and 398, which includes Thr229 (see below) and Thr389, is required for activation. Consistent with this, the double truncation mutant, lacking both termini, can be activated by serum, is resistant to rapamycin, but is inhibited by wortmannin 3~ While data obtained from the relatively drastic truncation mutants must be treated with caution, as they differ substantially from the intact protein, it must be pointed out that these data do not agree with the idea that Thr389 plays the key role in the rapamycinsensitive activation of p70 s6k. For example, the amino-terminal truncation and the double truncation both contain this site, but are not sensitive to rapamycin, while the ACT104 mutant (which also contains Thr389) is only partially sensitive to this drug 3~
The target of rapamycin is homologousto PI 3-kinase Despite the identification of the target of rapamycin in yeast (target of rapamycin, TOR31) and in mammalian cells
REVIEWS (mTOR, also called RAFT or FRAp32), there is little information on the mechanism of action of this drug. It should be noted that rapamycin does not itself interact with mTOR; rather, rapamycin binds to a protein termed FKBP12 Ras PI 3-kinase Others? (FK506-binding protein 12, where FK506 is another immunosuppressant) and the rapamycin-FKBP12 complex PKB mTOR actually interacts with mTOR Rapamycin (Fig. 2). It is interesting to Unknown kinases FKBP12 note that TOR shows homology with PI 3-kinases, whereas the mammalian protein has Activation of p70 s6k been reported to possess phosphoinositide 4-kinase acFigure 2 tivity33, although this remains Mechanism of activation of p70 s6k. Ras does not play controversial. a role in p70 s6k activation. However, most (but not Recent data demonstrate all) of the available evidence suggests that phosthat mTOR is indeed a rapaphoinositide 3-kinase (PI 3-kinase) is important, and mycin-sensitive regulator of that it might act through protein kinase B (PKB). Wortmannin blocks activation of p70 s6k presumably p70 s6k in vivo 28 and that by inhibiting PI 3-kinase. The target of rapamycin in mTOR undergoes autophosmammalian cells (mTOR) confers rapamycin sensitivphorylation, which is blocked ity to the activation of p70 s6k, but lies on a pathway by rapamycin. This suggests that provides an input into p70 s6k activation that is that the kinase activity of separate from that provided by the PI 3-kinase. mTOR is essential for its Rapamycin does not bind directly to mTOR, but instead forms a complex with FKBP12, which in turn binds to function in the activation of and blocks mTOR. Activation of p70 s6k is associated p70 sBk, an idea that is conwith phosphorylation at multiple sites, probably catafirmed by 'kinase-dead' mulysed by several distinct protein kinases. It is not entants of TOR that cannot tirely clear whether PKB acts upstream of roTOR or function to activate p70 s6k parallel to it (the variant shown here). The obser(Ref. 28). By contrast, autovation that rapamycin and wortmannin affect differphosphorylation of mTOR is ent inputs into p70 s6k is more consistent with this situation, assuming that wortmannin is inhibiting the not blocked by wortmannin, PI 3-kinase upstream of PKB. Not shown is the actiagain showing that this comvation of p70 s6k by phorbol esters, which is insensipound acts differently from tive to wortmannin and could bypass PKB (see text). rapamycin and might not be merely acting to inhibit the links and suggested possible roles for putative kinase activity of mTOR. Deletion mutations of mTOR reveal others, while also providing some apthat the kinase domain alone is not parently conflicting data. Also, it is not enough for activation of p70 s6k and that clear which steps lie above, and which amino-terminal sequences are also re- act in parallel to, the step blocked by quired 28. Perhaps this region interacts rapamycin. with p70 s6k, although there is no direct Neither Ras nor Raf plays a role in the evidence for this. So far, mTOR has not activation of p70 s6k.The platelet-derived been shown to phosphorylate p70 s6k.As growth factor (PDGF) receptor contains rapamycin decreases the phosphoryl- a so-called 'kinase insert', which contains ation of p70 s6k, an additional com- Tyr residues that might act as docking ponent, most likely a protein kinase sites for SH2-containing proteins such or phosphatase, must lie in between as P1 3-kinase when phosphorylated 34. mTOR and p70 s6k(Fig. 2). In cells synthesizing PDGF receptors from which the kinase insert domain Upstream signallingevents has been deleted, Ras is still activated What links the rapamycin-sensitive normally, but p70 s6k is not 35. These and step to cell surface receptors such as other data (e.g. use of dominant-negathose with intrinsic tyrosine kinase ac- tive mutants of Ras) indicate that Ras is tivity? The short answer is that we do not involved in the activation of p70 s6k. not know, although recent work has Similarly, dominant-negative mutants of apparently rule(] out some potential Raf (the kinase immediately downstream
183
REVIEWS
TIBS 2 1 -
MAY 1996
by Chung e t al. 36 had provided several pieces of evidence supporting a role for PI 3-kinase in pT0s6k actiR a p a m y c i n - - t roTOR vation. In addition to finding that the Tyr740/751 mutant could not activate p70ssk,they Other also showed that two differp70 s6k . ~ kinases ent inhibitors of PI 3-kinase ' (wortmannin and LY294002) blocked activation of p70s6k p27Kip1 .,~,.~ by insulin as well as PDGF l CREM $6 4E-BP1 ~ef. 37). The explanation CDKs 1 for these discrepancies remains unclear. A more informative way of employing mutants of the PDGF receptor might be to carry out 'add-back' experiments: in this strategy, all Figure 3 five T~ phosphorylation The role of p70 s6k in cellular regulation, where it directly phosphorylates both the cyclic-AMP-responsivesites in the PDGF receptor element modulator (CREM), a transcription factor inare mutated to Phe, and one volved in the control of expression of specific genes, or more of the Tyrs are then and ribosomal protein $6, whose role in controlling 'added back' so that one can translation remains unclear. Rapamycin interferes with examine the roles of individthe control of the cyclin-dependent kinase (CDK) p27 Kip1 ual residues against a backand the elF-4E-binding protein (4E-BP1), implying links to p70 s6k, but it is not known if p70 s6k is itself inground in which the other volved in their regulation, or whether the relevant sigphosphotyrosines have been nalling pathways merely share common features with eliminated. those controlling p70 s6k (indicated by broken lines). Chung et al. 36 provided the Abbreviation: rnTOR, mammalian target of rapamycin. first evidence that wortmannin and rapamycin inhibit of Ras in the MAP kinase cascade) p70s6k activation in different ways, as also fail to block activation of p70s6k activation by phorbol esters is sensitive (Ref. 35). to rapamycin, but not to wortmannin, suggesting that protein kinase C ~KC)Role of PI 3-kinase in the activation of p70m mediated activation bypasses PI 3-kiMing e t al. 35 individually mutated the nase. two Tyrs of the PDGF receptor, which Recent work has provided further are thought to act as docking sites for strong evidence for a role for PI 3PI 3-kinase. Mutation of Tyr740 causes kinase in activating p70s6k. Weng e t al. 3~ complete abolition of activation of PI showed that synthesis of a constitu3-kihase, but leaves activation of p70s6k tively active PI 3-kinase causes actiintact, thus apparently ruling out vation of either p70s6k or the ACT104 involvement of PI 3-kinase. As mutation mutant. These data show that PI 3of the Tyr751 residue blocked both, it kinase acts at sites outside set 2, and seems likely that another SH2-domain Thr229 was subsequently identified as protein is the key here, but its identity a key residue for the control of p70s6k, is unknown. its phosphorylation increasing or deThe findings of Ming e t al. 3s came as a creasing in response to PI 3-kinase overconsiderable surprise, as earlier work production or wortmannin treatment.
This site also undergoes phosphorylation in response to serum, and this is blocked by wortmannin (see above2'). Incidentally, Thr229 is phosphorylated even in a 'kinase-dead' p70s6k mutant, showing that it is not a target for autophosphorylation and suggesting that the Thr229 kinase could be a key player in the control of p70s6k. The hunt for this kinase is on. It will provide muchneeded further information on the events involved in the upstream control of p70s6k. Additional evidence for a link to PI 3-kinase comes from the demonstration that protein kinase B (PKB, the cellular homologue of the transforming kinase v-Akt) lies downstream of PI 3-kinase but upstream of p70s6k ~ef. 38). Intriguingly, Franke e t a l ) 9 have shown that PKB can be activated in vitro by phosphatidylinositol 3-phosphate, perhaps by binding to the amino-terminal pleckstrin-homolo~ domain of PKB, thus providing a clear potential link to PI 3-kinase. Activation of PKB by PDGF or insulin is accompanied by its phosphorylation and is sensitive to treatments that block or prevent activation of PI 3-kinase (e.g. wortmannin)39,4~ Burgering and Coffer38 conclude that PKB is either upstream of, or acts in parallel to, the rapamycin-sensitive step involved in the activation of p70S6k: the latter would be consistent with the data, discussed above, which indicate that wortmannin and rapamycin block p70s6k activation by inhibiting different inputs (Fig. 2). Mutants of PKB can block activation of the wild-type ldnase4~ This provides the possibility of using such dominant-negatives to assess the requirement for PKB for activation of p70s6k,although this has not yet been done. Information on the requirement for Ras in the activation of PKB is conflicting3s,39. Incidentally, phorbol esters activate PKB only very weakly38,4~ This suggests that they feed into p70s6k activation by an alternative route and could explain why wortmannin does not block the activation of p70s6k by these agents36.
Table I. Selected examples of rapamycin-sensitive signalling pathways
Other substrates and other roles of p70 ~ p70s6k is implicated in the regulation of cellular processes other than $6 phosphorylation (Fig. 3; and Table I) by data showing that rapamycin interferes with the control of the process in question. For reasons of space, and because none of these examples is yet fully characterized, they will not be discussed in detail. Briefly, rapamycin
Hormones, growth factors
/
/
\
\
Target
Physiological role
Comments
Ref.
4E-BP1a
Control of translation initiation
Not a direct substrate for p70s6k
41
CREMb
Regulation of transcription
Physiological substrate for p70s6k
13
Regulatory role of p70s6k unclear
48
p34cdc2and p33cdk2 Cell-cycle control (G1/S transition) aelF-4E-bindingprotein. bCyclic-AMP-responsive-elementmodulator.
REVIEWS
TIBS 2 1 - M A Y 1 9 9 6
blocks changes in the phosphorylation/activity of another protein involved in the control of mRNA translation. This is the initiation factor elF-4E-binding protein 4E-BP1 (Refs 41, 42). Insulin (and other stimuli) lead to changes in the phosphorylation of 4E-BP1, which is linked to activation of translation, and this is blocked by rapamycin, suggesting that the p70 s6k pathway or a related one is involved. The initiation factor elF-4E appears to play a crucial role in the regulation of cell growth and the control of the translation of specific mRNAs with highly structured 5' untranslated regions (for review, see Ref. 43). In this case, p70 s6k is not directly involved itself (i.e. it does not phosphorylate 4E-BP1)41. The above findings might help explain the observation that rapamycin blocks the activation of protein synthesis by angiotensin II in vascular smooth muscle cells 44. Rapamycin also blocks the translational upregulation by serum of mRNAs that possess a polypyrimidine tract at their extreme 5' ends (next to the cap) 45,46.Initially this was interpreted in terms of a role for phosphorylation of $6, which lies close to the mRNA in the ribosome. However, as rapamycin also influences the phosphorylation of 4E-BP1 (Refs 41, 42), which in turn regulates the cap-binding protein elF-4E, other mechanisms are clearly possible. In fact, elF-4E is implicated in the control of the translation of certain members of the class of mRNAs47. It remains unclear why microinjection of antibodies to p70 s6k causes more generalized and profound inhibition of translation than would be expected from these specific types of regulation of mRNA translation 27. The finding that p70 s6k phosphorylates one isoform of the transcription factor cyclic-AMP-responsive element modulator (CREM"0 at Ser117, the site in CREM phosphorylated in response to serum in vivo, also implicates it in the control of transcription. Furthermore, rapamycin blocks both CREM phosphorylation and transactivation in response to serum ~3. Thus, p70 s~k might be important in transcriptional activation by mitogens.
Concludingremarks There is clearly still much to do to tie up the many loose ends in this story. These include the identification of p70 s6k kinase(s) and their links, presumably by mTOR, to cell surface receptors.
The role of PI 3-kinase in p70 s6k 23 Kunz, J. and Hall, M. N. (1993) Trends Biochem. Sci. 18, 334-338 activation still remains enigmatic. The 24 Terada, N. et al. (1993) J. Biol. Chem. 268, basis of the G1 block induced by 12062-12068 rapamycin also remains unexplained: 25 Kuo, C. J. et al. (1992) Nature 358, 70-73 rapamycin-induced growth arrest oc- 26 CaNo, V., Crews, C. M., Vik, T. A. and Bierer, B. E. (1992) Proc. Natl Acad. Sci. USA curs very late in G1 and is associated 89, 7571-7575 with inhibition of the cell-cycle-control 27 Lane, H. A., Fernandez,A., Lamb, N. J. C. and Thomas, G. (1993) Nature 363, 170-172 kinases p34 r and p33 cdk2(Ref. 48), but 28 Brown, E. J. et al. (1995) Nature 377, whether p70 s6k is directly involved is 441-446 not known. Rapamycin has been shown 29 Pearson, R. B. et al. (1995) EMBO J. 14, to block the inactivation of the CDK in5279-5287 hibitor p27 Kip', which normally occurs 30 Weng, Q. R eta/. (1995) Proc. Natl Acad. Sci. USA 92, 5744-5748 in response to interleukin 2 in T cells 49. 31 Kunz, J. et al. (1993) Cell 73, 585-596 The upstream control of 4E-BP1 needs 32 Sabatini, D. M. et aL (1994) Cell 78, 35-43 to be addressed to establish whether 33 Sabatini, D. M. et al. (1995) J. Biol. Chem. 270, 20875-20878 p70 s6k itself is involved, or whether 34 Pawson, T. (1995) Nature 373, 573-580 there is bifurcation (or trifurcation) in 35 Ming, X. F. et al. (1994) Nature 371, 426-429 rapamycin-sensitive signalling pathways. 36 Chung, J. eta/. (1994) Nature 370, 71-75 37 Cheatham, B. et al. (1994) Mol. Cell. Biol. 14,
Acknowledgements l wish to thank G. Thomas and J. Avruch for providing information on the regulation of p70 s6k before publication and G. Welsh for his careful reading of this article.
References 1 Bandi, H. R. et al. (1993) J. Biol. Chem. 268, 4530-4533 2 Kozma, S. C. and Thomas, G. (1994) Cancer Biol. 5, 406-412 3 Lavoinne, A. et al. (1991) Eur. J. Biochem. 199, 721-728 4 Flotow, H. and Thomas, G. (1992) J. Biol. Chem. 267, 3074-3078 5 Chung, J., Kuo, C. J., Crabtree, G. R. and Blenis, J. (1992) Ceil 69, 1227-1236 6 Price, D. J. et al. (1992) Science 257, 973-977 7 Kozma, S. C. et al. (1990) Proc. Natl Acad. Sci. USA 87, 7365-7369 8 Banerjee, P. et al. (1990) Proc. Natl Acad. Sci. USA 87, 8550-8554 9 Harmann, B. and Kilimann, M. W. (1990) FEBS Lett. 273, 248-252 10 Grove, J. R. et al. (1991) Mol. Cell. Biol. 11, 5541-5550 11 Reinhard, C., Thomas, G. and Kozma, S. C. (1992) Proc. Natl Acad. Sci. USA 89, 4052-4056 12 Reinhard, C., Fernandez,A., Lamb, N. J. C. and Thomas, G. (1994) EMBO J. 13, 1557-1565 13 de Groot, R. P., Ballou, L. M. and SassoneCorsi, P. (1994) Cell 79, 81-91 14 Weng, Q-P. et al. (1995) Mol. Cell. Biol. 15, 2333-2340 15 Ballou, L. M., Siegmann, M. and Thomas, G. (1988) Proc. Natl Acad. Sci. USA 85, 7154-7158 16 Ballou, L. M., Jeno, P. and Thomas, G. (1988) J. Biol. Chem. 263, 1188-1194 17 Kozma, S. C. et al. (1993) J. Biol. Chem. 268, 7134-7138 18 Ferrari, S. et al. (1992) Proc. Natl Acad. Sci. USA 89, 7282-7286 19 Ballou, L. M., Luther, H. and Thomas, G. (1991) Nature 349, 348-350 20 Mukhopadhyay, N. K. et al. (1992) J. Biol. Chem. 267, 3325-3335 21 Han, J. W., Pearson, R. B., Dennis, P. B. and Thomas, G. (1995) J. Biol. Chem. 270, 21396-21403 22 Ferrari, S. et al. (1993) J. Biol. Chem. 268, 16091-16094
4902-4911 38 Burgering, B. M. T. and Coffer, P. J. (1995) Nature 376, 599-602 39 Franke, T. F. et al. (1995) Cell 81, 727-736 40 Kohn, A. D., Kovacina, K. S. and Roth, R. A. (1995) EMBO J. 14, 4288-4295 41 Lin, T-A. et al. (1995) J. Biol. Chem. 270,
18531-18538 42 Graves, L. M. eta/. (1995) Proc. Natl Acad. Sci. USA 92, 7222-7226 43 Sonenberg, N. (1994) Biochimie 76, 839-846 44 Giasson, E. and Meloche, S. (1995) J. Biol. Chem. 270, 5225-5231 45 Jefferies, H. B. J., Reinhard, G., Kozma, S. C. and Thomas, G. (1994) Proc. NatlAcad. Sci. USA 91, 4441-4445 46 Terada, N. et al. (1994) Proc. Natl 4cad. Sci. USA 91, 11477-11481 47 Bommer, U. A. et al. (1994) Cell. Mol. Biol. Res. 40, 633-641 48 Morice, W. G. et al. (1993) J. Biol. Chem. 268, 22737-22745 49 Nourse, J. et al. (1994) Nature 372, 570-573
Computer Corner Have you come across any applications (freeware or commercially available), CDs, servers or tips recently that might be of interest to other biochemists and molecular biologists? If so, why not let us know so that we can review them in
Computer Comer? Please contact: Jo McEntyre Tel: +44 1 2 2 3 3 1 5 9 6 1 Fax: +44 1 2 2 3 4 6 4 4 3 0 Email: [email protected] 185