[Cell Cycle 5:23, 2729-2732, 1 December 2006]; 2006 Landes Bioscience

Perspective

The Association of Tap42 Phosphatase Complexes with TORC1

Another Level of Regulation in Tor Signaling
Charles J. Di Como1 Yu Jiang2,3
1Aureon Laboratories, Inc; Yonkers, New York USA 2Department

AbstrACt
In the budding yeast Saccharomyces cerevisiae, rapamycin has been known to induce a rapid dephosphorylation of many downstream targets of Tor. The key components mediating this dephosphorylation process are the Tap42associated phosphatases, which become active upon rapamycin treatment. However, the mechanism by which rapamycin rapidly activates phosphatases is unclear. A recent report has provided evidence demon strating a physical association of the Tap42phosphatase complexes with TORC1, which is sensitive to rapamycin treatment or nutrient starvation. This association adds another level of regulation in Tor signaling, and explains why rapamycin or nutrient availability is able to initiate a rapid and robust response in the cell.

of Pharmacology; University of Pittsburgh School of Medicine; Pittsburgh, Pennsylvania USA *Correspondence to: Yu Jiang; Department of Pharmacology; University of Pittsburgh School of Medicine; E1357 Biomedical Science Tower; 200 Lothrop Street; Pittsburgh, Pennsylvaia 15213 USA; Tel.: 412.648.3390; Fax: 412.648.1945; Email: jiang@server.pharm.pitt.edu Original manuscript submitted: 10/09/06 Manuscript accepted: 10/11/06 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3516

IntroDuCtIon

Tap42, Rapamycin, Tor kinases, PP2A, Sit4

20

06

LA

ND

ES

www.landesbioscience.com

BIO

SC

IEN

tAp42 AnD the phosphAtAses

In the budding yeast, Saccharomyces cerevisiae, the major event downstream of TORC1 is the transcriptional regulation of genes involved in nutrient metabolism and ribosomal biogenesis.5,6 TORC1 mediates this event by regulating a group of serine/threonine protein phosphatases, including Sit4 and protein phosphatase 2A (PP2A).7 The activity of these phosphatases is normally repressed in growing cells where TORC1 is active, but becomes activated upon TORC1 inactivation. The activated phosphatases in turn dephosphorylate several transcription factors, including Gln3, resulting in their activation. This Tor-dependent regulation of the phosphatases is mediated through a phosphatase-associating protein termed Tap42, which associates with the PP2A and 2A-like phosphatases in a nutrient and Tor-dependent manner.8 It has been shown that Tor phosphorylates Tap42 and promotes its interaction with phosphatases. Upon Tor inactivation, Tap42 is dephosphorylated and consequently, phosphatases are released from its association.9 Interestingly, Tor inactivation is accompanied by a rapid activation of the phosphatases associated with Tap42. As such, the correlation between the release of the phosphatases from Tap42 and their activation advocates that Tap42 acts as a phosphatase inhibitor. According to this role, Tap42 is alleged to bind to and inhibit phosphatases upon phosphorylation by Tor. When Tor is inactive, Tap42 is dephosphorylated, leading to the release of the phosphatases and their activation.10,11

InhIbItor or ACtIvAtor?
One major fault with the above model is that Tap42 dephosphorylation occurs much more slowly than phosphatase activation, and hence can hardly be the cause for the Cell Cycle 2729

CE

.D

ON

KeY worDs

The microlide antibiotic rapamycin possesses potent growth inhibitory activity against virtually all types of eukaryotic cells. It does so by inhibiting Tor, a protein kinase that is highly conserved from yeast to humans.1 Tor controls a broad spectrum of cellular processes by integrating signals originating from changes in nutrient conditions and growth factor stimulation. Rapamycin, in complex with the immunophilin FKBP12, specifically binds to Tor and interferes with its function, thus inhibiting cell growth.2 Recent findings from yeast and mammalian cells have revealed that Tor elicits its pleiotropic effects via two distinct multi-protein complexes, termed Tor complex 1 (TORC1) and Tor complex 2 (TORC2), or mTORC1 and mTORC2 in mammalian cells. These findings raise numerous questions concerning how Tor elicits its pleiotropic effects in the context of the multi-protein complexes and how rapamycin interferes with its function.3,4

OT D

IST

RIB

UT E

The Association Between Tap42 and TORC1

activation. It has thus been suggested that other factors may be involved in the release of the phosphatases from Tap42. One of such factors is Tip41, which interacts with Tap42 and precludes its association with phosphatases.10 Similar to Tap42, Tip41 undergoes Tor-dependent phosphorylation; however phosphorylated-Tip41 is incapable of interacting with Tap42. Therefore, in order for Tip41 to interfere with the Tap42-phosphatase interaction, it has to be dephosphorylated first. Nevertheless, there is no evidence that Tip41 dephosphorylation precedes phosphatase activation, as it would be the case should Tip41 be the primary factor in regulating phosphatase activation. Additionally, since inactivation of Tip41 does not significantly affect cell growth, it is unlikely for this protein to play a vital role in regulating the Tap42-associated phosphatases. As such, how inactivation of TOR by rapamycin leads to phosphatase activation remains unsolved. However, a recent finding from Yan et al has provided an effective explanation. Yan et al established that the Tap42-phosphatase complexes exist only in association with TORC1.12 This finding reveals a further level of regulation to the Tor-signaling cascade, suggesting that rapamycin may interfere with Tor function by perturbing the association of the Tap42-phosphatase complexes with TORC1. Indeed, when cells are treated with rapamycin, the Tap42-phosphatase complexes are rapidly released from membrane-bound TORC1 into the cytosol. Interestingly, the Tap42-phosphatase complexes do not immediately fall apart upon release from TORC1. Instead, the Tap42-phosphatase complexes linger in the cytosol for ~30 minutes prior to disassembly. When the timing of the release and disassembly is compared with that of phosphatase activation, which occurs within minutes of the drug treatment, it is evident that the release of the Tap42-phosphatase complexes from TORC1, but not the disassembly of the complexes, correlates with the activation of the Tap42-associated phosphatases. This correlation indicates that rapamycin induces phosphatase activation simply by dislodging the Tap42-phosphatase complexes from TORC1. Interestingly, Tap42 is phosphorylated only when it is associated with TORC1, and dephosphorylation ensues after the Tap42-phosphatase complexes are released into cytosol. The rate of Tap42-dephosporylation mirrors that of the disassembly of the Tap42-phosphatase complexes, suggesting that the dephosphorylation of Tap42 causes its dissociation from phosphatases. Release of the Tap42-phosphatase complexes from TORC1 also occurs under physiological conditions. When cells are shifted from nitrogen-rich medium to nitrogen-starvation medium, the complexes are rapidly released from TORC1, similar to what has been demonstrated when cells are treated with rapamycin. This observation suggests that regulation of the association between Tap42-phosphatase complexes and TORC1 represents an important mechanism by which nutrient conditions control the signaling activity of Tor. In this regard, rapamycin acts as a surrogate of nitrogen starvation.

Figure 1. Model for rapamycininduced phosphatase activation (see text for detail).

A posItIve regulAtorY role reveAleD

Taken together, the findings from Yan et al support the model depicted in Figure 1. In actively growing cells, the Tap42-phosphatase complexes are associated with TORC1 residing on membrane structures. Nitrogen starvation or rapamycin treatment disrupts the association and releases the complexes into the cytosol, where the Tap42-associated phosphatases become active. Once released from TORC1, Tap42 is dephosphorylated by the PP2A holoenzyme in the cytosol. It is this dephosphorylation that results in the disassembly 2730

of the Tap42 complexes and termination of phosphatase activity12 (Fig. 1). A prediction from this model is that Tap42 is a positive regulator of the phosphatases to which it associates, a notion that challenges the previously ascribed negative role of Tap42 in phosphatase regulation. As mentioned above, the chief support for Tap42 being a negative regulator of phosphatases is the correlation between phosphatase activation and dissociation of the Tap42-phosphatase complexes, which upon closer inspection, does not exist. Conversely, substantial evidence has accumulated supporting Tap42 as a positive regulator of phosphatases. Wang and Jiang have previously shown that Tap42 is required for the activity of the phosphatases to which it associates.13 Similarly, Duvel et al have recently demonstrated that inactivation of Tap42 severely attenuates the rapamycin-induced expression levels of genes under control of the Gln3 transcription factor, a target for one of the Tap42-associated phosphatases.14 Furthermore, an earlier study has shown that overexpressing Tap42 in concert with phosphatases generates a growth inhibitory activity that is more pronounced than when phosphatases are expressed alone.8 These findings, together with the latest insight gained from Yan et al., demonstrate that Tap42 is a positive regulator of phosphatases. If Tap42 is a positive regulator for its associated phosphatases, then dissociation from Tap42 is expected to result in phosphatase inactivation. It is thus conceivable that the rapamycin-induced phosphatase activation is a transient process. In this scenario, the phosphatases are activated when the Tap42-phosphatase complexes are released from TORC1 and become inactive upon disassembly of the complexes. Direct evidence supporting this notion is absent at present. However, analysis of rapamycin-induced gene expression profiles did reveal a dynamic change in gene expression levels, whose increase and decrease mirror the release and disassembly of the Tap42 complexes.15 It would be of interest to determine whether there is a causal connection between these two events. Another important implication of the above positive regulator model for Tap42 is that the initial action of rapamycin is not Tor inhibition, but the release of the Tap42-phopshatase complexes and subsequent phosphatase activation. This mechanism clarifies why the drug is able to initiate a rapid and robust response in the cell. However, the initial effect of rapamycin is likely to be transient, which cannot account for the irreversible growth arrest induced by the drug. As such, the mode of action of rapamycin comprises two steps: (1) release of Tap42-phosphatase complexes and (2) inhibition of Tor. The effect of the first action is rapid yet short lasting, while that of the second is slow but irreversible. Since nitrogen starvation also causes a rapid release of Tap42-phosphatase complexes and eventual cell growth arrest, it is apparent that the same mechanisms are utilized inherently to deal with stressful growth conditions. Interestingly, release of the Tap42-phosphatase complexes occurs only when cells are under nitrogen starvation conditions. When 2006; Vol. 5 Issue 23

Cell Cycle

The Association Between Tap42 and TORC1

shifting cells from rich nitrogen- to poor nitrogen-containing medium, the Tap42-phosphatase complexes remain associated with TORC1 and the Tap42-associated phosphatases are not activated (Yan and Jiang, unpublished observation). This observation is contradictory to the notion that the Tor pathway responds to a downshift in nutrient conditions, raising the possibility that the Tap42-dependent branch of the Tor pathway is not involved in nitrogen discrimination. Consistent with this view, recent findings from Coopers group have suggested that the effects of nitrogen downshift are different from those induced by rapamycin treatment.16,17

whAts next for tor/tAp42?

The finding that the Tap42-phosphatase complexes are part of TORC1 begs two immediate questions. How do Tap42-phosphatase complexes associate with TORC1? And how does rapamycin trigger their release? The Tap42-phosphatase complexes may associate with TORC1 via Tap42 or phosphatases or both. Because mutations in Tap42 render the association resistant to rapamycin, it is likely that Tap42 mediates the link.12 However, Yan and Jiang have found that in the absence of Sit4 or PP2Ac, the two major Tap42-associated phosphatases, the association of Tap42 with TORC1 is significantly reduced (unpublished observation). This suggests that phosphatases may be required to stabilize the association. Tap42 may associate with TORC1 through direct interaction with Tor itself, in which case, rapamycin may simply dislodge Tap42 by competing with it for Tor binding. Alternatively, Tap42 may associate with TORC1 through an adaptor protein. It has been demonstrated that TORC1 comprises two essential components, Lst8 and Kog1, in addition to Tor1 or Tor2.3 Tap42 differs from these two components in that the association of Tap42 with TORC1 is rapamycin sensitive, while the association of both Lst8 and Kog1 with Tor is not. Hence, it is unlikely that these two components serve as adapters to bridge the association of Tap42 with Tor. One possibility is that Tap42 interacts with Tor through an unidentified adaptor protein, whose association with Tor is rapamycin sensitive. In support of this, a counterpart of this putative protein has been identified in mammalian cells. The adaptor protein raptor, whose interaction with mTOR is sensitive to rapamycin, acts in targeting mTOR to downstream substrates.18-20 It remains to be determined whether the yeast TORC1 contains additional components that act in a way similar to raptor. While rapamycin may act by simply dislodging the Tap42-phosphatase complexes from TORC1, the mechanism by which nitrogen starvation brings about the release of the complexes is hard to fathom. Tor may sense, directly or indirectly, nitrogen availability and adopts a new conformation that leads to the release of the Tap42 complexes. Alternatively, an inherent inhibitor of Tor may interfere with the association in a way similar to that of the rapamycin-FKBP12 complex. Clearly, a critical step in resolving these questions lies in understanding the molecular basis that mediates the association of the Tap42 complexes with TORC1. The finding that the Tap42-phosphatase complexes are associated with TORC1 also raises several other questions. How are Tap42 and phosphatases recruited to TORC1? Is Tap42-phosphatase association with TORC1 required for TORC1 function under normal conditions, or might the complexes be hitchhikers that come into play only under emergence conditions? Several proteins have been shown to interact with Tap42, including Tip41, Rrd1 and Rrd2. What might be their roles in recruiting, associating and/or releasing Tap42-phosphatase complexes? Is all TORC1 occupied by www.landesbioscience.com

the Tap42-phopshatase complexes under normal growth conditions? If not, what other factors might associate with TORC1 in lieu of the Tap42-phosphatase complexes? Resolving these issues would allow us to decipher how Tor exerts its function in the context of TORC1. The counterparts of Tap42 and TORC1 have been identified in mammalian cells.3,21 Akin to S. cerevisiae Tap42, the mammalian Tap42 (mTap42, also known as the a-4 protein) is associated with PP2Ac and 2A-like phosphatases.22-24 Nevertheless, how mTOR regulates mTap42, as well as its interaction with phosphatases, is not clear. Interestingly, both amino acid starvation and rapamycin treatment have been suggested to induce phosphatase activation.25,26 It would be appealing to see if the mechanism found in yeast also exists in mammalian cells.
References
1. Abraham RT. Mammalian target of rapamycin: Immunosuppressive drugs uncover a novel pathway of cytokine receptor signaling. Curr Opin Immunol 1998; 10:330-6. 2. Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell 2000; 103:253-62. 3. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D, Oppliger W, Jenoe P, Hall MN. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 2002; 10:457-68. 4. Reinke A, Anderson S, McCaffery JM, Yates IIIrd J, Aronova S, Chu S, Fairclough S, Iverson C, Wedaman KP, Powers T. TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem 2004; 279:14752-62. 5. Powers T, Dilova I, Chen CY, Wedaman K. Yeast TOR signaling: A mechanism for metabolic regulation. Curr Top Microbiol Immunol 2004; 279:39-51. 6. Crespo JL, Hall MN. Elucidating TOR signaling and rapamycin action: Lessons from Saccharomyces cerevisiae. Microbiol Mol Biol Rev 2002; 66:579-91, (table of contents). 7. Duvel K, Broach JR. The role of phosphatases in TOR signaling in yeast. Curr Top Microbiol Immunol 2004; 279:19-38. 8. Di Como CJ, Arndt KT. Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev 1996; 10:1904-16. 9. Jiang Y, Broach JR. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. Embo J 1999; 18:2782-92. 10. Jacinto E, Guo B, Arndt KT, Schmelzle T, Hall MN. TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol Cell 2001; 8:1017-26. 11. Schmidt A, Beck T, Koller A, Kunz J, Hall MN. The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. Embo J 1998; 17:6924-31. 12. Yan G, Shen X, Jiang Y. Rapamycin activates Tap42-associated phosphatases by abrogating their association with Tor complex 1. Embo J 2006; 25:3546-55. 13. Wang H, Jiang Y. The Tap42-protein phosphatase type 2A catalytic subunit complex is required for cell cycle-dependent distribution of actin in yeast. Mol Cell Biol 2003; 23:3116-25. 14. Duvel K, Santhanam A, Garrett S, Schneper L, Broach JR. Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. Mol Cell 2003; 11:1467-78. 15. Hardwick JS, Kuruvilla FG, Tong JK, Shamji AF, Schreiber SL. Rapamycin-modulated transcription defines the subset of nutrient- sensitive signaling pathways directly controlled by the Tor proteins. Proc Natl Acad Sci USA 1999; 96:14866-70. 16. Cox KH, Kulkarni A, Tate JJ, Cooper TG. Gln3 phosphorylation and intracellular localization in nutrient limitation and starvation differ from those generated by rapamycin inhibition of Tor1/2 in Saccharomyces cerevisiae. J Biol Chem 2004; 279:10270-8. 17. Tate JJ, Rai R, Cooper TG. Methionine sulfoximine treatment and carbon starvation elicit Snf1-independent phosphorylation of the transcription activator Gln3 in Saccharomyces cerevisiae. J Biol Chem 2005; 280:27195-204. 18. Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga C, Avruch J, Yonezawa K. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002; 110:177-89. 19. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002; 110:163-75. 20. Nojima H, Tokunaga C, Eguchi S, Oshiro N, Hidayat S, Yoshino K, Hara K, Tanaka N, Avruch J, Yonezawa K. The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem 2003; 278:15461-4. 21. Murata K, Wu J, Brautigan DL. B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci USA 1997; 94:10624-9. 22. Kunz J, Schneider U, Howald I, Schmidt A, Hall MN. HEAT repeats mediate plasma membrane localization of Tor2p in yeast. J Biol Chem 2000; 275:37011-20. 23. Nanahoshi M, Nishiuma T, Tsujishita Y, Hara K, Inui S, Sakaguchi N, Yonezawa K. Regulation of protein phosphatase 2A catalytic activity by alpha4 protein and its yeast homolog Tap42. Biochem Biophys Res Commun 1998; 251:520-6.

Cell Cycle

2731

The Association Between Tap42 and TORC1 24. Prickett TD, Brautigan DL. Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits. J Biol Chem 2004; 279:38912-20. 25. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 1998; 273:14484-94. 26. Peterson RT, Desai BN, Hardwick JS, Schreiber SL. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci USA 1999; 96:4438-42.

2732

Cell Cycle

2006; Vol. 5 Issue 23