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

Perspective

The Association of Tap42 Phosphatase Complexes with TORC1Another Level of Regulation in Tor Signaling

Ca J. Di Cm1

Y Jia2,3

1Aureon Laboratories, Inc; Yonkers, New York USA

2Department 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: [email protected]

Original manuscript submitted: 10/09/06

Manuscript accepted: 10/11/06

Previously published online as a Cell CycleE-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3516

KeY worDs

Tap42, Rapamycin, Tor kinases, PP2A, Sit4

AbstrACt

In the budding yeast Saccharomyces cerevisiae, rapamycin has been known to inducea rapid dephosphorylation of many downstream targets of Tor. The key componentsmediating this dephosphorylation process are the Tap42associated phosphatases, whichbecome active upon rapamycin treatment. However, the mechanism by which rapamycinrapidly activates phosphatases is unclear. A recent report has provided evidence demonstrating a physical association of the Tap42phosphatase complexes with TORC1, whichis sensitive to rapamycin treatment or nutrient starvation. This association adds anothelevel of regulation in Tor signaling, and explains why rapamycin or nutrient availability isable to initiate a rapid and robust response in the cell.

IntroDuCtIon

The microlide antibiotic rapamycin possesses potent growth inhibitory activity againsvirtually all types of eukaryotic cells. It does so by inhibiting Tor, a protein kinase thatis highly conserved from yeast to humans.1 Tor controls a broad spectrum of cellularprocesses by integrating signals originating from changes in nutrient conditions andgrowth factor stimulation. Rapamycin, in complex with the immunophilin FKBP12specifically 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) andTor complex 2 (TORC2), or mTORC1 and mTORC2 in mammalian cells. These findingsraise numerous questions concerning how Tor elicits its pleiotropic effects in the context othe multi-protein complexes and how rapamycin interferes with its function.3,4

tAp42 AnD the phosphAtAses

In the budding yeast, Saccharomyces cerevisiae, the major event downstream of TORC1is the transcriptional regulation of genes involved in nutrient metabolism and ribosomalbiogenesis.5,6 TORC1 mediates this event by regulating a group of serine/threonineprotein phosphatases, including Sit4 and protein phosphatase 2A (PP2A).7 The activityof these phosphatases is normally repressed in growing cells where TORC1 is active, butbecomes activated upon TORC1 inactivation. The activated phosphatases in turn dephosphorylate several transcription factors, including Gln3, resulting in their activation. ThisTor-dependent regulation of the phosphatases is mediated through a phosphatase-associating protein termed Tap42, which associates with the PP2A and 2A-like phosphatases ina 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 InterestinglyTor inactivation is accompanied by a rapid activation of the phosphatases associated withTap42. As such, the correlation between the release of the phosphatases from Tap42 andtheir activation advocates that Tap42 acts as a phosphatase inhibitor. According to this roleTap42 is alleged to bind to and inhibit phosphatases upon phosphorylation by Tor. WhenTor is inactive, Tap42 is dephosphorylated, leading to the release of the phosphatases andtheir activation.10,11

InhIbItor or ACtIvAtor?

One major fault with the above model is that Tap42 dephosphorylation occurs muchmore slowly than phosphatase activation, and hence can hardly be the cause for the

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The Association Between Tap42 and TORC1

activation. It has thus been suggested that other factors may beinvolved in the release of the phosphatases from Tap42. One ofsuch factors is Tip41, which interacts with Tap42 and precludes itsassociation with phosphatases.10 Similar to Tap42, Tip41 undergoesTor-dependent phosphorylation; however phosphorylated-Tip41 isincapable of interacting with Tap42. Therefore, in order for Tip41to interfere with the Tap42-phosphatase interaction, it has to bedephosphorylated first. Nevertheless, there is no evidence that Tip41

dephosphorylation precedes phosphatase activation, as it would bethe case should Tip41 be the primary factor in regulating phospha-tase activation. Additionally, since inactivation of Tip41 does notsignificantly affect cell growth, it is unlikely for this protein to playa vital role in regulating the Tap42-associated phosphatases. As such,how inactivation of TOR by rapamycin leads to phosphatase activa-tion remains unsolved. However, a recent finding from Yan et al hasprovided an effective explanation.

Yan et al established that the Tap42-phosphatase complexes existonly in association with TORC1.12 This finding reveals a furtherlevel of regulation to the Tor-signaling cascade, suggesting thatrapamycin may interfere with Tor function by perturbing theassociation of the Tap42-phosphatase complexes with TORC1.Indeed, when cells are treated with rapamycin, the Tap42-phosphatasecomplexes are rapidly released from membrane-bound TORC1 intothe cytosol. Interestingly, the Tap42-phosphatase complexes donot immediately fall apart upon release from TORC1. Instead, theTap42-phosphatase complexes linger in the cytosol for ~30 minutesprior to disassembly. When the timing of the release and disas-sembly is compared with that of phosphatase activation, whichoccurs within minutes of the drug treatment, it is evident that therelease of the Tap42-phosphatase complexes from TORC1, but notthe disassembly of the complexes, correlates with the activation ofthe Tap42-associated phosphatases. This correlation indicates thatrapamycin induces phosphatase activation simply by dislodging theTap42-phosphatase complexes from TORC1.

Interestingly, Tap42 is phosphorylated only when it is asso-

ciated with TORC1, and dephosphorylation ensues after theTap42-phosphatase complexes are released into cytosol. The rateof Tap42-dephosporylation mirrors that of the disassembly of theTap42-phosphatase complexes, suggesting that the dephosphorylationof Tap42 causes its dissociation from phosphatases.

Release of the Tap42-phosphatase complexes from TORC1 alsooccurs under physiological conditions. When cells are shifted fromnitrogen-rich medium to nitrogen-starvation medium, the complexesare rapidly released from TORC1, similar to what has been demon-strated when cells are treated with rapamycin. This observationsuggests that regulation of the association between Tap42-phosphatasecomplexes 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.

A posItIve regulAtorY role reveAleD

Taken together, the findings from Yan et al support the modeldepicted in Figure 1. In actively growing cells, the Tap42-phosphatasecomplexes are associated with TORC1 residing on membranestructures. Nitrogen starvation or rapamycin treatment disrupts theassociation and releases the complexes into the cytosol, where theTap42-associated phosphatases become active. Once released fromTORC1, Tap42 is dephosphorylated by the PP2A holoenzyme in thecytosol. It is this dephosphorylation that results in the disassembly

of the Tap42 complexes and termination of phosphatase activity12

(Fig. 1).A prediction from this model is that Tap42 is a positive regulato

of the phosphatases to which it associates, a notion that challenges thepreviously ascribed negative role of Tap42 in phosphatase regulation

As mentioned above, the chief support for Tap42 being a negativeregulator of phosphatases is the correlation between phosphataseactivation and dissociation of the Tap42-phosphatase complexes

which upon closer inspection, does not exist. Conversely, substantiaevidence has accumulated supporting Tap42 as a positive regulator ophosphatases. Wang and Jiang have previously shown that Tap42 irequired for the activity of the phosphatases to which it associates.13

Similarly, Duvel et al have recently demonstrated that inactivation oTap42 severely attenuates the rapamycin-induced expression levelsof genes under control of the Gln3 transcription factor, a target forone of the Tap42-associated phosphatases.14 Furthermore, an earliestudy has shown that overexpressing Tap42 in concert with phosphatases generates a growth inhibitory activity that is more pronouncedthan when phosphatases are expressed alone.8 These findingstogether with the latest insight gained from Yan et al., demonstratethat Tap42 is a positive regulator of phosphatases.

If Tap42 is a positive regulator for its associated phosphatasesthen dissociation from Tap42 is expected to result in phosphataseinactivation. It is thus conceivable that the rapamycin-induced

phosphatase activation is a transient process. In this scenario, thephosphatases are activated when the Tap42-phosphatase complexesare released from TORC1 and become inactive upon disassemblyof the complexes. Direct evidence supporting this notion is absentat present. However, analysis of rapamycin-induced gene expressionprofiles did reveal a dynamic change in gene expression levels, whoseincrease and decrease mirror the release and disassembly of the Tap42complexes.15 It would be of interest to determine whether there is acausal connection between these two events.

Another important implication of the above positive regulatormodel for Tap42 is that the initial action of rapamycin is not Torinhibition, but the release of the Tap42-phopshatase complexes andsubsequent 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 transientwhich cannot account for the irreversible growth arrest induced bythe drug. As such, the mode of action of rapamycin comprises twosteps: (1) release of Tap42-phosphatase complexes and (2) inhibitionof Tor. The effect of the first action is rapid yet short lasting, whilethat of the second is slow but irreversible. Since nitrogen starvationalso causes a rapid release of Tap42-phosphatase complexes andeventual cell growth arrest, it is apparent that the same mechanismsare utilized inherently to deal with stressful growth conditions.

Interestingly, release of the Tap42-phosphatase complexes occursonly when cells are under nitrogen starvation conditions. When

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

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24. Prickett TD, Brautigan DL. Overlapping binding sites in protein phosphatase 2A forassociation 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 suf-ficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effectormechanism. J Biol Chem 1998; 273:14484-94.

26. Peterson RT, Desai BN, Hardwick JS, Schreiber SL. Protein phosphatase 2A interacts withthe 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated pro-tein. Proc Natl Acad Sci USA 1999; 96:4438-42.

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