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Bub1 phosphorylates Cdc20, which is a key reg-ulatory protein of APC/C, and found that it did.By contrast, a kinase-deficient form of Bub1failed to phosphorylate Cdc20. When theauthors next tested the effects of Bub1-medi-ated phosphorylation of Cdc20 on the ubiqui-tylation activity of APC/CCdc20, they found thatsubstoichiometric amounts of Bub1 were suffi-cient to inhibit APC/CCdc20. Also, the kinase-defective Bub1 mutant failed to inhibitAPC/CCdc20. Together, these observations indi-cate that Bub1 catalytically inhibits APC/CCdc20

by phosphorylating Cdc20.Using mass spectrometry, the Yu team

mapped the in vivo phosphorylation sites ofendogenous Cdc20, which had been purifiedfrom nocodazole-treated cells. They identifiedsix sites, all of which were also phosphorylatedby Bub1 in vitro. That Bub1 phosphorylatesCdc20 in vivo was confirmed by checking thephosphorylation status of Cdc20 in Bub1-depleted cells — the slower-migrating, phos-phorylated forms of Cdc20 that were present inwild-type, nocodazole-treated cells were absentin Bub1-depleted cells.

Next, the authors prepared a Cdc20 mutant(Cdc20BPM) in which all six phosphorylationsites had been mutated. Bub1 failed to inhibitthe ubiquitylation activity of APC/CCdc20BPM,which shows that Cdc20 — rather than APC/C

The mechanisms that underlie biologicalprocesses often turn out to be more complexthan was initially thought. However, a recentstudy of the spindle checkpoint, which wasreported by Hongtao Yu and colleagues inMolecular Cell, hints at a regulatory mechanismof striking simplicity.

The spindle checkpoint ensures the accurateseparation of chromosomes by blocking theubiquitin-ligase activity of the anaphase-pro-moting complex/cyclosome (APC/C) inresponse to improper chromosome alignmentsand inappropriate tension of the microtubularspindle. Many proteins are involved in control-ling this checkpoint, so its regulation is com-plex. In particular, the Bub1 kinase interactswith and regulates several other checkpointproteins and was expected to be an upstreamcomponent of the checkpoint. But, Yu and col-leagues now show that, surprisingly, Bub1inhibits the activity of APC/C directly.

The authors first showed that a significantfraction of HeLa cells that had been depleted forBub1 using RNA interference (RNAi) failed toundergo mitotic arrest after treatment withnocodazole (a spindle-damaging agent). Thisindicates that Bub1 is indeed required for thespindle checkpoint.

Because Bub1 contains a protein-kinasedomain, Yu and co-workers tested whether

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If you tread on someone’s toe, the response isusually quite vocal. But, beyond the yelps,what might be happening when force isapplied at the cellular level? Tamada, Sheetzand Sawada investigated how forces on theextracellular matrix (ECM) are transducedinto intracellular biochemical signals, using

cells that have been stripped of their cellmembrane and soluble proteins. Stretchingthe resultant ‘Triton cytoskeletons’ initiatedsignalling to the small GTPase Rap1, asoutlined in Developmental Cell.

When cells were extracted using Triton X-100, the remaining complex containedmainly cytoskeletal and adhesion proteins, andonly a few membrane lipids or cytoplasmicproteins. Nevertheless, the authors found that,when these Triton cytoskeletons werestretched, they could activate Rap1 that waspresent in added cytoplasmic extracts. Suchstretch-mediated Rap1 activation is known tooccur in intact cells.

On investigating potential upstreamcandidates, the authors found that twocomponents of an added soluble cytoplasmicextract, the Rap1 guanine nucleotide-exchange factor C3G and the adaptor proteinCrkII — which are both involved inactivating Rap1 — bound to the Tritoncytoskeletons in a stretch-dependent manner.Activation of Rap1 was prevented if C3G wasdepleted from the extracts, so the Rap1response to Triton-cytoskeleton stretchingdepended on C3G.

In response to various stimuli, CrkII–C3Gbinds to tyrosine-phosphorylated proteinsthrough the Src-homology-2 (SH2) domain

of CrkII. And, accordingly, Tamada, Sheetzand Sawada noticed an increase inphosphotyrosine levels in several proteincomponents of Triton cytoskeletons inresponse to stretch. The prime suspects forthis phosphorylation were Src-family kinases(SFKs), and a selective SFK inhibitorprevented this stretch-inducedphosphorylation response and the ability offluorescently tagged CrkII to bind tostretched Triton cytoskeletons. A candidatesubstrate for such phosphorylation was Cas(Crk-associated substrate), a significantamount of which remained on Tritoncytoskeletons. The authors’ assumptionsproved correct — tyrosine phosphorylationof Cas in Triton cytoskeletons increased inresponse to stretch. And CrkII no longerbound to Triton cytoskeletons of Cas–/– cells,which indicates that CrkII binds directly totyrosine-phosphorylated Cas.

Does this affect all parts of the cell in thesame way? The greatest stresses are expectedto occur at the regions of cell–ECM contact.At these points, Tamada, Sheetz and Sawadaobserved an increase in tyrosinephosphorylation in Triton cytoskeletons inresponse to externally applied force — nocytosolic molecules were required. A similarlocalized increase in tyrosine

Simply effective

C E L L C Y C L E

The stretch effect

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | JANUARY 2005 | 7

Ubiquitylation is a reversible post-translationalmodification that has important roles inprocesses such as protein degradation andintracellular signalling. Yet, despite a growinginterest in ubiquitylation, a tool for studying thedynamics of this process has been lacking. Nowthough, in Nature Methods, Bouvier and col-leagues describe a technique that can be used todetect in situ changes in protein ubiquitylation.

Their work revolved around biolumines-cence resonance energy transfer (BRET), whichallows protein–protein interactions to bedetected in real time in vivo, and β-arrestin — aprotein that is ubiquitylated in response toG-protein-coupled receptor (GPCR) activation.

They made a Renilla-luciferase–β-arrestinconstruct (Rluc–β-arrestin) and a green-fluo-rescent-protein–ubiquitin construct (GFP–Ub;mutant ubiquitin was used to prevent polyubiq-uitin-chain formation), and added a specificRluc substrate (substrate-1) to cells co-express-ing these constructs. Substrate-1 hydrolysis byRluc emits light that overlaps with the excitationspectrum of GFP. So, if GFP–Ub is covalentlyconjugated to Rluc–β-arrestin, BRET occursand a fluorescent signal that originates fromGFP should be detected.

Indeed, a BRET signal was detected, and thesignal increased with increasing concentrationsof GFP–Ub until it reached a plateau (weaker,linear signals were obtained using GFP alone ora GFP–Ub construct that could not be conju-gated to Rluc–β-arrestin). Furthermore, no sig-nal was obtained when GFP–Ub and Rluc wereco-expressed. These data therefore confirm thatthe signal reflects the covalent attachment ofGFP–Ub to β-arrestin in Rluc–β-arrestin.

Next, the authors showed that this techniquecan be used to study receptor-regulated ubiqui-tylation by monitoring energy transfer betweenGFP–Ub and Rluc–β-arrestin in the presence ofGPCRs. When they activated the receptors

using selective agonists, the BRET signalincreased in a dose-dependent manner.

It has been proposed that GPCR activationmight regulate β-arrestin ubiquitylation and itsrecruitment to the GPCR. By using two differ-ent substrates for Rluc and a GPCR–yellow-flu-orescent-protein construct (V

2R–YFP), Bouvier

and colleagues were able to monitor these twoevents simultaneously. They split a cell cultureexpressing GFP–Ub, Rluc–β-arrestin andV

2R–YFP into two samples, and added sub-

strate-1 to one sample (substrate-1 hydrolysisproduces light that excites GFP–Ub) and sub-state-2 to the other (substrate-2 hydrolysis emitslight that excites V

2R–YFP). They found that the

activation of V2R–YFP produced BRET signals

indicative of the concomitant ubiquitylationand recruitment of β-arrestin.

Finally, Bouvier and co-workers followed thereal-time kinetics of agonist-promoted β-arrestinubiquitylation in cells co-expressing GPCRs thatinteract with β-arrestin transiently (class A) orstably (class B). They found that class-B-GPCRactivation resulted in a more stable ubiquityla-tion of β-arrestin than class-A-GPCR activation.The nature of the interaction between the recep-tor and β-arrestin therefore effects the dynamicsof β-arrestin ubiquitylation.

So, using β-arrestin as a model, theseauthors have shown that BRET can specificallydetect basal and regulated ubiquitylationprocesses in living cells. Their assay will there-fore be useful “…for studying the dynamicubiquitination of proteins and for understand-ing which cellular functions are regulated bythis post-translational event.”

Rachel SmallridgeReferences and links

ORIGINAL RESEARCH PAPER Perroy, J. et al. Real-timemonitoring of ubiquitination in living cells by BRET. Nature Meth.1, 203–208 (2004)WEB SITEMichel Bouvier’s laboratory:http://www.mapageweb.umontreal.ca/bouvier/

itself — is required for the inhibition ofAPC/CCdc20.

Yu and colleagues isolated Bub1 fromnocodazole-treated (metaphase-arrested) cellsand from thymidine-treated (G1/S-arrested)cells. The kinase activity of Bub1 that waspurified from the former cells was muchhigher than from the latter, which shows thatthe kinase activity is specifically activated afterspindle-checkpoint activation. Overexpressionof the non-phosphorylatable Cdc20BPM

mutant caused 40–50% of cells to arrest inmitosis, compared with >90% in the case ofwild-type protein. So, elimination of the Bub1phosphorylation of Cdc20 causes a substan-tial, yet partial, spindle-checkpoint defect.This is consistent with the existence of otherAPC/C-inhibitory mechanisms.

The spindle checkpoint is extremely sensi-tive, and Yu and colleagues propose that the cat-alytic mechanism for Bub1-mediated APC/Cinhibition “…might be partially responsible forthis remarkable sensitivity…”

Arianne HeinrichsReferences and links

ORIGINAL RESEARCH PAPER Tang, Z. et al. Phosphorylationof Cdc20 by Bub1 provides a catalytic mechanism for APC/Cinhibition by the spindle checkpoint. Mol. Cell 16, 387–397(2004) FURTHER READING Margolis, R. L. Bub1, a gatekeeper for Cdc20-dependent mitotic exit. Dev. Cell 7, 634–635 (2004)

phosphorylation was seen when intact,non-Triton-treated cells were stretched.And fluorescently tagged CrkII was seen tomove to adhesion sites and to colocalizewith Cas in response to stretching in intactcells.

So stretching Triton cytoskeletons andintact cells induces CrkII–C3G–Rap1signalling. How the signalling is initiated isunknown, but it’s likely that proteins areunfolded or distorted when force is applied,which could create new binding sites forother proteins. As Rap1 is known to induceintegrin-mediated adhesion, its activationat cell–ECM sites could well stabilize suchcontacts. Recent insights into the activationof integrins by Rap1 come from the studiesof Lafuente et al., who cloned andcharacterized RIAM, a Rap1–GTP-interacting adaptor molecule. Whenoverexpressed, RIAM induced β

1- and β

2-

integrin-mediated adhesion and influencedactin dynamics.

Katrin BussellReferences and links

ORIGINAL RESEARCH PAPER Tamada, M., Sheetz, M.& Sawada, Y. Activation of a signaling cascade bycytoskeleton stretch. Dev. Cell 7, 709–718 (2004)FURTHER READING Lafuente, E. M. et al. RIAM, anEna/VASP and profilin ligand, interacts with Rap1–GTPand mediates Rap1-induced adhesion. Dev. Cell 7,585–595 (2004)

Monitoring modification

T E C H N I Q U E

Plasma membrane

Substrate-1

BRETBRET

GPCR

β-arrestin

β-arrestin

Substrate-2

Ub

GFP Ub

GFP

YFPRluc

Rluc