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Common sense signallingRichard K. Assoian

Extracellular stimuli transmit information to cells by activating receptor-dependent signal-transduction cas-cades, most typically involving tyrosine and serine/threonine kinases. The cell’s response to the activationof these kinases depends on the duration of their signals. New work now shows how cells sense the dura-tion of an ERK signal in the G1 phase of the cell cycle. The study suggests that a common mechanism forsensing ERK signal duration may be involved in diverse cellular functions.

Cellular functions as diverse as prolif-eration, differentiation and morpho-genesis are regulated by changes in

the extracellular environment and the ensu-ing receptor-mediated stimulation or inhi-bition of signal-transduction cascades.There has been an intense interest in under-standing how activation of these signallingcascades is translated into particular cellu-lar responses, and results obtained with theERK subfamily of MAP kinases provide agood example of the issues that have fasci-nated so many of us. ERK1 and ERK2 areactivated within minutes in response to alarge number of extracellular stimuli, andtheir activation typically induces a commonset of immediate-early genes (IEGs). Butthis common response to the initial ERKsignal does not always translate into thesame cellular outcome; ERK activation canlead to proliferation, cell-cycle arrest or dif-ferentiation. How do cells interpret the ERKsignal to generate the appropriate response?One important parameter seems to be theduration of the signal (reviewed in refs 1,2).In this issue of Nature Cell Biology, Murphyet al.3 use the induction of c-Fos (a proto-typic IEG) as a read-out to describe a mech-anism that senses the duration of an ERKsignal in the G1 phase of fibroblasts.

There are three central features of themodel proposed by Murphy et al.3. First, theinitial response to ERK activation (whethertransient or sustained) involves inductionof IEGs such as c-Fos (Fig. 1, step 1). TheBlenis group and others previously showedthat ERK and its downstream effector, RSK,phosphorylate the carboxy-terminal regionof c-Fos at its Ser 374 and Ser 362 residues,respectively, to stabilize it4,5. Results inMurphy et al.3 now show that productionand/or nuclear localization of c-Fos canalso be viewed as a regulatory step becausethe time required for translation and trans-port to the nucleus is more than the timeassociated with transient ERK activity. Soonly a sustained ERK signal will result in C-terminal phosphorylation and stabilizationof c-Fos (Fig. 1, step 2).

Murphy et al.3 then proceed to show thatthe C-terminal phosphorylations of c-Fosdo more than just stabilize the protein. Infact, they prime the protein for additionalphosphorylation events by exposing a novel

DEF domain (an FXYP-like sequence)6 thatacts as an ERK-binding site. Although theauthors did not address whether inactiveERK would bind to the DEF domain, activeERK interacts with the DEF domain, at leastin vitro6. And the authors show that target-ing active ERK to the DEF domain allowsfor additional ERK-dependent phosphory-lation of c-Fos at its Thr 325 and Thr 331residues (Fig. 1, step 3). So c-Fos acts as asensor for sustained ERK activity becausethe stabilizing phosphorylations, therecruitment of ERK to the DEF domain,and the DEF-domain-dependent phospho-rylations all require sustained ERK activity.

Mutations in the DEF-dependent ERKsites (Thr 325 and/or Thr 331) and the DEFdomain itself were made to assess whatthese c-Fos regions do; the effects of thesemutations were assessed by measuring acti-vation of a c-fos-dependent promoter (AP-1) and the induction of anchorage-inde-pendent growth as read-outs. Surprisingly,mutating the DEF domain inhibited c-Fosfunction to a greater degree than mutatingthe Thr 325/Thr 331 residues. The muta-tions were made in a wild-type c-Fos back-

ground and expressed ectopically, so thestabilizing C-terminal phosphorylationswere not variables; these c-Fos mutantswere expressed at similar levels. It seems,therefore, that recruitment of active ERK tothe DEF domain must have effects that gobeyond c-Fos phosphorylation at Thr325/Thr 331. The main purpose of phos-phorylating the two serine residues andconsequent protein stabilization might beto expose the DEF domain and allow ERKto dock, rather than to increase the half-lifeof the c-Fos protein per se.

Sustained ERK signalling is associatedwith progression through the G1 phase ofthe cell cycle in fibroblasts7, so an obviousquestion is whether the effects reported byMurphy et al.3 are connected to activationof the cyclin-dependent kinases (cdks), theenzymes that mediate progression throughthe cell cycle. Although this is a big ques-tion, sustained ERK activity has been linkedto the mid-G1-phase induction of cyclinD1 (refs 8–10), which activates cdk4 andcdk6. The human cyclin D1 promoter con-tains an AP-1 site11,12 and, because AP-1sites are occupied by Fos–Jun complexes, it

(AAA)n

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and other functions

Figure 1 A working model of how c-Fos functions as a sensor for sustained ERK activity.Although both transient and sustained ERK signals stimulate c-Fos gene expression(step 1), the transient signal decays before c-Fos can be produced and translocated tothe nucleus. Sustained ERK activity allows c-Fos’s Ser 374 and Ser 362 residues to bephosphorylated in ERK- and RSK-dependent manners, respectively (step 2). In additionto stabilizing the protein, these phosphorylations allow ERK to interact with the DEFdomain. Recruited ERK then phosphorylates c-Fos at Thr 325 and Thr 331, amino-termi-nal of the DEF domain (step 3). These DEF-domain-directed phosphorylations, as well asother uncharacterized ERK/DEF-domain-associated effects, regulate c-Fos function.

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NATURE CELL BIOLOGY VOL 4 AUGUST 2002 http://cellbio.nature.comE188

is tempting to speculate that a stabilizedand DEF-domain-activated c-Fos couldmore effectively participate in the mid-G1-phase induction of cyclin D1. Only onestudy has looked directly at this question,but it concluded that c-Fos is probably notinvolved because its half-life (presumablyeven after stabilization by sustained ERKactivity) is too short8. On the surface, thisconclusion would seem to detract from thesignificance of the story proposed byMurphy et al.3, but the authors have per-formed an insightful in silico analysis thataddresses this discrepancy and extends thepotential significance of their findings dra-matically. In particular, they identified puta-tive DEF domains in a number of IEGsincluding the Fos gene family (Fra-1 andFra-2) and the Jun gene family (JunB andJunD). And the same study that concluded c-Fos is not involved in cyclin D1 gene induc-tion proposes that Fra-1 and Fra-2 areinvolved (ref. 8). So whether c-Fos itself oranother Fos family member is playing themain role, the ability of sustained ERK activ-ity to stabilize these proteins and confer newDEF-domain-dependent functions may welllie at the heart of cyclin D1 gene control.

The potential ramifications of Murphyet al.3 do not stop here. One of the intrigu-ing aspects of ERK signalling is that thesustained ERK signal associated with cellproliferation in fibroblasts has also beenassociated with neuronal differentiation inPC12 cells. Last year, Harada et al.13, whostudied ERK signalling in PC12 cells,

reported that sustained ERK activity wasnecessary and sufficient to induce p35, aneuron-specific activator of cdk5.Cdk5/p35 activity is required for neuronaldifferentiation. Sustained ERK signallingled to a relatively stable induction of Egr-1(another IEG) which, in turn, induced p35.Although Harada et al.13 showed the con-nection between sustained ERK activityand Egr-1, they did not provide a mecha-nism by which sustained ERK activity regu-lated Egr-1. It was particularly satisfyingtherefore to see that Egr-1 scored positivewhen Murphy et al.3 searched for DEF-domain-containing IEGs. In addition, theidentification of a putative DEF domain inmPer-1 (the mammalian homologue of theDrosophila clock gene, period) provides newinsight into the possible connectionsbetween sustained ERK activity and circa-dian rhythms. Murphy et al.3 put forth theprovocative hypothesis that a commonmechanism for sensing ERK signal dura-tion could be involved in very different cel-lular functions.

Finally, a word of caution for thoseintrigued enough to begin thinking aboutthe broader issues outlined here. There isno standard definition of transient and sus-tained ERK signalling, and the times thathave been associated with these descrip-tions can vary widely. For example, in PC12cells, the sustained signal is typically 1–2hours and the transient signal is just a fewminutes13. In fibroblasts, the sustained sig-nal (if defined as the induction of cyclin

D1) is typically 5–6 hours and the transientsignal can persist for more than an hour9.Do these differences reflect mere details ofexperimental conditions and cell lines, oremphasize that we need a deeper under-standing of the biology? The answer to thisquestion will probably come from elucidat-ing the molecular events specifically associ-ated with transient and sustained ERKactivity. Blenis and colleagues3 have provid-ed just the kind of mechanistic insight thatthis field needs as we strive to understandthe logic behind cellular decisions.Department of Pharmacology, University of

Pennsylvania School of Medicine, Philadelphia, PA

19104-6084, USA

e-mail: rka@pharm.med.upenn.edu

1. Marshall, C. J. Cell 80, 179–185 (1995).

2. Roovers, K. & Assoian, R. K. Bioessays 22, 818–826. (2000).

3. Murphy, L. O., Smith, S., Chen, R.-H., Fingar, D. C. Blenis, J.

Nature Cell Biol. 4, 556–564 (2002).

4. Chen, R. H., Juo, P. C., Curran, T. & Blenis, J. Oncogene 12,

1493–1502 (1996).

5. Okazaki, K. & Sagata, N. EMBO J. 14, 5048–5059 (1995).

6. Jacobs, D., Glossip, D., Xing, H., Muslin, A. J. & Kornfeld, K.

Genes Dev. 13, 163–175 (1999).

7. Meloche, S., Seuwen, K., Pages, G. & Pouyssegur, J. Mol

Endocrinol 6, 845–854 (1992).

8. Balmanno, K. & Cook, S. J. Oncogene 18, 3085–3097 (1999).

9. Roovers, K., Davey, G., Zhu, X., Bottazzi, M. E. & Assoian, R. K.

Mol. Biol. Cell 10, 3197–3204 (1999).

10. Weber, J. D., Raben, D. M., Phillips, P. J. & Baldassare, J. J.

Biochem. J. 326, 61–68 (1997).

11. Herber, B., Truss, M., Beato, M. & Muller, R. Oncogene 9,

2105–2107 (1994).

12. Albanese, C. et al. J. Biol. Chem. 270, 23589–23597 (1995).

13. Harada, T., Morooka, T., Ogawa, S. & Nishida, E. Nature Cell

Biol. 3, 453–459 (2001).

GEF without a Dbl domain?Vania M. M. Braga

Guanine nucleotide-exchange factors (GEFs) interact with and activate members of the Rho family of smallGTPases through Dbl homology (DH) domains. A new domain has been found in DOCK180 and shown tospecifically activate Rho proteins. Interestingly, this module does not contain any homology to Dbl sequences.

The Rho family of small GTPases (Rho,Rac and Cdc42) is important in a vari-ety of different cellular processes.

Activation of Rho small GTPases isachieved by interaction with GTP, a processfacilitated by regulatory proteins known asGEFs. These exchange factors are character-ized by the presence of a Dbl domain (DH,identified by its homology with the Dbloncogene) in tandem with a pleckstrinhomology (PH) domain, which interactswith lipids and proteins. Until recently, itwas believed that the DH domain is essen-tial for binding and activation of Rho pro-teins and that every GEF contained thecombination of DH and PH modules. Now

this consensus view has been challenged bythe report of Brugnera et al.1 in this issue ofNature Cell Biology. The authors identify anew domain in DOCK180 called Dockerthat is both necessary and sufficient for Racactivation and is nothing like DH domains.

Over the past few years, there has beengreat deal of interest in understanding howRho small GTPases are activated. So farmore than 50 GEFs have been isolated, andthey all contain various combinations ofprotein–protein interaction domains thatinclude at least one DH and PH domain2.The Dbl domain is responsible for bindingto Rho proteins and facilitating theexchange reaction, in which GDP is

replaced by GTP. It is thought that GEFs notonly facilitate the loading of GTP, but alsoprovide spatial information by means of thePH-domain interactions. GEFs may, there-fore, contribute to the specificity of the sig-nalling pathways activated downstream ofthe small GTPases by restricting the activa-tion to a particular intracellular site.

DOCK180 was originally identified byits ability to interact with Crk, an adaptorprotein3. Crk is involved in signallingprocesses activated by tyrosine-kinasereceptors and cell adhesion to the extracel-lular matrix4. DOCK180–Crk complexes arefound at focal-adhesion sites and ruffles,and are also implicated in the regulation of

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