Báo cáo khoa học: Control of transforming growth factor b signal transduction by small GTPases pot

All members of the TGFb superfamily signal via a ‘canonical’ pathway that involves a heterotetrameric complex of two type I and two type II Ser⁄ Thr kinase receptors on the plasma membra

Control of transforming growth factor b signal

transduction by small GTPases

Dimitris Kardassis1,2, Carol Murphy3, Theodore Fotsis3,4, Aristidis Moustakas5and

Christos Stournaras1

1 Department of Biochemistry, University of Crete Medical School, Heraklion, Greece

2 Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology-Hellas, Heraklion, Greece

3 Biomedical Research Institute, Foundation for Research & Technology-Hellas, Ioannina, Greece

4 Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece

5 Ludwig Institute for Cancer Research, Uppsala University, Sweden

Transforming growth factor b (TGFb) is the prototype

member of a large, evolutionarily conserved,

superfam-ily of pleiotropic cytokines that also includes activins,

bone morphogenetic proteins (BMPs) and growth and

differentiation factors, among others [1] TGFb

con-trols various physiological processes during

embryo-genesis and is an important homeostatic regulator in

various cell types, for example, epithelial and

endo-thelial cells in adult organisms [1–3] TGFb is a growth

suppressor because of its cytostatic program [4]

However, during the late stages of cancer and metasta-sis, TGFb acts as a tumor promoter because of its ability to enhance processes such as epithelial to mesenchymal transition (EMT), cell motility and inva-sion, immunosuppreinva-sion, angiogenesis and extracellu-lar matrix production [4–7]

All members of the TGFb superfamily signal via a

‘canonical’ pathway that involves a heterotetrameric complex of two type I and two type II Ser⁄ Thr kinase receptors on the plasma membrane and downstream

Keywords

actin cytoskeleton; activin; non-Smad

signaling; Rab ⁄ Ran ⁄ Ral; receptor

endocytosis; Rho; Smad signaling; small

GTPases; TGFb; trafficking

Correspondence

C Stournaras, Department of Biochemistry,

School of Medicine, University of Crete,

GR-71110 Heraklion, Greece

Fax: +30 2810 394530

Tel: +30 2810 394563

E-mail: cstourn@med.uoc.gr

(Received 6 February 2009, revised 11

March 2009, accepted 31 March 2009)

doi:10.1111/j.1742-4658.2009.07031.x

The integrated roles of small GTPases in executing the transforming growth factor b (TGFb) signaling pathway have attracted increasing atten-tion in recent years In this review, we summarize recent findings on TGFb signaling during receptor endocytosis, Smad trafficking and actin cytoskele-ton remodeling, and emphasize the role of small GTPases in these pro-cesses First, we give an overview of the different endocytic routes taken by TGFb receptors, their impact on active TGFb signaling versus degradation and their regulation by the small GTPases Rab, RalA⁄ Ral-binding protein

1 and Rap2 Second, we focus on the mechanisms and regulation of Smad trafficking in the cytoplasm, through the nuclear pores and into the nucleus, and the contribution of Ran GTPase to these events Third, we summarize the role of Rho small GTPases in early and late cytoskeleton remodeling in various cell models and diseases, and the positive and nega-tive cross-talk between Rho GTPases and the TGFb⁄ Smad pathway The biological significance of this exciting research field, the perspectives and critical open questions are discussed

Abbreviations

AP1, activating protein 1; ARIP2, activin receptor interacting protein 2; BMP, bone morphogenetic proteins; CCVMR, clathrin-coated vesicle-mediated route; CRM1, chromosome region maintenance 1; EH, Eps15 homology; EMT, epithelial to mesenchymal transition; Endofin, endosome-associated FYVE-domain protein; GAP, GTPase activating protein; GEF, guanine exchange factor; NES, nuclear export signal; NLS, nuclear localization signal; RalBP1, Ral-binding protein 1; ROCK, Rho coiled-coiled kinase; SARA, Smad anchor for receptor activation; TGFb, transforming growth factor b; TbRI, TGFb type I receptor; TbRII, TGFb type II receptor.

cytoplasmic effector proteins termed Smads [8,9].

TGFb promotes receptor oligomerization which leads

to the phosphorylation of its type I receptor (TbRI) by

the constitutively active type II receptor (TbRII)

Acti-vated TbRI (also called ALK5), phosphorylates Smad2

and Smad3 at their C-terminal SSXS motifs [8–10]

The R-Smads, in turn, oligomerize with the common

partner Smad4 and rapidly translocate to the nucleus

where they bind to the promoters of a large variety of

target genes and regulate their expression in a positive

or negative manner [8–10] TGFb target genes code for

proteins involved in cell-cycle regulation, apoptotic

regulation, extracellular matrix production, cytokine

signaling, transcriptional regulation, differentiation

control and autoinhibitory loops [4] The best

under-stood example of a negative feedback loop involves

Smad7, an inhibitory Smad, which blocks Smad

phos-phorylation by TbRI and directs receptor

ubiquitina-tion and degradaubiquitina-tion via the ubiquitin ligases Smurf1

and Smurf2, thus ensuring that the pathway is shut off

[9,11]

Proteins have been identified which recruit Smads to

the activated type I receptor for phosphorylation

Smad anchor for receptor activation (SARA) recruits

Smad2 into the vicinity of the receptor

Phosphoryla-tion of Smad2 increases its affinity for Smad4 and

decreases its affinity for SARA, promoting the

dissoci-ation of Smad2 from SARA, unmasking a nuclear

localization signal in Smad2 and allowing signaling to

occur [12,13] In the BMP pathway,

endosome-associ-ated FYVE-domain protein (Endofin) functions as a

Smad anchor for receptor activation [14,15]

Interest-ingly, both SARA and Endofin are

FYVE-domain-containing proteins [16] and localize predominantly to

the early endocytic compartment [17–20], thereby

underscoring the importance of this compartment in

the signaling cascades of both pathways Hrs, another

FYVE domain protein, also localizes to the early

end-ocytic compartment, binds to Smad2 via its C-terminal

domain and cooperates with SARA to stimulate

acti-vin receptor-mediated signaling via the efficient

recruit-ment of Smad2 to the receptor [21] It is therefore

evident that receptor endocytosis is an important early

step in TGFb signal transduction

To date, five emerging transport routes for proteins

that become internalized have been identified: the

clathrin-coated vesicle-mediated route (CCVMR),

macropinocytosis⁄ phagocytosis, the APPL route, the

caveolar route and the nonclathrin and noncaveolar

pathways [22] Therefore, it is clear that understanding

the endocytic route followed by TGFb receptor–ligand

complexes will allow a systems-level molecular

dissec-tion of the signaling regulators of TGFb

Since the discovery and molecular cloning of Smad proteins, it has been known that Smads rapidly accu-mulate in the cell nucleus upon activation of the TGFb receptors [23–26] The original studies gave a static view of the pathway, whereby Smads were thought to reside firmly in the cytoplasm and translocate rapidly into the nucleus upon activation via receptor-mediated phosphorylation Twelve years later, we appreciate that Smad proteins show a very dynamic behavior within the cell because they constantly shuttle in to and out

of the nucleus [27]

Furthermore, both TGFb receptor endocytosis and Smad trafficking seem to rely on interactions and cross-talk with the cytoskeleton, including micro-tubules and actin-based microfilaments [10] Such cross-talk facilitates the timely movement and accurate transport of signaling components to their various des-tinations In addition, TGFb signaling has a profound impact on the regulation of the actin cytoskeleton, which supports various physiological and developmen-tal processes such as cell motility, differentiation changes and tissue organization [10] The regulatory enzymes of the Ras family, namely Rab, Ran and Rho GTPases are pivotal components in the regulation of TGFb signaling during receptor endocytosis, Smad trafficking and cross-talk with the actin cytoskeleton, respectively [28] Here, we provide a detailed review

of the specific and integrated roles of small GTPases

in the control and execution of the TGFb signaling pathway

Interconnection between TGFb signaling and receptor trafficking-regulation by small GTPases

Endocytosis has long been considered a way of termi-nating signaling processes via receptor degradation This was challenged in recent years when activated epi-dermal growth factor receptors and their effectors were found in what was considered to be the endosomal compartment [29] It is now evident that endomem-brane structures serve as signaling platforms [30], and there are signaling endosomes or hermesomes which may be specialized for this process [31] The endomem-brane system is divided into functionally and composi-tionally specialized subdomains [32,33], which determine the strength and duration of signaling responses by controlling recruitment of the down-stream effectors of signaling complexes and sorting events such as recycling and transport to the lysosomal compartment for degradation The endocytic pathway itself is controlled by signaling, demonstrating the extent to which signaling and trafficking are

interlinked [34,35] Furthermore, transport from early

to late endocytic compartments is controlled by the

cargo, and activated receptors may alter the kinetics to

modulate their signaling duration [36]

Is internalization required for TGFb family

signaling?

The presence of SARA, Hrs and Endofin in early

end-ocytic compartments questions whether signaling can

occur from the plasma membrane or whether

internali-zation is required to bring activated receptors to the

endosome which is enriched in SARA and Endofin

This issue remains controversial, reflecting differences

in experimental approaches and their limitations

TbRII undergoes constitutive internalization in the

absence of ligand via clathrin-coated pits This process

is dependent on a short sequence (I218-I219-L220) that

conforms to the di-leucine family of internalization

sig-nals [37,38] and the direct binding of type I and II

receptors to b2-adaptin [39] No di-leucine motifs have

been found in type I receptors Interestingly, the

NANDOR box is well conserved throughout type I

receptors [40] and appears to play a role in type I

receptor endocytosis

Indeed, TbRI (ALK5) is internalized rapidly via

CCVMR [41,42] Ligand stimulation has no effect on

the initial internalization rate or receptor recycling

[42] Using a range of techniques including potassium

(K+) depletion, which inhibits clathrin-mediated

endo-cytosis [43], and a dominant-negative form of the

dyn-amin GTPase, K44A dyndyn-amin II, which inhibits both

clathrin- and caveolar-mediated endocytosis [44],

vari-ous groups have addressed the requirement for

inter-nalization in TGFb signaling Lu et al [41] found no

involvement, however, several other groups have

demonstrated the need for internalization [45,46]

Further studies showed that TGFb receptors localize

to both raft and nonraft membrane domains and the

internalization route dictates whether signaling or

deg-radation will ensue [11,47] Internalization of TGFb

receptors, via the CCVMR, into an EEA1- and

SARA-positive endosome promoted signaling

How-ever, internalization via the raft–caveolar pathway,

where Smad7 and Smurf2 are localized, promoted

ubiquitin-dependent receptor degradation and

inhibi-tion of this pathway led to receptor stabilizainhibi-tion,

suggesting that trafficking of receptors to the

SARA-positive early endosome functions to sequester

recep-tors from the rafts and caveolae, thereby stabilizing

the receptors [11]

In support of the above model, hyaluronan, an

extracellular matrix polysaccharide, attenuated TGFb

signaling by increasing the segregation of TGFb recep-tors into a lipid raft–caveolar compartment [48], whereas ADAM12 (a disintegrin and metalloprotein-ase) facilitated signaling by inducing the accumulation

of TbRII in early endosomal vesicles and counteract-ing the internalization of TbRII into a caveolin1-posi-tive compartment [49] Likewise, interleukin-6 augmented TGFb signaling by increasing partitioning

of TGFb receptors to the nonlipid raft fraction (early endosomal) [50] No significant caveolar internalization was observed in the study by Mitchell et al [42], in which nystatin (used at lower, more specific doses) had

no effect on receptor internalization and degradation Moreover, TGFb receptors did not exhibit consi-derable co-localization in compartments positive for caveolin-1 [42]

What about the role of endocytosis in the signaling

of other members of the TGFb receptor family? With regard to activin A signaling, an ALK4 mutant, Alk4W477A, that was unable to undergo activin-dependent internalization, retained the ability to signal, demonstrating that ALK4 can signal without receptor internalization [51] However, in another detailed study addressing the memory of Xenopus embryonic cells to activin A exposure, the critical step in determining the duration of activin A signaling was the time spent by the ligand⁄ receptor complexes in the endo-lysosomal pathway Activin A internalization was required for correct signaling, suggesting that the localization of ligand to the endosomes was also required for a signaling step upstream of Smad2 activation Dynam-in-dependent endocytosis was necessary to generate signaling complexes, whereas delayed targeting to the lysosome ensured the persistence of signaling by such internalized complexes [52]

In agreement with the results with endosomal signal-ing of activin A⁄ receptor complexes in Xenopus, work

in Drosophila has shown that mutations in spinster (spin) [53], hrs⁄ vps27p [54] and vps25 [55], which impair endosome-to-lysosome trafficking, cause an increase in BMP signaling, accompanied in some cases by increased levels of Thick Veins (an ortholog

of ALK3⁄ 6) By contrast, Spichthyin (Spict), the Drosophila ortholog of the SPG6 and ichthyin protein family, which causes segregation (without degradation)

of Wit (an ortholog of BMPRII) in early endosomes (Rab5-positive compartment), inhibits BMP signaling [56] Further work in Drosophila revealed that Nervous Wreck interacts with Thick Veins and the endocytic machinery to attenuate BMP signaling Because Ner-vous Wreck co-localizes with Rab11, the authors suggested that Nervous Wreck might regulate the rate

at which vacant Thick Veins receptors are recycled

back to the plasma membrane following activation

and internalization [57] Indeed, as mentioned below,

TGFb receptors are recycled via a Rab11-dependent

mechanism independent of ligand binding, possibly as

a means of rapidly and dynamically regulating surface

receptor number and thus sensitivity to TGFb [42]

Recent biochemical data has shed more light on the

link between BMP signaling and endocytic trafficking

BMPRI and BMPRII appear to be continuously

inter-nalized via CCVMR endocytosis, and BMPRII is also

endocytosed via a caveolae- and cholesterol-dependent

route [58] Smad1⁄ 5 phosphorylation seems to occur at

the plasma membrane; however, continuation of

Smad1⁄ 5-dependent signaling requires internalization

via the CCVMR The BMP receptor population that

resides in cholesterol-enriched, detergent-resistant

membrane fractions is required for Smad-independent

BMP signaling [58] However, downregulation of

cave-olin-1 via siRNA resulted in a loss of BMP-dependent

Smad phosphorylation and gene regulation, and was

not linked only to Smad-independent signaling [59]

Rab GTPases

Rab GTPases are master regulators of vesicular

trans-port and are distributed in distinct intracellular

com-partments Rab5 is a key regulator of endocytosis that,

by interacting with multiple effectors [60], regulates

organelle-tethering, fusion and motility Rab7 localizes

to the late endocytic compartment and controls the

trafficking of late endosomes [61] Therefore,

conver-sion of Rab5 to Rab7 controls the progresconver-sion of

cargo from the early to the late endocytic

compart-ment, but the cargo itself can also modulate the

kinet-ics of this transport step [36] Thus, inputs from the

Rab5⁄ 7 machinery or cargo (activated growth-factor

receptors) may modulate the extent of downstream

sig-naling by altering early⁄ late endosome transport

kinet-ics, thereby allowing activated receptors to access

and⁄ or reside for longer in an environment that allows

productive signaling, especially in the case of

TGFb⁄ activin A pathways in which SARA is enriched

in the early endosome

Indeed, RIN1, a Rab5 guanine exchange factor

(GEF), via the activation of Rab5, directs TbRs into

an endocytic pathway that promotes TGFb signaling

through Smads [62] (Fig 1A) Silencing of RIN1, in

turn, reduces TbR signaling efficiency A negative

feed-back loop exists, whereby TbR signaling induces

SNAI1, which in turn represses RIN1 expression

Interestingly, RIN1 promotes clathrin-dependent

endo-cytosis of RTKs, such as MET and epidermal growth

factor receptor, through direct binding to activated

receptors and the stimulation of Rab5 proteins [62] This serves principally to direct RTK receptors to deg-radation, thereby leading to reduced signaling [63–65] The differential signaling outcome of RTK versus TbR signaling by RIN1, however, suggests that Rab5-medi-ated endocytosis is not inextricably linked to a particu-lar signaling outcome Multiple endocytic complexes, each containing RIN1 and Rab5, and also other distinct components, may help explain different signaling outcomes during and following receptor internalization

An additional consideration is the length of time TGFb family receptors reside in early endosomes [55] once trafficked there This is important because the signaling outcome is proportional to the residence time

in this compartment Trafficking of TGFb family receptors via early endosomes with extremely fast kinetics will most likely have a minimal enhancing effect on signaling compared with early endosomal trafficking that is accompanied by blocking of further trafficking Indeed, several studies on activin A and BMPs have revealed that the enhancing effect on sig-naling of various proteins was dependent on how long the relevant receptors resided on early endosomes [52– 54] Whether conversion of Rab5 to Rab7 or other mechanisms are responsible remains open Our previ-ous results suggest that Rab5 cycling between the GTP and GDP forms may influence the length and intensity

of TGFb⁄ activin signaling cascades by regulating TGFb–activin type I⁄ II receptor trafficking via the early endocytic compartment [17] Indeed, in endothe-lial cells, Rab5S34N, a Rab5 mutant locked in the GDP form, caused augmented Smad3-dependent tran-scription in the absence of ligand Because RN-tre, a specific Rab5 GTPase-activating protein (GAP) that blocks plasma membrane endocytosis, did not influ-ence Smad3-dependent transcription, we concluded that the effect of Rab5S34N should have been the con-sequence of decreased degradative or recycling traffick-ing, leading to an accumulation of constitutively formed TGFb–activin type I⁄ II receptor complexes on early endosomal membranes

Certainly, the station after early endosomes in the trafficking route of TGFb family receptors is critical Recycling back to the plasma membrane will influence signaling differently compared with trafficking towards late endosomes⁄ lysosomes This issue has been investi-gated by overexpressing dominant-negative forms of Rab4 (Rab4S22N) and Rab11 (Rab11S25N) and assessing TGFb receptor trafficking [42] Rab4 regu-lates recycling from sorting⁄ early endosomes to the plasma membrane, whereas Rab11 controls recycling through the perinuclear recycling endosomes [66] and

trans-Golgi network to plasma membrane transport

[67] Only Rab11S25N caused significant intracellular

retention of TGFb receptors, in both the presence and

absence of ligand Because co-localization of TGFb

receptors with Rab11 has been reported [11], it seems

that, after clathrin-dependent internalization, TGFb

receptors recycle (irrespective of their activation state)

in a Rab4-independent and Rab11-dependent manner

(Fig 1A) To date, the effect of Rab4 and Rab11

mutants or siRNA silencing on TGFb signaling has

not been investigated However, it is expected to

influ-ence TGFb signaling, especially its developmental

aspects

RalA⁄ Ral-binding protein 1

RalA is a multifunctional GTPase that is activated by

receptor-activated Ras via recruitment of Ral GEFs

[68–70] Activated Ral associates with the Ral effector

Ral-binding protein 1 (RalBP1), a cytosolic protein that is recruited to membranes following Ral activa-tion [71] and activates hydrolysis of GTP bound to Rac1 and Cdc42 RalA has been implicated in many intracellular trafficking events [72] from the regulation

of the endocytosis of EGF and insulin receptors [73]

to secretion [74] Indeed, RalA, via its effector protein RalBP1, interacts with the l2 subunit of the AP-2 complex [75] as well as with REPS1 [76] and POB1 [77] which are EGF receptor substrates containing Eps15 homology (EH) domains POB1 interacts directly with the EH-containing proteins epsin and eps15, which have been reported to be involved in the regulation of EGF and transferin receptor endocytosis [67,78,79] Thus, activation of RalA by EGF and insu-lin suggests that RalA⁄ RalBP1 and its interactions with the l2 chain of AP-2, REPS1, POB1, epsin and eps15 act as a scaffold that conveys signals from recep-tors to the endocytic machinery, thereby regulating

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Fig 1 Control of TGFb and activin ⁄ nodal receptor trafficking by small GTPases (A) The role of Rab5 and Rab11 in TGFb receptor endocyto-sis and recycling The cycling of Rab5 between the GTP and GDP forms may influence the length and intensity of TGFb ⁄ activin signaling cascades by regulating TGFb–activin type I ⁄ II receptor trafficking via the early endocytic compartment RIN1, a Rab5 GEF, via activation of Rab5, directs TbRs into an endocytic pathway that promotes TGFb signaling through Smads SNAI1, which represses RIN1 expression, is induced by TGFb thus creating a negative feedback loop Following clathrin-dependent internalization, TGFb receptors recycle (irrespective of their activation state) in a Rab4-independent and Rab11-dependent manner (B) The role of ARIP2, RalA and RalBP1 in activin A receptor internalization ARIP2 interacts with ActRII and triggers their endocytosis via RalA ⁄ RalBP1 and POB1 POB1 interacts directly with the EH-containing proteins Epsin and Eps15 This protein complex acts as a scaffold to convey signals from the activin receptor to the endocytic machinery (C) The role of Rap2 in activin⁄ nodal receptor trafficking in Xenopus embryos In the absence of ligand, Rap2 directs activin ⁄ nodal receptors into a Rab11-dependent recycling compartment, thereby avoiding degradation and maintaining cell-surface levels of receptors Upon ligand addition, Rap2 competes with the Smad7 ⁄ Smurf1 complex and delays receptor degradation, thus enhancing signaling.

ligand-dependent receptor-mediated endocytosis.

Moreover, REPS1 interacts with Rab11-FIP2 [80]

a Rab11 effector that may couple REPS1-containing

vesicles originating from clathrin-coated vesicles (and

the early endocytic compartment) to the recycling

endosomes

RalA and RalBP1 appear to be involved in activin

A receptor trafficking and signaling (Fig 1B) It has

been shown that activin receptor interacting protein 2

(ARIP2) interacts with ActRIIs and regulates their

endocytosis via a PDZ domain-mediated interaction,

concentrating them in a perinuclear compartment

Thus, ARIP2 reduces the response to ligands by

decreasing the levels of ActRII at the plasma

membrane [81] ARIP2 triggers the endocytosis of

ActRIIs via Ral⁄ RalBP1 Indeed, ARIP2 associates

with ActRIIA and RALBP1 via its PDZ domain and

C-terminal region, respectively Because ARIP2C, the

C-terminal deletion mutant of ARIP2 that does not

bind RalBP1, failed to induce ActRII endocytosis, it

appears that endocytosis of ActRIIs by ARIP2 is

RalA⁄ RalBP1 dependent Moreover, activin A

acti-vates GDP–GTP exchange in RalA [81] Activation of

RalA⁄ RalBP1 by activin A is calcium dependent, in

contrast to activation by EGF and insulin, which

occurs via a Ras-dependent cascade [73] Interestingly,

because only ActRIIs among all the serine⁄ threonine

kinase receptors for BMP⁄ TGFb ⁄ activin have the

PDZ-binding sequence (ESSL for ActRIIA and ESSI

for ActRIIB) [82], PDZ protein-regulated endocytosis

and sorting is expected to influence only ActRIIs

Because ActRIIs bind both activins and also nodal

and BMP7, ARIP2 is likely to play a role in shaping

the activin⁄ nodal ⁄ BMP gradient by regulating the

endocytosis of ActRIIs

Rap2

Rap2 is a member of the Ras family of small GTPases

whose effector domain is almost identical to that of

Ras, and can therefore bind most Ras effectors Rap2

inhibits many Ras pathways including Ras-induced

Raf activation at the plasma membrane [83] Rap2 also

binds to the Ral GEFs, Ral GDS, RGL and RLF [84]

These proteins are also Ras effectors and induce

nucle-otide exchange leading to the formation of active

RalA As discussed above, Ral has been implicated in

activin A receptor trafficking and may be linked to the

molecular mode of action of Rap2 in Xenopus, as

explained below

In a very elegant study in Xenopus embryos, Rap2

was shown to regulate activin⁄ nodal signaling by

mod-ulating receptor trafficking [85] (Fig 1C) In the

absence of ligand, Rap2 directs activin⁄ nodal receptors into a Rab11-dependent recycling compartment, thereby avoiding degradation and maintaining cell-sur-face levels of receptors Upon ligand addition, Rap2

no longer directs the receptors for recycling, but rather competes with Smad7 and delays receptor degradation, thus enhancing signaling Moreover, Rap2 is initially enriched in the dorsal region of the blastulae, then as gastrulation proceeds, it decreases dorsally and increases ventrally However, Smad7 is expressed uni-formly across the dorso–ventral axis in early gastrula-tion and as gastrulagastrula-tion proceeds, Smad7 is restricted

to the ventral region Thus, Smad7 and Rap2 levels appear to regulate Smad2 activation along the dorso– ventral axis of the developing embryo

Growing evidence links the progression of TGFb receptor signaling to key regulatory steps in endocytic trafficking These steps involve the active regulation of GDP-to-GTP exchange by various small GTPases of the Rab⁄ Ral and Rap families These mechanisms ensure optimal signal transduction from active receptor complexes to activated Smads

Intracellular Smad trafficking – the role

of the Ran GTPase

Most current evidence on the mechanisms that govern the dynamic shuttling of Smad proteins in the cell is based on the behavior of engineered GFP–Smad2 and GFP–Smad4 fusion proteins which are stably expressed in human cells cultured in vitro The evi-dence supports a model whereby Smads shuttle con-stantly, although each specific Smad seems to obey distinct kinetic properties during its movements [86] Mathematical modeling of Smad protein shuttling has recently suggested that the strength of Smad signaling depends directly upon the length of time a certain Smad molecule spends in the nucleus [87] Such kinetic analysis also emphasized that the nuclear export of Smads is highly regulated, whereas the nuclear import

of Smads may act as a default pathway

The evidence from the in vitro cell system is comple-mented by pioneering in vivo studies first developed in Xenopus embryos [88,89] Continuous shuttling of Smad2 could be observed in developing Xenopus and zebrafish embryos [88] Furthermore, Smad2 and Smad4 proteins fused to fluorescent protein fragments fluoresce only when a Smad2–Smad2 homo-oligomer, Smad4–Smad4 homo-oligomer or Smad2–Smad4 hetero-oligomer forms inside the living cells of Xenopus embryos caused by trans-complementation of the fused fragments [89] Cells in the developing Xenopus embryo are responding to the TGFb members nodal or activin

and show accumulation of Smad4 homo-oligomers

only in the cytoplasm, whereas Smad2 homo-oligomers

and Smad2–Smad4 hetero-oligomers accumulate in the

nucleus These experiments demonstrated that Smad2–

Smad4 oligomers can be observed in the nuclei of

developing embryonic cells only when these cells

reached the proper developmental stage This

observa-tion suggested that factors independent of nodal⁄

acti-vin signaling regulate the ‘competence’ of the

embryonic cell to accumulate nuclear Smad2–Smad4

oligomers Smad trafficking may be classified

accord-ing to the cellular compartment where this specific

movement occurs Thus, we can consider Smad

traf-ficking in the cytoplasm, Smad traftraf-ficking through the

nuclear pores and Smad trafficking inside the nucleus

Smad trafficking in the cytoplasm

When Smad2 moves inside the cytoplasm it associates

with the motor protein kinesin-1 and the integrity of

the microtubular network is essential to support this

type of motility [88] This new evidence is compatible

with an older study that first identified an inherent

ability of all Smad proteins to associate and localize

on microtubules [90] Another motor-like protein that

associates with Smad2 is the dynein light chain km23-1,

which assists in the nuclear accumulation of Smad2,

and also regulates trafficking of the TbRI [91]

Accord-ing to this new evidence, cytoplasmic Smads traffic

towards the signaling receptors with the help of kinesin

motors that slide on microtubules The signaling

recep-tors most likely reside on endosomes, as discussed

above However, cytoplasmic Smad trafficking towards

the nucleus involves the dynein motor–microtubule

machinery Although it makes sense to consider

micro-tubules as trafficking highways that facilitate the

movement of Smad proteins, microtubules have also

been shown to act as cytoplasmic traps for Smads [92]

According to this model, connexin 43 is a regulatory

protein that competes with Smads for binding to

microtubules However, the latter mechanism needs to

be further clarified as it is important to understand

which factor regulates the residence of Smads on

microtubules versus their mobility along microtubules

and towards neighboring cellular locations

The association of Smads with microtubules

pro-vides additional insight into the functional regulation

of these proteins In dividing cells, such as those of the

Xenopusembryo, Smads can associate with the spindle

and decorate the metaphase chromosomes [89] This

evidence is compatible with a role for microtubules in

trapping Smads and protecting their integrity, thus

delivering them safely to the daughter cells after

mito-sis It remains unclear as to whether Smad signaling may also regulate mitosis or cytokinesis However, in addition to protecting Smad integrity, microtubules may also guide a pool of Smads towards their ultimate turnover The site of assembly of the microtubular net-work is known to be the centrosome, a subcellular structure in which Smads that are phosphorylated in their linker domain can also localize and undergo ubiquitin-dependent proteasomal degradation [93] It appears that Smads may slide along microtubules to reach the centrosomes and become degraded [94] Interestingly, when cells divide, the pool of linker-phosphorylated Smads that traffic towards the centro-some segregates together with other ubiquitinated proteins on the mitotic spindle towards only one of the two daughter cells [94] This mechanism ensures that proteins targeted for disposal go to only one of the two daughter cells, leaving the other relatively clear of such signaling byproducts A deeper understanding of the role of microtubules in the regulation of Smad trafficking and signaling is clearly warranted

Smad trafficking through nuclear pores The entry of Smad proteins to the nucleus is regulated

by specific interactions with transporters and nucelo-porins A lysine-rich nuclear localization signal (NLS) located in the N-terminal Mad homology 1 domain of all Smads binds to importin-b in the case of Smad3 and importin-a in the case of Smad4, while mutation

of the NLS blocks the ability of these proteins to enter the nucleus [95–98] Although the functional role of the Smad2 NLS has not yet been determined, the long Smad2 isoform that incorporates exon 3 fails to bind

to importin-b, whereas the shorter Smad2 isoform that lacks exon 3 binds to importin-b similar to Smad3 [95] In addition, the importin moleskin mediates the nuclear entry of the Drosophila R-Smad Mad, and its human orthologues, importin-7 and importin-8, mediate the nuclear translocation of Smad1, Smad2, Smad3 and Smad4 in human cancer cells in response

to BMP or TGFb signaling [99] Future work may explain why Smads utilize multiple importins for their entry to the nucleus (Fig 2)

Importins are known to move through the pore by consecutive contacts with the phenylalanine⁄ glycine (F⁄ G)-rich repeats of specific nucleoporins Such step-wise translocation is energetically demanding and requires GTP expenditure Similar to the role of Rab GTPases that control the trafficking of endocytic vesi-cles during TGFb signaling in the cytoplasm (Fig 1), the small GTPase Ran controls Smad3 trafficking via the nuclear pore (Fig 2) [95] Ran is a small GTPase

dedicated to the control of nucleocytoplasmic

traffick-ing and chromosomal segregation durtraffick-ing mitosis [100]

A Ran activity gradient is established through the

nuclear pore with high Ran–GDP concentrations in

the cytoplasmic phase of the pore which gradually

decrease along the pore [101] In the nuclear phase of

the pore, the Ran-specific GEF RCC1 loads Ran with

GTP, thus establishing a high Ran–GTP concentration

in the nucleus GDP-bound Ran drives the transport

of Smad3 through the pore, whereas Ran–GTP

induces the allosteric change needed to dissociate

Smad3 from importin-b (Fig 2) [95] Ran also

medi-ates importin-b trafficking back into the cytoplasmic

phase of the pore [102]

In addition to binding to importins, Smad2 can also

bind directly to the F⁄ G-rich repeats of nucleoporins

Nup214 and Nup153 of the nuclear pore (Fig 2)

[103,104] However, whether Smad3 and Smad4 bind to

the nucleoporins directly or via the importins remains

unclear [103,104] In addition, it would be interesting to

examine whether Smad2–nucleoporin interactions are

regulated by the Ran GTPase gradient along the

nuclear pore Analysis of importin-7 and importin-8 as

Smad carriers suggested that continuous Smad

shut-tling in the absence of ligand activation is independent

of the action of transportins, and is presumably

facili-tated by direct contacts with nucleoporins [99] By

con-trast, when TGFb receptor activation leads to R-Smad phosphorylation, nuclear import seems to depend on the activity of specific transportins Thus, different mechanisms of nuclear import might operate at differ-ent stages of the TGFb signaling pathway

The cytoplasmic distribution of Smads in the resting cell seems to be regulated by the dominant role of Smad nuclear export [86,87] Upon ligand-dependent signal-ing, nuclear Smad complexes prevail but eventually shuttle back to the cytoplasm, thus providing a way of dampening the strength of the signal or alternatively replenishing the cytoplasmic pool of Smads with mole-cules that are ready to become activated again, as long

as the receptors remain active The importance of nuclear export is underscored by the presence of nuclear export signals (NES) in all Smads examined to date Smad4 carries a leucine-rich NES in its linker domain, which mediates export via exportin-1⁄ chromosome region maintenance 1 (CRM1) (Fig 2) [105,106] Muta-tion of hydrophobic amino acids within the Smad4 NES

or exposure of cells to the pharmacological inhibitor of CRM1 leptomycin-B, lead to an exclusive nuclear distri-bution of Smad4, independent of the presence or absence of ligand Smad3 is exported from the nucleus

in a CRM1-independent manner and an extended peptide surface of the MH2 domain has been identified

as critical for this export by exportin-4 [107] In the case

4

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Fig 2 Smad trafficking through nuclear pores Smad2, Smad3 and Smad4 are shown to interact with importins (Imp) in the cytoplasm and start their nuclear import via additional contacts with nucleoporins (Nup) Smads are released in the nucleoplasm and importins recycle back

to the cytoplasm (not shown) Nuclear Smads associate with exprotins (Exp) and Ran–GTP and translocate to the cytoplasm by making con-tacts with nucleoporins The cytoplasmic Smad–exportin–Ran–GTP complex is disrupted by the action of RanGAP, which releases Smad, exportin and Ran–GDP, and free orthophosphate after the hydrolysis of GTP Completion of the Ran cycle is shown in the middle for Smad3 because the role of Ran has only been analyzed in detail in the case of Smad3 Cytoplasmic Ran–GDP (grey symbol) diffuses through the nuclear pore where it meets the nuclear GEF RCC1, which exchanges GDP for GTP and restores nuclear Ran–GTP (black symbol) levels.

of Smad3, the role of Ran has been studied and it was

clearly demonstrated that, similar to many other

exported proteins, Ran supports the movement of

Smad3 via the nuclear pore towards the cytoplasm

(Fig 2) The Smad3 NES has no obvious resemblance

to a bipartite leucine-rich motif identified in the MH2

domain of Smad1, the R-Smad of the BMP pathways,

which is thought to be recognized by CRM1 based on

leptomycin-B inhibitor experiments [108] The role of

Ran in mediating the export of proteins from the

nucleus follows the inverse biochemical steps used for

import of proteins to the nucleus [100,102] Ran–GTP

promotes the association of Smad3 with exportin-4 in

the nuclear phase of the pore [107] Upon trafficking

via the nuclear pore, Ran–GTP in complex with cargo

is attacked by Ran GAP, which is associated on the

cytoplasmic phase of the nuclear pore, and activates

the GTPase activity of Ran so that GTP is hydrolyzed

to GDP and orthophosphate (Fig 2) [100,102] This

leads to conformational changes in Ran that facilitate

disruption of the complex between exportin and

its cargo, and the ultimate release of cargo to the

cytoplasm

Smad trafficking in the nucleus

Although a growing understanding of the mechanisms

that guide bidirectional Smad trafficking in the

cyto-plasm and through the nuclear pores is now

estab-lished, nothing is known about Smads trafficking

within the nucleoplasm Classically, the native, yet

weak, ability of Smads to bind to DNA has suggested

that upon entry to the nucleus, Smads might tether

chromatin However, the current dynamic shuttling

model of Smads necessitates a more dynamic view of

the nuclear residence of these proteins The dynamic

shuttling model disfavors long-lasting and very stable

tethering mechanisms, however, it allows for the highly

regulated formation of protein complexes between

Smads and nuclear residents In fact, nuclear Smads

are known to bind to a high number of nuclear

tran-scription factors and the role of such interactions in

the timing and shuttling behavior of Smads remains

unexplored [27] One nuclear factor that seems to fulfill

the criteria for a tethering factor and which might

coordinate the nuclear residence time of Smads and

the process of transcription is the newly reported

pro-tein transcriptional coactivator with PDZ-binding

motif (TAZ) [109] TAZ is a transcriptional regulator

containing a WW domain and promotes the nuclear

accumulation of Smads Loss of TAZ perturbs the

ability of Smads to accumulate in the nucleus TAZ

binds the transcriptionally active Smad complex and

anchors it to ARC105, a central component of the transcriptional mediator complex TAZ has a close homolog, the WW domain protein YAP, which might also be involved in a similar mechanism Thus, we await significant developments in Smad nuclear traf-ficking that might provide a more comprehensive view

of how the entry and exit of Smads from the nucleus coordinates with transcription It will also be interest-ing to examine the role of additional nuclear small GTPases as regulators of nuclear Smad function, because this class of proteins offers a versatile regula-tory system that empowers biological processes with the ability to switch on and off

The role of small GTPases of the Rho subfamily in TGFb-induced actin cytoskeleton remodeling

Actin cytoskeleton remodeling is one of the earliest cellular responses to extracellular stimuli [110–115] Binding of ligands to the appropriate receptors triggers specific signaling cascades, which may generate rapid and long-term modifications of actin polymerization dynamics and microfilament organization [116–120] Among the specific signaling effectors regulating actin architecture, the family of small Rho GTPases has a prominent role Classically, plasma membrane recep-tors activate specific guanine-exchange facrecep-tors often via phosphorylation, which leads to the subsequent activation of Rho GTPases [121] Rho GTPases have been implicated in many cellular processes, including actin and microtubule cytoskeleton organization, cell division, motility, cell adhesion, cell-cycle progression, vesicular trafficking, phagocytosis and transcriptional regulation [122,123] Rho proteins cycle constantly between GTP-bound active forms and GDP-bound inactive forms, and this process is regulated by various factors including GEFs, guanine nucleotide dissocia-tion inhibitors and GAPs [124] As well as contributing

to physiological processes, Rho GTPases have been found to contribute to pathological processes includ-ing cancer cell migration, invasion, metastasis, inflammation and wound repair [122,123] Although Rho proteins do not seem to be mutated in cancer cells, their expression is often elevated, indicating that Rho dysregulation promotes malignant phenotypes [125]

Rho proteins can be subdivided into three major groups: Rho (RhoA, RhoB, RhoC), Rac (Rac1, Rac2) and cdc42 proteins [123] Active Rho GTPases trans-mit signals via downstream effectors such as Rho coiled-coiled kinase 1 (ROCK1), p21-activated kinase

1 and neural Wiskott–Aldrich syndrome protein

[126,127] Activated p21-activated kinase 1 and

ROCK1 phosphorylate and activate LIM-kinases 1

and 2, respectively [128–132] Eventually, LIM-kinases

1 and 2 phosphorylate actin-depolymerizing proteins

such as cofilin, destrin and actin-depolymerizing factor,

which are inactivated and thus permit actin

polymeri-zation to occur [128–130,133,134]

Mechanisms of TGFb-induced actin cytoskeleton

remodeling – short- and long-term events

The ability of TGFb to regulate actin cytoskeleton

remodeling has been demonstrated in a variety of cell

systems, and specific members of the Rho subfamily of

small GTPases including RhoA, RhoB, Rac and cdc42

have been found to play essential roles (Fig 3) The

contribution of individual Rho GTPases and their

downstream effectors in TGFb-induced actin

remodel-ing has been studied usremodel-ing a variety of experimental

tools These tools include constitutively active and

dominant-negative mutants of Rho proteins or their

target proteins, siRNA-mediated gene silencing or

gen-eral inhibition of Rho function using molecules such

as the C3 exoenzyme, which selectively ADP-ribosy-lates and inactivates low molecular mass G proteins of the Rho subfamily at an asparagine residue within the effector domain Rho GTPase activation is generally measured by affinity precipitation using appropriate GST–fusion peptides that bind only to GTP-bound Rho proteins such as GTP–Rhotekin binding domain for RhoA and RhoB or GST–p21-activated kinase and GST–Wiskott–Aldrich syndrome protein for Rac1 and cdc42 [135] Changes in the actin cytoskeleton are monitored by immunofluorescence microscopy of rhodamin⁄ phalloidin-labelled actin or by calculating the ratio of total versus polymerized actin by immuno-blotting soluble (globular actin) and Triton-insoluble (filamentous actin) cell extracts [136]

TGFb-induced cytoskeleton rearrangements involving Rho activation in EMT

The most extensively investigated TGFb-induced cyto-skeleton rearrangements are the differentiation of epi-thelial to mesenchymal cells, a process that is called epithelial to mesenchymal transition or

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Fig 3 The role of small Rho GTPases in short- and long-term actin cytoskeleton reorganization in response to the TGFb signaling pathway TGFb induces short-term actin cytoskeleton remodeling via the activation of various Rho GTPases including RhoA, RhoB, Rac and Cdc42 (generally termed Rho) Activation of these GTPases causes actin polymerization via the ROCK1 ⁄ LIMK2 ⁄ cofilin, as well as by MAPK ⁄ PKN ⁄ PRK2 pathways In long-term cytoskeletal reorganization, which involves nuclear events, TGFb receptor activation causes the phosphorylation of Smads and their subsequent translocation to the nucleus In the nucleus, R-Smad ⁄ Smad4 complexes bind to the promot-ers of various target genes such as the smooth muscle-specific genes a-SMA, SM-22a or SM-MHC, the Rho GEF NET1, the inhibitory Smad7 protein and the RhoB gene Activation of cofactors such as serum response factor, AP1, GATA and myosin enhancer factor 2 via p38 MAPK or other pathways facilitates these transcriptional responses Actin remodeling in turn facilitates processes such as smooth muscle cell differentiation, EMT and others.