[advances in immunology] volume 93 || regulation of immune responses and hematopoiesis by the rap1...

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Regulation of Immune Responses and Hematopoiesis by theRap1 Signal

Nagahiro Minato, Kohei Kometani, and Masakazu Hattori

Department of Immunology and Cell Biology, Graduate School of Biostudies,Kyoto University, Kyoto, Japan

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229ances in immunology, vol. 93 0065-2776/07 $

007 Elsevier Inc. All rights reserved. DOI: 10.1016/S0065-2776(06)93

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1. I ntroduction ....................................................................................................... 2 29 2. G eneral Biology of the Rap1 Signal ........................................................................ 2 30 3. R ap1 Signal in Lymphocyte Development and Immune Responses............................... 2 37 4. R ap1 Signal in Hematopoiesis and Leukemia............................................................ 2 48 5. R ap1 Signal in Malignancy: New Aspects in Cancer................................................... 2 53 6. C onclusions and Perspectives ................................................................................ 2 55
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eferences ......................................................................................................... 2 56
Abstract

Rap1 (Ras‐proximity 1), a member of the Ras family of small guanine tripho-sphatases (GTPases), is activated by diverse extracellular stimuli. While Rap1 hasbeen discovered originally as a potential Ras antagonist, accumulating evidenceindicates that Rap1 per se mediates unique signals and exerts biological functionsdistinctly different from Ras. Rap1 plays a dominant role in the control of cell–celland cell–matrix interactions by regulating the function of integrins and otheradhesionmolecules in various cell types. Rap1 also regulatesMAP kinase (MAPK)activity in a manner highly dependent on the context of cell types. Recent studies(including gene‐targeting analysis) have uncovered that the Rap1 signal isintegrated crucially and unpredictably in the diverse aspects of comprehensivebiological systems. This review summarizes the role of the Rap1 signal in develop-ments and functions of the immune and hematopoietic systems as well as inmalignancy. Importantly, Rap1 activation is tightly regulated in tissue cells, anddysregulations of the Rap1 signal in specific tissues result in certain disorders,including myeloproliferative disorders and leukemia, platelet dysfunction withdefective hemostasis, leukocyte adhesion‐deficiency syndrome, lupus‐like system-ic autoimmune disease, and T cell anergy. Many of these disorders resemblehuman diseases, and the Rap1 signal with its regulators may provide rationalmolecular targets for controlling certain human diseases including malignancy.

1. Introduction

Rap1 (Ras‐proximity 1), a member of the Ras family of small guanine tripho-sphatases (GTPases), displays high overall homology (�50%) to the classicalK‐, H‐, and N‐Ras with an identical effector domain (Pizon et al., 1988). Rap1

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230 NAGAHIRO MINATO ET AL .

is conserved in eukaryocytes from yeasts through mammals. In budding yeasts,Rap1 (Rsr1) is essential for the proper determination of budding sites formating(Bender and Pringle, 1989). Rap1 in Drosophila melanogaster (DRap1) is alsoan essential gene that plays crucial roles in various aspects of morphogenesis(Asha et al., 1999). The prototype, or the so‐called ‘‘roughened eyes,’’ is causedby a gain‐of‐function mutation of the Rap1 gene (Hariharan et al., 1991). Inmammals, there are two Rap1 isoforms coded by distinct genes (Rap1A andRap1B) with limited differences in constitutional amino acids and redundantactivities (Bos et al., 2001). It was first reported that overexpression of Rap1(originally called K‐rev) could revert the characteristic ‘‘malignant’’ contours ofthe fibroblasts transformed by oncogenic K‐Ras to a flat shape similar to normalfibroblasts (Kitayama et al., 1989). This raised the initial idea that Rap1 mightact as a functional antagonist of oncogenic Ras. The exact roles of Rap1 inmammalian cells, however, have remained rather enigmatic for nearly a decade.In the late 1990s, two distinct biological activities mediated by the Rap1 signal(independent of Ras) emerged, viz., the activation of MAP kinases (MAPKs)and control of cell adhesion via integrins. Since then, numerous findings on theroles of Rap1 in many cell types of various tissues have accumulated, and it hasbecome evident that the Rap1 signal mediates highly diverse cellular activitiesdepending on the cellular contexts. In the present chapter, we first summarizerecent advances on the general biology of Rap1, including regulation andfunction of the Rap1 signal, before proceeding to discuss how a ubiquitousmolecular switch (Rap1) is integrated into the signaling pathways to controlhighly sophisticated and specified cellular functions, with particular emphasison the immune/hematopoietic systems and malignancy.

2. General Biology of the Rap1 Signal

One of the most intriguing features of Rap1 is that it is activated by anextensive variety of external stimuli delivered to the cell, including numerousgrowth factors, peptide hormones, neurotransmitters, cytokines, chemokines,antigens, cell‐adhesion molecules, and physical stimuli such as cell stretch/contraction.

2.1. Regulation of Rap1 Activation

Similar to many other small G‐proteins, Rap1 binds with guanine nucleotides toform Rap1GTP (an active form) or Rap1GDP (an inactive form) that interactsor dissociates with a number of downstream effector molecules, respectively.Due to its intrinsic GTPase activity, GTP bound to Rap1 is autonomouslyhydrolyzed to GDP. Therefore, activation of Rap1 requires specific enzymes

RAP1 IN IMMUNE RESPONSE AND HEMATOPOIESIS 231

that dissociate GDP from Rap1 to facilitate repetitive GTP loading, that is, theguanine nucleotide exchange factors (GEFs). A number of distinct Rap1 GEFssharing a catalytic GEF domain have been identified, and they are coupled withvarious receptors or intracellular second messengers (Fig. 1; Bos et al., 2001;Hattori andMinato, 2003). C3G, which is a Rap1GEFubiquitously expressed inmost cell types, binds to the SH3 domain of Crk adaptor proteins, and is re-cruited to the plasma membrane and phosphorylated on activation of receptor‐associated protein tyrosine kinases (PTKs; Gotoh et al., 1995; Ichiba et al.,1999). Phosphorylated C3G is a major Rap1 activator in the plasma membrane.CalDAG‐GEF harbor the Ca2þ ion‐ and diasylglycerol (DAG)‐binding sites.Activation of CalDAG‐GEF I is regulated by the Ca2þ ion, while CalDAG‐GEF III is translocated to the membrane by binding DAG, and thus theseGEFs may mediate Rap1 activation downstream of PLC activation (Kawasakiet al., 1998). On the other hand, the Epac family of Rap1GEFs has specificcyclic AMP (cAMP)‐binding domains at the N‐terminal region. Binding cAMPinduces conformational changes in Epacs to release the inhibitory constraintcovering the catalytic GEF domain, thus allowing interaction with the substrate

Proliferation, survivalgene activation

Cell adhesion, migrationpolarity

Smgcross-talk

GTP

GDP

Pi

C3G

Epac (1,2)

CD-GEF (I,III)

PDZ-GEF

DOCK-4

Crk (L)PTK

A-cyclase cAMP

PLC Ca2+ DAG

GEFsGAPs

SPA-1 family(SPA-1, E6TP1,SPA-L2,3)

RapGAs (1,2)

Rap1-GDP

Rap1-GTP

Extracellularstimuli

Ras-GTP

c-Raf-1

MEK1,2

ERK

B-Raf

MEK3,6

p38MAPK

RapL

Integrins

RIAM

ProfillinEna/VASP

F-actin

RalGDS

Ral, Rac

AF-6

Cadherins

Figure 1 Regulation and functions of the Rap1 signal. Refer to the text for the details.


Rap1 (Bos, 2003; de Rooij et al., 1998). It has been demonstrated that certaincAMP‐induced biological activities, such as cell adhesion and insulin secretion, aremediated by the Epac/Rap1 rather than by cAMP‐dependent protein kinasepathway (Bos, 2003). Epacs may play a major role in the cytosolic Rap1 activationdownstream of trimeric G‐protein–coupled receptors (GPCRs). Thus, distincttypes of Rap1 GEFs are tightly coupled with the major signaling pathways toinduce Rap1 activation at the different intracellular compartments via diverseextracellular stimuli (Fig. 1).Rap1 does not share a conserved catalytic residue with other small

G‐proteins, such as Ras, Rho, or Cdc42, and displays much lower intrinsicGTPase activity. The swift inactivation of Rap1GTP to terminate the signal,therefore, is crucially dependent on Rap1 GTPase‐activating proteins (GAPs).Rap1GAPs specifically bind to GTP‐bound Rap1 to provide a catalytic residue(asparagine) for Rap1 thereby enhancing the GTPase activity by multipleorders (Daumke et al., 2004). In contrast to the diverse types of Rap1GEFs,there are only two groups of Rap1GAPs (i.e., RapGAs and SPA‐1 family)that share a catalytic domain called the GAP‐related domain (GRD). WhileRapGA1 is expressed rather ubiquitously (most prominently expressed in thebrain), RapGA2 is distributed exclusively in platelets (Kurachi et al., 1997;Schultess et al., 2005). An isoform of RapGA1 binds to Ga via the N‐terminalregion and may be translocated to the plasma membrane following the activa-tion of GPCRs, thus attenuating Rap1 activation at the membrane (Mochizukiet al., 1999). The SPA‐1 family of Rap1GAPs consists of SPA‐1, SPA‐1‐like(SPA‐L) 1 (also called E6TP1 or SPAR), SPA‐L 2, and SPA‐L 3, all of whichshare a PDZ domain in addition to a GRD. SPA‐1 is most abundantly exp-ressed in lymphohematopoietic tissues and certain cancer cells, while SPAR isdistributed in epithelial tissues and the brain (Gao et al., 1999; Hattori et al.,1995; Pak et al., 2001). They are located in various intracellular compartments,such as the synaptic vesicles, actin cytoskeleton, plasma membranes, andpossibly nuclei, depending on the cell type and specific protein interactionvia the PDZ domain (Farina et al., 2004; Roy et al., 2002; Tsukamoto et al.,1999). All the Rap1GAPs are constitutively active without any protein modi-fication. As such, the expression levels of Rap1GAPs per se may determinethe threshold and degree of Rap1 activation at any given compartment (seebelow). Notably, E6TP1 is specifically degraded by human papillomavirus E6oncoprotein via E6AP ubiquitin ligase similarly to p53 in a fashion closelycorrelated with E6‐mediated epithelial cell transformation (Gao et al., 2001,2002). Rap1GAPs have no structural similarities to GAPs for other smallG‐proteins, such as Ras, Rho, Arf, or Rab, and a study has indicated that themode of GAP activity for Rap1 is also different from that of GAPs for otherG‐proteins (Daumke et al., 2004).


2.2. Biological Function of Rap1

2.2.1. Regulation of MAPK Activation

Although it has been a matter of argument for almost a decade whether theRap1 signal activates extracellular signal‐regulated protien kinase (ERK),accumulating evidence has indicated that the Rap1 signal does activate theMAPK kinase‐1 (MEK‐1), 2–ERK pathway selectively via B‐Raf (Vossler et al.,1997), which is expressed in only selected tissue cells (Barnier et al., 1995).This finding probably explains why Rap1‐mediated ERK activation has beenobserved only in certain selected cell types. While ubiquitous c‐Raf‐1 needs tobe phosphorylated to activate MEK‐1, 2, B‐Raf is constitutively phosphory-lated at the corresponding sites (Mason et al., 1999) and activates MEK‐1, 2 bybeing recruited to the plasma membrane by Rap1GTP. This was confirmedgenetically in Drosophila melanogaster, which has only one Raf isoform (DRaf)corresponding to the mammalian B‐Raf. Thus, DRap1 that has been activateddownstream of torso receptor tyrosine kinase binds to DRaf and induces ERKactivation, which in turn incites activation of tailless and huckebein genescontrolling the terminal structures in embryos (Mishra et al., 2005). AlthoughRap1GTP binds to c‐Raf‐1 with an affinity even higher than RasGTP inmammalian cells, it may not lead to the activation of MEK‐1, 2–ERK pathway,partly because Rap1GTP is incapable of inducing c‐Raf‐1 phosphorylationrequired for the activation of the kinase activity (Mishra et al., 2005).

ERK activation by Rap1, however, shows a unique feature distinctly differentfrom Ras‐mediated ERK activation. In PC12 neuronal cell line that stronglyexpresses B‐Raf, the epithelial growth factor (EGF) and nerve growth factor(NGF) specifically induce the proliferation and differentiation, respectively(Marshall, 1995; Qui and Green, 1992; Traverse et al., 1992). NGF rapidlyelicits peak followed by sustained activation of ERK, while EGF induces onlya transient ERK activation, suggesting that a persistent ERK activation isrequired for PC12 cell differentiations. Although Ras mediates the rapid andtransient ERK activation elicited by both factors, Rap1 is responsible for theNGF‐induced sustained activation of ERK (Kao et al., 2001; York et al., 1998).Sophisticated analyses have suggested that different ERK activation kineticsmight reflect the different regulatory mechanisms of Ras and Rap1 activations(Sasagawa et al., 2005). While Ras is activated rapidly by SOS recruited to theplasma membrane via Grb2 following stimulation before recruitment of phos-phorylated RasGAP, activated ERK induces phosphorylation of SOS and thedissociation from Grb2. Because of the recruitment of RasGAP and the tightnegative feedback by ERK, the activation dynamics of Ras may primarilydepend on the temporal rate, rather than the magnitude, of stimuli. On theother hand, a negative feedback mechanism for Rap1 activation has not been


defined to date, and there is little evidence advocating that Rap1GAPs arespecifically recruited following stimulation. Thus, Rap1 activation may continueas long as the stimuli persist potently enough to overcome the basal Rap1GAPactivity, and the activation dynamics of Rap1 may directly reflect the durationand degree of stimuli. Ligand‐occupied EGF receptors (EGFRs) are inter-nalized and rapidly degraded thereafter to likely induce only transient Rasactivation with limited Rap1 activation, that is, the transient ERK activation.In contrast, NGF receptors (TrkAs) occupied with ligands are trapped andprevail in the endosomal membrane to induce sustained ERK activation viaRap1 activation. Thus, Rap1 and Ras may mediate and yield different biologicaleffects due to their distinctly different kinetics in ERK activation.Another aspect of the Rap1 signal in ERK activation is its possible effect on

Ras‐mediated ERK activation. As mentioned earlier, Rap1GTP may not con-tribute to ERK activation via ubiquitous c‐Raf‐1 but may rather competitivelyinterfere with Ras‐mediated ERK activation at the level of c‐Raf‐1 whenoverexpressed (Boussiotis et al., 1997). While ambiguity of such an effectoccurring under normal physiological conditions remains unresolved, recentreports have indicated its involvement under certain in vivo conditions (seebelow). In short, the Rap1 signal, depending on the cell contexts, may regulateERK activation in different ways.Recent evidence has demonstrated that the Rap1 signal also activates the

MEK‐3, 6–p38MAPK pathway. Rap1 is reportedly activated by cell‐stretchforce to induce MEK‐3, 6‐mediated p38MAPK activation, while Ras‐signalingis inactivated (Sawada et al., 2001; Tamada et al., 2004). On the contrary, cellcontraction force activates theRas‐mediatedMEK‐1, 2–ERKpathwayswith littleRap1 activation (Sawada et al., 2001). Similar complementary activations ofRap1‐p38MAPK and Ras–ERK pathways have also been reported in other systems.In hippocampal neurons, for instance, Rap‐1‐mediated p38MAPK activationinduces nonmetabolic glutamate receptor‐mediated removal of synaptic AMPA(alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) receptors duringlong‐term depression (Zhu et al., 2002). On the other hand, delivery of AMPAreceptors to the synaptic sites during long‐term potentiation is dependent onthe Ras‐mediated ERK activation. Under such circumstances, it appears that theRap1–MKK3, 6–p38MAPK and Ras–MKK1, 2–ERK pathways form parallel butopposing signaling modules.Since Rap1 shares the effector domain with Ras, Rap1GTP is expected

to bind other Ras effectors such as RalGDS and PI3Kp110 subunit (Boset al., 2001). The PI3K–AKT pathway is activated by receptor PTKs in aRas‐dependent or Ras‐independent manner (Shaw and Cantley, 2006). Forinstance, the regulatory effect of cAMP on cell proliferation is in part mediatedby the control of Rap1‐mediated activation of the PI3K–AKT pathway


(Tsygankova et al., 2001; Wang et al., 2001). It has been shown that the prolifer-ation and survival of hematopoietic cells induced by IL‐3 or B‐cell receptors(BCR)‐ABL oncoprotein are influenced by Rap1‐mediated activation of thePI3K–AKT pathway (Jin et al., 2006).

2.2.2. Control of Cell Adhesion

SPA‐1 overexpression (abrogating the endogenous Rap1 activation) inducedrounding and eventual detachment of inherently adherent cells from extra-cellular matrix, while overexpression of membrane‐targeted C3G (C3G‐F)enhanced cell‐spreading on extracellular matrix (Tsukamoto et al., 1999). Thiswas one of the first indications to show involvement of the Rap1 signal inregulating cell adhesion. Since then, many reports have confirmed that theRap1 signal regulates the integrin‐mediated cell adhesion induced by variousstimuli in many cell types; viz., b1 (VLA‐4) and b2 (LFA‐1) integrin activationsof lymphocytes by CD31 (Reedquist et al., 2000) and CD98 (Suga et al., 2001)stimulation; b2 (Mac‐1) integrin activation of macrophage for phagocytosis byLPS (Schmidt et al., 2001); LFA‐1 activation of lymphocytes by chemokinestimulation (Shimonaka et al., 2003); and aIIb b3 integrin activation of plateletsby thrombin or ADP (Crittenden et al., 2004). Normal resting T cells initiallyexpress a low affinity or an ‘‘inactive’’ form of LFA‐1 before conversion to the highaffinity or ‘‘active’’ form by stimulation with antigens via a process called ‘‘inside‐out’’ activation (Carman and Springer, 2003). Expressing an active form of Rap1in Tcells rapidly increases the affinity of LFA‐1, while overexpression of SPA‐1 ora dominant‐negative Rap1 mutant completely inhibits LFA‐1 activation byantigen‐receptor stimulation (Katagiri et al., 2000). These findings clearly indicatethat Rap1 is a major mediator of ‘‘inside‐out’’ activation of integrins in Tcells (seebelow). These results have clarified that Rap1 serves as amajor integrin‐activatingsignal, unveiling a novel and important functional aspect of Rap1.

The overall cell adhesiveness induced by integrins is controlled by distinctelements such as the ‘‘affinity’’ of each monomeric integrin molecule, ‘‘valency’’defined by the diffusivity or local density of integrins and ligands, and ‘‘adhesionstrengthening’’ induced following integrin interaction with ligands (Carman andSpringer, 2003). Crystal structure analyses have revealed that affinity regulationof integrins is based on their conformational changes (Xiong et al., 2001). Thus,the extracellular stork of a and b chains in a low‐affinity state is sharply bent sothat the ligand‐binding head is juxtaposed to the membrane portion of thestork (closed posture), while the binding site is free from constraints andunfurls openly (open posture) in a high‐affinity state. The conformationalchange is regulated by proteins (such as talin), which bind to the cytoplasmicdomain of integrins (Carman and Springer, 2003). In lymphoid cells, RapL (aspecific effector of Rap1) associates directly with the cytoplasmic tail of LFA‐1


a‐chain on binding to Rap1GTP to likely induce an open posture of LFA‐1(Katagiri et al., 2003). In addition, a study has indicated that activation ofintegrins by the Rap1 signal also induces redistribution and polarity of integrinexpression (Shimonaka et al., 2003). Thus, the Rap1 signal may affect theoverall integrin‐mediated cell adhesion through multiple mechanisms.In this aspect, Rap1GTP has been found to bind also with a new adaptor called

the Rap1‐interacting adaptor molecule (RIAM), which displays high homologyto lamellipodin (Lpd; Lafuente et al., 2004). Knockdown of RIAM displacesRap1GTP from the plasma membrane and reverts integrin‐mediated cell adhe-sion induced by Rap1, while RIAM overexpression enhances integrin‐dependentcell adhesion and facilitates cell spreading. Interestingly, RIAM interacts directlywith profillin and Ena/VASP family proteins to maintain the cellular content ofF‐actin (Lafuente et al., 2004). Profillin and Ena/VASP are important regulatorsof the actin cytoskeleton, and thus Rap1 not only induces integrin activation butalso may directly regulate actin dynamics required for cell spreading and migra-tion, viz., lamellipodia formation (Bailly, 2004). These results may place Rap1 in acrucial position linking cell‐signaling and actin‐cytoskeleton changes.Cumulative evidence has further advocated that Rap1 plays an important

role in the maintenance of integrity of intercellular adhesion in epithelial andendothelial cells. Convincing data on the role of the Rap1 signal in controllingadherence junctions have been derived again from a genetic study ofDrosophila(Knox and Brown, 2002). During development of the wing, where mediation byeven cell adhesion among adjacent cells via the circumferentially distributedDE‐cadherin is involved, expanding epithelial cells of the related lineages normallystay in a coherent group. The epithelial cells withmutant Rap1, however, lose thecircumferential expression of DE‐cadherin and selectively form an adherencejunction to the adjacent cells ipsilaterally, resulting in disrupted epithelial cellbehavior (Knox and Brown, 2002). This may, in part, explain the abnormalmorphogenesis in Rap1‐mutant Drosophila (Hariharan et al., 1991). The rolesof Rap1 in the formation and maintenance of E‐cadherin‐mediated adherencejunctions in epithelial cells and VE‐cadherin‐mediated endothelial barrier func-tion have been reported also in mammalian cells. For instance, the Rap1 signalplays an important role in protecting the endothelial cell barrier function againstfactors (e.g., thrombin and so on) that cause barrier dysfunctions (Cullere et al.,2005; Fukuhara et al., 2005). In epithelial cells, Rap1 regulates the endocytosis ofE‐cadherin and controls the accumulation and distribution of E‐cadherin on cellsurfaces by specific binding to a scaffold protein afadin (or AF‐6) (Hoshino et al.,2005). Interestingly, AF‐6 also binds SPA‐1 and regulates Rap1‐GAP activity atthe adherence junction (Su et al., 2003). A component of tight junction (TJ),JAM1, has been found to constitutively deliver the Rap1 signal on intercellularepithelial cell adhesion, and disruption of the Rap1 signaling results in marked


changes in epithelial cell morphology and impairment of b1‐integrin‐mediatedadhesion to extracellular matrix (Mandell et al., 2005). These results imply thatthe Rap1 signal plays an important role in the functional cross talk betweenintercellular adhesion (adherence junction) and the adhesion to extracellularmatrix in epithelial cells.

3. Rap1 Signal in Lymphocyte Development and Immune Responses

Small G‐proteins of the Ras, Rho, and Rac families play crucial roles in variousaspects of lymphocyte development and function (Cantrell, 2003). Recentstudies, including those using gene‐engineered mice, have begun to unveilunique roles of the Rap1 signal in lymphocyte development and immuneresponses (Table 1).

3.1. Thymic T Cell Development: Distinct Roles of Rap1 and Ras

Using transgenic mice expressing the RapV12 mutant gene under a humanCD2 promoter, it was shown that excessive Rap1 signals barely affected overallthymic T cell development (Sebzda et al., 2002). However, we have recentlyfound that mice conditionally expressing the RapE63 (another dominant activemutant of Rap1) transgene, driven by a more potent CAG promoter, exhibitsignificant decreases in double‐positive (DP) thymocytes (Kometani et al.,manuscript in preparation). The discrepancy may reflect the fact that V12mutation of Rap1 may not be an ideal dominant active form unlike in othersmall G‐proteins such as Ras, Rho, and Rac (Daumke et al., 2004). Theexpansion and subsequent positive selection of DP thymocytes is reportedlydependent on the Ras signal (Swan et al., 1995). Thus, it appears that Rap1activation surpassing a certain level interferes with Ras‐dependent expansionor positive selection of DP thymocytes, although the physiological significanceremains to be verified.

Amore important question of whether the endogenous Rap1 signal is requiredfor the normal thymic Tcell development warrants attention. To shed light on thisambiguity, we innovated an experimental model—the SPA‐1 transgenic mice—for further investigation. Since the mice expressing SPA‐1 transgene driven by aubiquitous CAG promoter were embryonically lethal, we generated LoxP‐franked SPA‐1 transgenic mice and then crossed them with lck‐Cre transgenicmice. The conditional transgenic mice revealed severe thymic hypoplasia inwhich thymic T cell development was arrested at the late double‐negative (DN)stage (Kometani et al., manuscript in preparation). Consistently, in fetal thymicorgan cultures (FTOC), generation of DP thymocytes from the pro‐T cells ofRag2�/� mice in the presence of anti‐CD3 antibody was suppressed by the

Table 1 Phenotypes of Gene‐Engineered Mice Related to the Rap1 Signal

Mice Phenotypes References

C3G KO Embryonic lethal (at E5) Ohba et al., 2001C3Ggt/gt mutanta Embryonic lethal (at E15) Voss et al., 2003

Vascular defectIncreased cerebral neural cells Voss et al., 2006

SPA‐1 KO Myeloproliferative disordersof late onset

Ishida et al., 2003a

Memory T cell anergy Ishida et al., 2003bLupus‐like autoimmune

disease and B1 cell leukemiaIshida et al., 2006

Diabetes insipidus Noda et al., 2004CalDAG‐GEF1 KO Impaired platelet aggregation

and adhesionCrittenden et al., 2004

Bleeding diathesisRap1A KO Reduced adhesiveness of

T and B cells(probably redundant dueto intact Rap1b)

Duchniewicz et al., 2006

RapV12 Tg (hCD2) Enhanced T cell adhesion Sebzda et al., 2002RapE63 Tg (hCD2) Reduced antibody response

to TD‐antigensLi et al., 2005b

RapGA1 Tg (hCD2) Compromised CTLA‐4‐mediatedsuppression of T cell activation

Dillon et al., 2005

SPA‐1 Tg (CAG) Embryonic lethal UnpublishedSPA‐1 Tg (LoxP/lck‐Cre)b Severe thymic hypoplasia

(impaired b‐selection)Unpublished

aMice with mutant C3Ggt allele in which pGT1.8geo has been integrated in the first intron ofC3G gene, producing less than 5% normal C3G protein.

bTransgenic mice of SPA‐1 gene franked by LoxP under a CAG promoter were crossed withlck‐Cre transgenic mice.KO, knockout; Tg, transgenic; hCD2, human CD2 promoter; CAG, CMVearly enhancer‐chicken

b‐actin hybrid promoter.


retroviral transduction with SPA‐1 or RapA17 (a potent dominant‐negativemutant of Rap1; Dupuy et al., 2005). These results strongly suggested that theRap1 signal was essential for transition from pre‐T cells to abTCR‐expressingDP‐Tcells, that is, the b‐selection.While the Ras signal does not reportedly playa major role in b‐selection (Swan et al., 1995), a report has indicated that ERKactivation is crucially involved in the process (Crompton et al., 1996; Fischeret al., 2005). Therefore, Rap1 may serve as a major signal that mediates ERKactivation required for the thymic pre‐T cell proliferation and differentiation(Fig. 2).

Rap1Pre-TCR

ERK

abTCR

MHC:peptides

Rap1

Ras

ERK

pro-T pre-T DP SP

b -Selection

SPA-1 Tg

Positiveselection

RapE63 TgNegativeselection

NaiveCD4 T

Rap1

Ras

ERK

APC

LFA-1Rap1

Memory (CD44high)

Anergy

Rap1

Ras ERK

SPA-1 KO

Thymus

Immunological synapse

TCR TCR/CD28

Figure 2 Involvement of the Rap1 signal in development and activation of T cells. The Rap1signal is crucial for pre‐TCR‐mediated b‐selection, and conditional expression of SPA‐1 transgenein T cell lineage results in the arrest of thymic T cell development at the DN3 stage. On thecontrary, excess Rap1 activation in RapE63 transgenic (Tg) mice results in compromised expansionof double‐positive (DP) thymocytes and this is most likely due to interference with Ras signaling,which is essential for proliferation and positive selection of DP thymocytes via abTCR. In periph-eral T cells, Rap1 plays an important role in the initiation of immunological synapse formationwith antigen‐loaded antigen‐presenting cells (APC) via TCR‐mediated inside‐out activation ofLFA‐1. Such activated Rap1, however, has to be downregulated because persistent Rap1 activationmay cause T cell anergy. In SPA‐1 knockout (KO) mice, a proportion of CD44high CD4þ memoryT cells becomes progressively nonresponsive or anergic to TCR‐stimulation.


Pre‐T‐cell receptors (TCR)‐mediated b‐selection is distinctly different fromabTCR‐mediated positive selection in a few aspects: (1) the pre‐TCR‐mediatedsignal is triggered by self‐polymerization of receptors without specific ligands(Irving et al., 1998; Yamasaki et al., 2006) and (2) the signal threshold inb‐selectionis extremely low as compared with positive selection. The ‘‘hyperexcitability’’is ascribed to the intrinsic feature of thymocytes at the DN stage rather than to


that of the pre‐TCR (Erman et al., 2004; Haks et al., 2003). It may be possible thatthe higher excitability of pre‐T cells than DP‐T cells partly reflects the feature ofRap1‐mediated (as opposed to Ras‐mediated) ERK activation in the former(Section 1.2.1). Expression profiles of c‐Raf‐1 and B‐Raf at different developmen-tal stages may warrant further investigation. All in all, Rap1 plays significant rolesin the thymic abTcell development. In contrast, development of gdT‐lineage cellswas completely normal in the SPA‐1/lck‐Cre transgenic mice, suggesting that theRap1 signal displays a nonessential role in thymic gdT cell development if any.

3.2. Immunological Synapse and T Cell Activation

Interaction of T cells with antigen‐presenting cells (APCs) loaded by specificantigens results in the formation of three‐dimensional molecular clusters at thecontact site, that is, supramolecular activation clusters (SMAC). In SMAC,while TCR and costimulatory molecules are clustered in the central region(cSMAC), LFA‐1 is located at the periphery (pSAMC), and CD45 is excludedfrom the clusters (dSMAC; Huppa and Davis, 2003). Being maintained bycontinuous TCR signaling, this synaptic structure is quite stable and may lastmore than 10 h (Huppa and Davis, 2003). Although the exact function of sucha stable synaptic structure remains controversial, it may serve as a platform formolding architectural complexities to: (1) attain the cumulative TCR‐signalingeffect for full development of the T cell effector function and (2) accommodateregulatory mechanisms for guiding T cell activities. A hallmark of synapseformation is the ring‐cluster formation of LFA‐1 with ICAM‐1 on the APCaround TCR clusters, a process initiated by the initial contact of TCR with thespecific peptide‐loaded MHC (Huppa and Davis, 2003).It has been indicated that the Rap1 signal delivered by TCR ligation plays a

crucial role in clustering, reorganization, and activation of LFA‐1 for synapseformation. Thus, overexpression of SPA‐1 or RapN17 (another dominant‐negative mutant of Rap1) in a T cell clone strongly suppresses TCR‐mediatedLFA‐1 activation and synaptic conjugation with specific antigen‐loaded APCs,whereas that of Rap1 enhances the conjugate formation (Katagiri et al., 2002).These effects were not observed in the absence of relevant antigens, indicatingthat TCR‐mediated activation of endogenous Rap1 was crucial for clusteringand activation of LFA‐1 at the contact sites (Dustin et al., 2004; Fig. 2).A report has demonstrated that Rap1GTP is almost exclusively detected at theplasma membrane of T cells in a manner dependent on endosomal recycling,while Rap1GDP is mostly associated with the cytoplasmic vesicular membrane(Bivona et al., 2004). Interestingly, in contrast to most of the TCR‐proximalsignaling molecules, SLP‐76 rapidly dissociates from the TCR‐complex andmoves to the cytoplasm on APC interaction (Dustin et al., 2004). Thus, an


intriguing model may be proposed such that SLP‐76 associates with recyclingendosomes containing Rap1GDP, LFA‐1, and RapL to subsequently exposethem to the plasma membrane, where Rap1 is rapidly activated to Rap1GTP bymembrane‐recruited GEFs (e.g., C3G, CalDAG‐GEF I, and so on). Rap1GTPthen causes conformational activation of LFA‐1 by inducing RapL binding tothe cytoplasmic portion of a chain of LFA‐1 (Section 1.1), thus leading to tightinteraction with APCs via ICAM‐1 binding.

3.3. T Cell Nonresponsiveness and Anergy

Full activation of T cells requires additional engagement of costimulatoryreceptors such as CD28 with CD80/CD86 ligands on the professional APC(Sharpe and Freeman, 2002). Although Ras is strongly activated by the concom-itant stimulation of TCR/CD3 and CD28 receptors to induce ERK activation, itis poorly activated by TCR/CD3‐stimulation alone (Carey et al., 2000).In contrast to Ras, Rap1 is potently activated by TCR/CD3‐stimulation alone,while concomitant costimulation with anti‐CD28 markedly reduces Rap1activation (Carey et al., 2000; Reedquist and Bos, 1998). The results implythat Rap1 activation may have to be downregulated after the initiation ofsynapse formation for optimal T cell activation. In fact, persistent activation ofRap1 in T cells results in marked decreases of IL‐2 production on interactionwith antigen‐loaded APCs, albeit enhanced conjugate formation may occur(Katagiri et al., 2002). The reduced IL‐2 response is associated with compro-mised ERK activation, and this is consistent with the concept that persistentRap1 activation interferes with Ras‐mediated ERK activation downstream ofTCR (Boussiotis et al., 1997; Ishida et al., 2003b). It has further been confirmedthat T cells harboring RapE63 transgene show significantly reduced cell prolif-eration and IL‐2 production via TCR‐stimulation in vitro, and the transgenicmice exhibit compromised antibody responses to TD antigens, but not to TIantigens, in vivo (Li et al., 2005b). We have observed that SPA‐1 in T cells isspecifically recruited to synaptic sites with antigen‐loaded APCs (Harazaki et al.,2004), and thus SPA‐1most likely plays a role in restraining Rap1 activation afterestablishing efficient conjugations with APCs to yield optimal T cell activation.

CTLA‐4 has a higher affinity for CD80/86 than CD28 and exerts a negativesignal for Tcell activation, hence playing an important role in terminating Tcellresponses (Greenwald et al., 2005). Although CTLA‐4 expression is enhanced atthe late stages following T cell activation, significant CTLA‐4 expression isobserved in naive T cells (Schneider et al., 2005). A study has reported thatCTLA‐4 stimulation on naive T cells results in strong Rap1 activation to inducepotent activation of LFA‐1‐mediated cell adhesion (Schneider et al., 2005).Thus, CTLA‐4 may also contribute to synapse formation of T cells with APCs.


In contrast to CD28, however, concomitant stimulations of T cells with anti‐CD3 and anti‐CTLA‐4 antibodies potently inhibit Tcell activation by recruitingprotein tyrosine phosphatases via ITIM motif in the cytoplasmic domain(Greenwald et al., 2001). A study has demonstrated that the T cells fromRapGAP transgenic mice, which show compromised Rap1 activation, displaysignificant ERK activation by CD3/CD28/CTLA‐4 coligation, while ERK acti-vation is strongly suppressed in control T cells by the same stimuli (Dillon et al.,2005). Accordingly, T cells from the transgenic mice showed significantly lowerinhibition of IL‐2 production than control T cells by CTLA‐4 coligation,although both exhibited comparable inhibition of PLC‐g1 phosphorylation(Dillon et al., 2005). The results clearly suggest that part of the negative effectson T cell activation by CTLA‐4 ligation, especially on ERK activation and IL‐2production, is mediated by Rap1 activation.T cell anergy is a unique state, where T cells are incapable of producing IL‐2

and expanding clonally in response to antigens and is thus considered to playa role in peripheral T cell tolerance to self‐antigens (Schwartz, 1997). T cellanergy has originally been described as an in vitro phenomenon, where TCRoccupancy in the absence of costimulatory signals renders the cell nonrespon-sive, even to properly presented antigens with costimulatory signals. Theanergic state can be reversed at least partially by the addition of exogenousIL‐2, thus indicating that a major defect is in the TCR‐mediated IL‐2 produc-tion (Schwartz, 1997). Intensive analyses of anergic T cells have revealed twodominant biochemical features distinctly different from those of normalT cells. First, TCR‐mediated activation of the Ras–ERK pathway is severelyimpaired in anergic T cells, resulting in defective generation of the AP‐1complex, while other pathways (e.g., PLC‐g1 activation and so on) remainlargely intact. It is controversial whether Ras activation bypassing the TCRsignal (e.g., PMA) would restore ERK activation (Schwartz, 1997). Second,there is evidence that IL‐2 gene transcription is strongly repressed by cis‐acting elements in anergic T cells, although the exact nature of repressionremains to be elucidated.Boussiotis et al. (1997) have first reported that in vitro anergized Tcells reveal

constitutive activation of Rap1, which in fact is responsible for the defectiveRas activation and IL‐2 gene activation on CD3 and CD28 stimulations. Theyhave further suggested that constitutive phosphorylation of Cbl by Fyn and itsassociation with CrkL‐C3G may be involved in Rap1 activation of anergic T cells(Boussiotis et al., 1997). However, it has been documented that Cbl, whichpossesses an E3 ubiquitin ligase activity (Joazeiro et al., 1999), elicits ubiquitinmodification of CrkL and negatively regulates C3G recruitment and Rap1 activa-tion (Shao et al., 2003). Thus, the involvement of Cbl in constitutive Rap1activation in anergic T cells remains to be verified. Nonetheless, constitutive


Rap1 activation has also been observed in T cell populations in vivo with aner-gic features such asCD4þCD25þ andCD4þCD103þ regulatoryTcells (Li et al.,2005a,b). Although normal T cells barely express B‐Raf, T cells ectopicallyexpressing B‐Raf apparently escape anergy induction in vitro by APC stimulationof low B7 expression in association with potent Rap1 and ERK activation(Dillon et al., 2005). In addition, CTLA‐4‐deficient T cells have been shown toresist anergy induction in vivo as well (Greenwald et al., 2001). Collectively,the sustained Rap1 signal downstream of TCR stimulation plays a role in in-ducing and maintaining an anergic state in T cells, which do not express B‐Raf.Downstream effects of Rap1 in maintaining the anergic state warrant furtherinvestigations.

We have previously reported that the T cells with CD44high memory pheno-type in aged SPA‐1�/� mice selectively exhibit constitutive accumulation ofRap1GTP in vivo and manifest markedly compromised proliferation and IL‐2production via TCR stimulation (Ishida et al., 2003b; Fig. 2). We have recentlyfound that the anergic CD44high CD4þ T cells are in fact accumulated innormal mice as well with aging, albeit the extent is less than that observed inSPA‐1�/� mice (our unpublished observation). The hyporesponsiveness ofCD44high SPA‐1�/� T cells is probably attributed to impaired ERK activationby TCR‐stimulation despite the induction of normal Ras activation (Ishidaet al., 2003b). The results reinforce that downregulation of the Rap1 signal bySPA‐1 following antigen stimulation may be critical in preventing an anergicstate from occurring in primed T cells. By bypassing TCR stimulation withPMA and Ca2þ ionophore, such anergic T cells still show compromised prolif-eration and IL‐2 production, and thus, Rap1‐mediated interference with theRas–ERK pathway alone may not be able to fully account for the anergic state.A report has suggested that the specifically expressed Tob (an antiproliferativeprotein familymember gene) in anergic Tcells may be responsible for IL‐2 generepression by acting as a cofactor of Smad2/4 (Tzachanis et al., 2001). Therefore,the involvement of persistent Rap1 activation in the constitutive repression ofIL‐2 gene warrants further studies.

Importantly, the anergic Tcells in SPA‐1�/� and normal agedmice are confinedto a specific subset of CD44high T cells, and the proportions are drasticallyincreased in SPA‐1�/� mice that have developed frank leukemia (our unpub-lished observation). Studies have demonstrated that tumor‐specific T cells areanergized in hosts bearing experimental tumors with potent immunogenicity,albeit they may be efficiently generated (Willimsky and Blankenstein, 2005).Understanding the mechanisms of T cell anergy in tumor‐bearing hosts may becrucial for controlling malignancy, and SPA‐1�/� mice should provide a reli-able model to investigate the interaction between the immune system and thenaturally occurring tumor factors in vivo.


3.4. B Cell Development and Self‐Tolerance

Rap1 is activated by BCR stimulation in B cells as well (McLeod et al., 1998).While it has been reported that BCR‐induced Rap1 activation inhibits PI3K‐dependent AKT activation without affecting ERK activation in a B cell line(Christian et al., 2003), the physiological role of the effect remains unknown.A unique role of the Rap1 signal in development and function of B‐lineage cellsin vivo has again been uncovered using SPA‐1 knockout (KO) mice. SPA‐1 KOmice show preferential increases in peritoneal B1a cells (CD5þ Mac‐1þ B220þ

IgMhigh) with aging, accompanied by development of antinuclear antibodiessuch as anti‐dsDNA antibody (Ishida et al., 2006). While autoantibodies areof the IgM class in young SPA‐1 KO mice, significant IgG and IgA autoanti-bodies develop in elder mice to eventuate characteristic lupus‐like immunecomplex glomerulonephritis (Ishida et al., 2006). It has been documented thatB1a cells are responsible for the production of anti‐dsDNA antibodies. Perito-neal B1a cells of SPA‐1�/� mice displayed marked accumulation of Rap1GTPand were actively cycling, indicating that the cells were activated by constitu-tive self‐antigens in vivo. Rather surprisingly, however, the SPA‐1�/� peritonealB1a cells did not show enhanced proliferation via BCR stimulation, instead arather compromised response was manifested. Thus, unlike hitherto reportedmany mutant mice that developed lupus‐like autoimmune diseases, autoim-munity in SPA‐1�/� mice is not attributed to intrinsic BCR‐hyperreactivity ofB cells.B cells of SPA‐1�/� mice, however, revealed significantly altered BCR reper-

toire of the Vk genes as compared to those of control mice (Fig. 3). Studies haveindicated that unexpectedly high proportions of the newly emerged immatureB cells (>50%) in BM are autoreactive (Wardemann et al., 2003) and receptor‐editing plays a major role in negating the autoreactivity (Casellas et al., 2001).Receptor‐editing primarily involves Ig light (L)‐chain genes taking advantage ofthe fact that Vk/Jk gene rearrangements may occur repetitively unlike Ig heavychain genes because of the absence ofD gene segments.OcaB,which controls therecombination and expression of selected Vk genes as a transcriptional cofactor ofOct1,2, plays a crucial role in receptor editing (Casellas et al., 2001, 2002). It hasbeen revealed that the Rap1 signal induces transcriptional activation of OcaB viap38MAPK‐dependentCreb activation inB cells, and in fact immatureBMBcellsof SPA‐1�/� mice with excessively enhanced Rap1GTP levels exhibit augmentedOcaB gene expression (Ishida et al., 2006). The results suggest that the Rap1signal generated by the ligation of BCR with self‐antigens in self‐reactive imma-ture BMB cells may play a role in receptor editing via OcaB gene activation. Theexpected consequences of excessive Rap1 activation in SPA‐1�/� immatureB cells might be twofold: (1) it may cause skewing of the Vk gene usage toward

Pro-B

Pre-B

Immature B

Non-self-reactive

Transitional B Follicular B

Self-reactive Rap1

p38MAPKOcaB

Complete receptor editing

B1a cellsperitoneal cavitymarginal zone

IgL allelicinclusion

Genetic hit?

Pathogenicautoantibodies(Anti-dsDNA IgG)

B-CLL

IgkIgl

Apoptosis

Partialreceptor editing

Self-antigens

Self-antigens

Class switchaffinity maturation

B2

B1a

B1a

Hemolyticautoantibody

Figure 3 Rap1 signal may function as a ‘‘self‐sensing’’ signal in immature bone marrow B cells andcontrol the editing of self‐reactive BCR. Self‐reactive immature B cells in BM are tolerated byseveral distinct mechanisms including clonal deletion and receptor editing. On stimulation withself‐antigens, they may undergo repetitive rounds of Vk‐gene expression and rearrangement untilthe self‐reactivity of BCR is negated by a new Igk chain (editor Vk), viz., complete receptorediting. Rap1‐mediated p38MAPK‐dependent OcaB gene activation plays an important role in theexpression and rearrangement of selective Vk gene. With excess Rap1 activation in the absence ofSPA‐1, repetitive Vk‐gene rearrangement may proceed to Vl‐gene rearrangement, leading toallelic inclusion of Ig light chain genes. Such partial receptor editing may generate B cells withsignificantly reduced yet potential self‐reactivity. Such partially receptor‐edited B cells are deliv-ered preferentially to certain privileged sites, such as the peritoneal cavity, to become B1a (CD5þ

CD11aþ) cells. Since these B1a cells retain potential self‐reactivity, they may progress to producepathogenic autoantibodies (such as anti‐dsDNA IgG) following class switching and affinitymaturation once triggered in the periphery. Repetitive stimulations of the B1a cells by constitutiveself‐antigens may also predispose them to B1‐cell leukemia resembling human B cell chroniclymphocytic leukemia (B‐CLL) associated with autoantibody production.


the Vk genes with higher OcaB dependency (such as the most frequently utilizedVk4 gene inmouse anti‐dsDNA antibodies; Liang et al., 2003), a finding which infact has been demonstrated in SPA‐1�/� mice (Ishida et al., 2006); and (2)excessive Rap1 signals may abnormally accelerate Vk gene recombination andexpression in SPA‐1�/� self‐reactive immature B cells. Normally, receptor editingis completed by rearrangement and expression of rare editor Vk genes to replace


the Vk genes involved in self‐reactive BCR. Excessive Rap1 signals and sustainedOcaB overexpression, however, may result in incessant Vk gene rearrangementswith ineffective editing (due to the preference for particular Vk genes), eventuallyleading to Vl gene rearrangement and expression. In fact, significant proportionsof the peritoneal B1 cells in the SPA‐1 KO mice revealed an allelic inclusion(Ishida et al., 2006), expressing both the Vk‐IgL and Vl‐IgL‐chains indicative of‘‘partial editing’’ (Fig. 3). The results suggest the important role of Rap1 inmediating the ‘‘self‐sensing’’ signal downstream of BCR in the newly derivedimmature BM B cells.The origin of B1 cells has long been a matter of argument. In mice, the

polyspecific B1 cells are supposed to have originated at the embryonic stageand subsequently segregated in the peritoneal cavity, where they can be self‐renewed (Hayakawa and Hardy, 1988). Such B1 cells play important roles ininnate immunity against bacterial infections by producing natural IgM anti-bodies broadly reactive to the various bacterial antigens (Coutinho et al.,1995). Although it has been known that pathogenic autoantibodies in systemicautoimmune diseases are also derived preferentially from B1 cells, their exactorigin remains largely unresolved. B cells expressing transgenic anti‐dsDNABCR, for instance, are segregated and distributed in marginal zones ratherthan in the follicles of spleen (Li et al., 2002). Previous report also suggestedthat VH gene usage might primarily determine the B1/B2 fates of the B celldevelopment using VH gene transgenic models (Lam and Rajewsky, 1999).Thus, the unique features of pathogenic autoreactive B cells being defined asB1 cells may be primarily attributed to BCR‐specificity per se rather than todistinct lineage. B1 cells in SPA‐1�/� mice exhibit remarkably high expressionlevels of b1‐integrin (unpublished results), and the Rap1 signal may as wellcontrol the unique distribution pattern of B1 cells having potentially pathogenicautoreactivities.Notably, a minor portion of SPA‐1�/� mice (ca. 10%) eventually developed

characteristic leukemia of CD5þ Mac‐1þ B220þ phenotypes corresponding toB1a cells with hemolytic autoantibodies (Ishida et al., 2006). Marked increasesin CD5þ B cells with high frequencies of autoimmunity (such as hemolyticanemia and autoimmune thrombocytopenia) are a hallmark of human B cellchronic lymphocytic leukemia (B‐CLL), and thus B cell leukemia in SPA‐1�/�

mice is highly reminiscent of human B‐CLL. Furthermore, leukemic B cells insome SPA‐1�/� mice revealed chromosomal translocation involving the Igl‐chain gene, t(2;6), and Igk‐chain gene, t(2;16) (Ishida et al., 2006). We there-fore propose that enhanced receptor editing and persistent stimulations byconstitutive self‐antigens of self‐reactive B1 cells may have predisposed themto the eventual leukemic transformation. Dysregulated Rap1 signals in theB‐lineage cells may provide a link between autoimmunity and B‐CLL (Fig. 3).


3.5. Lymphocyte Migration and Homing

Lymphocytes generated andmaturated in primary lymphoid organs continuouslyemigrate via the blood circulation to specific secondary lymphoid tissues.Homingof naive lymphocytes to particular areas in the secondary lymphoid tissues andmigration of primed or effector lymphocytes to local tissues where antigens existare crucial events in immunosurveillance. Attraction of lymphocytes by specificchemokines and transendothelial migration plays a major role in the lympho-cyte migration and homing to secondary lymphoid organs. The first step ofchemokine‐induced transendothelial migration is the rolling of lymphocytes viaselection followed by firm adhesion to endothelial cells against shear flow inresponse to specific chemokines (Butcher and Picker, 1996; Springer, 1995).The latter depends on integrin (LFA‐1, VLA‐4) activation and strong adhesionto their ligands (ICAM‐1, VCAM‐1) expressed on endothelial cells (Wittchenet al., 2005). Chemokine gradient induces the polarized accumulation of integrinsat the leading edge, while CD44 is mobilized to the uropod (Katagiri et al., 2003;Shimonaka et al., 2003). This polarity is vital for subsequent lymphocyte migra-tions across the endothelial cells (diapedesis). The direct interaction of LFA‐1with JAM‐1, another Ig superfamily (IgSF) protein located at the apical part of theendothelial adherence junction near the TJ, is also involved in diapedesis andmay‘‘unlock’’ the homotypic intercellular junction to guide the lymphocytes duringtransmigration (Ostermann et al., 2002).

Stimulation of the lymphocytes with specific chemokines (e.g., SLC andSDF‐1) causes rapid Rap1 activation, and all the events required for transen-dothelial migration (including adhesion, polarization, and diapedesis) arepotently inhibited by overexpression of SPA‐1 or a dominant‐negative Rap1mutant, indicating the essential role of Rap1 signals (Shimonaka et al., 2003).Recent reports have indicated that a Rap1 effector (RapL) plays a major role inlymphocyte migration (Section 1.2.2). RapL�/� mice show significant atro-phies of the secondary lymphoid organs associated with increased circulatinglymphocytes, indicating their impaired homing to lymphoid organs (Katagiriet al., 2004). Furthermore, RapL�/� mice have indicated reductions of matu-rated T cell migration from the thymus as well as impaired migration of theskin DCs to regional LNs by inflammatory stimuli (Katagiri et al., 2004). Theseresults thus clarify the essential roles of the Rap1 signal in constitutive andinflammation‐induced lymphocyte migrations and trafficking.

In a human‐inherited disease called leukocyte adhesion deficiency (LAD)syndrome, affected patients show persistent leukocytosis and life‐threateningbacterial infections due to defective leukocyte adhesion to blood vessels andtransmigration. Of the various subtype LAD patients, a majority (type‐I LAD)indicates germline mutations with impaired expression and function in the


b2‐integrin (CD18) gene (Anderson and Springer, 1987). A clinical encounterwith a rare autosomal recessive LAD syndrome (type‐III LAD), whereb2‐integrin expression and intrinsic adhesive activities of lymphocytes areapparently normal, has added an intriguing dimension to the complexity ofLAD syndrome (Alon and Etzioni, 2003). The lymphocytes from type‐III LADpatients display severely compromised Rap1‐mediated integrin activation inresponse to chemokines, although chemokine receptor signaling per se as wellas PMA‐induced Rap1 activation remains normal (Kinashi et al., 2004). Whilethe reasons for impaired Rap1 activation in response to chemokines and therelevant causative gene remain to be identified, the results strongly suggestthat functional defects in chemokine receptor‐coupled Rap1 activation areresponsible for type‐III LAD syndrome in humans.

4. Rap1 Signal in Hematopoiesis and Leukemia

SPA‐1 is most prominently expressed in the BM, in particular in the immaturehematopoietic cell population, implicating a requirement of tight control ofRap1 signal in them. Analysis of SPA‐1 KO mice disclosed unexpected yetimportant role of the Rap1 signal in regulating normal hematopoiesis (Table 1).

4.1. Hematopoietic Stem Cells and the Niche

Hematopoietic stem cells (HSCs) fulfill two opposing features: viz., per se self‐renewal without differentiation and the ability of differentiating to all lineagesof mature blood cells. Constitutive hematopoiesis depends on the homeostaticbalance of HSCs between self‐renewal and differentiation. Accumulatingevidence indicates that HSCs homeostasis is maintained by intimate interac-tions of HSCs with a specific hematopoietic microenvironment called theniche (Calvi et al., 2003; Whetton and Graham, 1999; Zhang et al., 2003).The role of Rap1 in maintaining stem cells in the niche has been wellillustrated by the male germ stem cells (GSCs) of Drosophila. In testes ofthe fruit fly, a cluster of 10–12 cells (or ‘‘hub’’) forms the niche for GSCs(Fuchs et al., 2004). When a GSC divides, one daughter cell remains anchoredto the hub cells, while the other drifts away from the hub and differentiates toform a gonialblast. Hub cells produce growth factors, such as Upd (unpaired)and Gbb (glass bottom boat), to regulate self‐renewal of GSCs, which havepreviously anchored to the hub through DE‐cadherin‐mediated cell adhesionto receive these signals (Yamashita et al., 2003). A genetic study has revealedthat the defect of Rap1 GEF (Gef26) or Rap1 causes the reduced formation ofadherens junctions at the hub–GSC interface, resulting in GSC loss due toexhaustive differentiation (Wang et al., 2006). Thus, the Rap1 signal plays amajor role in maintaining GSCs in the niche.


In mouse BM, spindle‐shaped N‐cadherinþ osteoblastic cells on the surfaceof cancellous or trabecular bone are the primary candidates of niche stroma forHSCs (Zhang et al., 2003). A previous study has demonstrated that c‐Myc‐deficient HSCs with enhanced expression of N‐cadherin and integrins aremarkedly accumulated in the niche with reduced differentiation. On thecontrary, c‐Myc overexpression accelerates the HSC release from the nicheand thereby promotes their differentiation to eventuate stem cell exhaustion(Wilson et al., 2004). Thus, the control of HSC adhesive molecules may play acrucial role in maintaining HSCs in a niche microenvironment to regulate thesignals for homeostatic balance between self‐renewal and differentiation ofHSCs (Fig. 4). SPA‐1�/� mice consistently display gradual increases of HSCcounts in BM with aging to eventually suffer overt myeloproliferative disorders(MPDs; see below). Furthermore, the diseased SPA‐1�/� mice have revealedmarked increases in the HSC population excessively expressing LFA‐1, result-ing in premature HSC mobilization out of BM to subsequently induce massiveextramedullary hematopoiesis (Kometani et al., 2006). These results indicatethat control of the Rap1 signal by SPA‐1 is crucially involved in the regulationof HSC interaction with the niche.

SDF‐1 produced by BM stroma cells is a major chemotactic factor involved inHSC migration and homing to a hematopoietic microenvironment (Nagasawaet al., 1996). The expression of SDF‐1 receptor (CXCR4) on human CD34þ

hematopoietic progenitors is reportedly enhanced by the cAMP‐induced Rap1-signal (Goichberg et al., 2006). TheRap1 signal also plays amajor role in SDF‐1‐mediated activation of b1‐integrins (Shimonaka et al., 2003), which is essentialfor migration and homing of HSCs to the hematopoietic microenvironment(Potocnik et al., 2000). We recently found that HSCs overexpressing SPA‐1 ordominant‐negative Rap17A mutant showed reduced engulfment when trans-planted into irradiated mice compared with control cells (our unpublisheddata). Therefore, Rap1 is most likely involved in HSC migration and homingto BM microenvironments or the niche, which is essential for the successfulhuman BM transplantation.

4.2. Dysregulated Rap1 Signal and Myeloproliferative Disorders

A vast majority of SPA‐1�/� mice eventually developed marked peripheral leuko-cytosis and massive splenomegaly with extensive extramedullary hematopoiesis intheir second year (Ishida et al., 2003a; Kometani et al., 2004; Fig. 4). Althoughwell‐differentiated granulocytes usually predominated in the blood of thesemice,CFU‐C assays have revealed increases in hematopoietic cells of all lineages, sug-gesting inductions of dysregulated expansion and differentiation of multipotenthematopoietic progenitors. A significant proportion of SPA‐1�/�mice additionally

HSC

MHPCHP

Matureblood cells

HSC

MHP

Blast

CML

Blast crisis

Nichestroma

Growth factors

Premature immobilization

N-Cad LFA-1

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metastasis

Normal medullary hematopoiesis

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Rap1

A

B

LFA-1N-Cad

LFA-1

CHP

BCR-ABL

Rap1

BM

BM

Spleen

Chronicphase

Figure 4 Control of homeostatic hematopoiesis by Rap1 and myeloid leukemia in SPA‐1 defi-ciency. Self‐renewal and differentiation of hematopoietic stem cells (HSCs) are controlled by theintimate interaction with niche stroma cells in BM via coordinated balance of adhesion molecules,including N‐cadherin (N‐Cad) and migratory integrins such as LFA‐1 (A). SPA‐1 is a principalRap1GAP expressed in HSCs and multipotent hematopoietic progenitors (MHPs), and persistentRap1 activation in SPA‐1�/� HSCs and MHPs results in their accelerated expansion and differen-tiation. In addition, SPA‐1�/� MHPs show strong expression of LFA‐1, and prematurely abandonthe bone marrow to initiate extensive extramedullary hematopoiesis in the spleen. SPA‐1�/� miceeventually develop myeloproliferative disorders (MPD) that resemble chronic myelogenous leuke-mia (CML) in the chronic phase. During the process, blast crisis may occur at any committedhematopoietic progenitors (CHP) to cause either myeloid or lymphoid acute leukemia. Rap1 isconstitutively activated in such blast cells as well and may play a significant role in their aggressive



accommodated variable extents of blastic cells of either myeloid or lymphoidlineages, which infiltrated in vital tissues (Ishida et al., 2003a). These featureshighly resemble human CML in the chronic phase and acute crisis. In addi-tion, a minor portion of mice developed severe anemia and pancytopenia oftenassociated with dysplastic or blastic leukocytes, and the phenotypes coincidedwell with the human myelodysplastic syndrome (MDS). The latent periods ofMPD were usually long (ca. 12 months), and secondary genetic events mightaffect the final disease phenotypes, especially in blast crisis. Despite theapparent diversity of MPD, diseased SPA‐1�/� mice share common features,including accumulation of Rap1GTP in the HSC‐enriched BM cell fraction,selective increase in HSC population with excessive LFA‐1 expression in BM,and marked HSC mobilization to the spleen (Kometani et al., 2006). Alto-gether, these findings strongly suggest that HSC disorders are the causativefactors underlying MPD (Fig. 4).

CML represents leukemia of HSCs, and leukemic stem cells generateincreasing numbers of various types of mature blood cells (Ren, 2005). Simila-rities and differences between normal and leukemic HSCs are the fundamen-tal issue in CML pathogenesis. Although it has been reported that enhancedself‐renewing capacity of HSCs in several mutant (such as Lnk�/�, c‐Myc�/�,and p18INK4C�/�) mice yields marked increases in the HSC population,these mice do not develop overt MPD (Takaki et al., 2002; Wilson et al.,2004; Yuan et al., 2004). On the other hand, a report has indicated thatconditional deletion of Pten gene in HSCs result in the development ofacute MPD (Yilmaz et al., 2006; Zhang et al., 2006a). Pten is a phosphatasethat converts PIP3 to PIP2 and negatively regulates the PI3K‐signalingpathway (Cully et al., 2006). Pten�/� mice indicate progressive reductions inself‐renewing HSCs due to their accelerated differentiation and peripheralmobilization, resulting first in massive extramedullary hematopoiesis rapidlyfollowed by blast crisis in association with frequent chromosomal transloca-tions (Yilmaz et al., 2006; Zhang et al., 2006a). The overall features of MPD inconditional Pten�/� mice resemble those of SPA‐1�/� mice, except for moreacute development in the former. Thus, the accelerated drive of HSCs indifferentiation and premature mobilization (with or without the reduced self‐renewing HSCs) seems to be the common features of CML‐like MPD, and itwould be of interest to investigate whether SPA‐1�/� and Pten�/� mice, inpart, share the dysregulation of signaling pathways in HSCs, particularly thePI3K–AKT pathway.

invasion into many vital tissues. In humans, the BCR‐ABL fusion gene from the Philadelphiachromosome is a major cause of CML and constitutive Rap1 activation downstream of BCR‐ABLoncoprotein may participate in molding phenotypes of human CML.


In humans, the vast majority of CML is caused by BCR‐ABL oncoproteingenerated by chromosomal translocation t(9;22). All lineages of maturatedperipheral leukocytes have an identical chromosomal anomaly, indicatingthat MPD is due to the leukemic transformation of an HSC clone (Wongand Witte, 2004). BCR‐ABL protein delivers a diverse array of signals viaconstitutive ABL tyrosine kinase activity, including the Ras–ERK, PI3K–AKT,and Stat5–Bcl2 pathways, as well as integrin activation (Jin et al., 2006;Sonoyama et al., 2002; Wong and Witte, 2004). Although all these signalsmay contribute to various aspects of leukemic features, the PI3K pathwayapparently plays a prominent role in terms of leukemogenesis in vivo becausethe BCR‐ABL mutant gene (lacking a domain responsible for PI3K activation)then loses the leukemogenic activity (Sattler et al., 2002). Evidence has indi-cated that Rap1 is activated constitutively by BCR‐ABL via recruitment andphosphorylation of C3G (Cho et al., 2005), or partial repression of SPA‐1 geneexpression (Kometani et al., 2006), or both. Most notably, SPA‐1 overexpressionhas significantly inhibited PI3K/AKT activation in BCR‐ABLþ cells (Jin et al.,2006). To directly examine the role of Rap1 signals in BCR‐ABL‐induced CML,we compared the leukemic phenotypes between the normal and SPA‐1�/�

progenitors transduced with BCR‐ABL oncogene in a mouse model. Thefindings demonstrated that, while both progenitors caused CML in the primaryrecipients, SPA‐1�/� leukemic progenitors persisted longer than the controlin vivo when judged by the serial transfer experiment (Kometani et al., 2006).In addition, significant proportions of the former showed blastic crisis, support-ing a role of the endogenous Rap1 signal in BCR‐ABL‐induced CML genesis inthe recipients. In some juvenile CML patients, loss of heterozygosity in NF1gene encoding a RasGAP has been observed (Shannon et al., 1994) and in factNF1þ/� mice have developed CML with a long latency of over a year (Jackset al., 1994). Due to enhanced activation of the Ras–ERK pathway, NF1�/�

cells consequently develop hyperresponsiveness to GM‐CSF (Bollag et al.,1996; Largaespada et al., 1996). In contrast, SPA‐1�/� CML cells show un-changed responsiveness to hematopoietic growth factors such as GM‐CSF(Ishida et al., 2003a), and thus deficiencies of SPA‐1 (Rap1GAP) and NF‐1(RasGAP) induce CML‐like via distinctly different mechanisms.Current literature advocates the dependence of CML‐genic potential of BCR‐

ABL on complex interactions with the intrinsic self‐renewing potential of HSCs(Huntly et al., 2004). This is of particular clinical significance because imatinibmesylate (Gleevec; a potent inhibitor of ABL kinase activity) that can rapidlyreduce the massive burden of leukemic leukocytes fails to eradicate the CMLstem cells, so‐called residual diseases (Michor et al., 2005). Under such circum-stances, most of the patients eventually develop recurrence of lethal aggressiveleukemia. SPA‐1 is among the gene set of the murine self‐renewal‐associated


signature and highly enriched in human leukemic stem cells as well (Krivtsovet al., 2006), and thus it seems likely that BCR‐ABL connects with the intrinsicRap1 signal in HSC, at least in part, to cause CML. The crucial target cells forcontrolling CML are the leukemic stem cells (Huntly and Gilliland, 2005), andRap1 may provide a rational molecular target for the eradication of CML inhumans.

4.3. Role of Rap1 in Generation and Function of Platelets

Among the various blood cells, the role of Rap1 signals in generation andfunction of platelets has been most extensively studied (Stork and Dillon,2005). Maturation of megakaryocytes depends on thrombopoietin acting onthe Mpl receptor to induce sustained ERK activation (Garcia et al., 2001).However, erythropoietin and GM‐CSF induce proliferation, but not matura-tion, of megakaryocytes, and such an action is associated with transient ERKactivation (Stork and Dillon, 2005). This represents another event where theRap1 and Ras signals mediate the distinct modes of ERK activation to inducedifferent effects (Section 1). BM stroma cells inhibit differentiation of mega-karyocyte progenitors by direct contact, an effect which has been elicited byinhibition of Rap1‐mediated persistent ERK activation (Delehanty et al.,2003). A specific type of b3‐integrin (aIIbb3), which is expressed selectivelyby platelets, plays essential roles in platelet function (e.g., aggregation andadhesion) related with homeostasis and thrombus formation. The abundantlyexpressed Rap1 in platelets is activated by many stimuli (e.g., turbulence,epinephrine, ADP, thrombin, thromboxane A2, and platelet‐activating factors)to cause platelet activation via aIIbb3 activation. CalDAG‐GEF I (RasGRP2)is responsible for Rap1 activation in platelets, and a report has revealeddefective aIIbb3‐mediated platelet aggregation in CalDAG‐GEF I�/� miceto induce markedly impaired haemostasis (Crittenden et al., 2004).

5. Rap1 Signal in Malignancy: New Aspects in Cancer

In spite of the highly convergent homology with classical Ras, there have beenlimited experimental findings implicating Rap1 to function as an oncoprotein.In 1998, however, Altschuler and Ribeiro‐Neto (1998) have revealed certainunique roles of the Rap1 signal in cell transformation; Rap1‐transfected Swiss3T3 fibroblasts are flatter and spread more with a higher saturation density thancontrol cells. Although cell growth could be strongly enhanced by cAMP or EGF,the Rap1‐transfected cells showed no anchorage‐independent growth, and thusthe cells revealed no evidence of transformation in vitro when viewed under aclassical criterion. Surprisingly, however, the cells formed tumors in nude mice


(Altschuler and Ribeiro‐Neto, 1998). This presents a very unusual phenomenon,where cells are anchorage‐dependent in vitro and yet tumorigenic in vivo. Theresults suggest that Rap1 caused tumors in vivo by constitutive interaction with,rather than bypassing, the intrinsic growth pathways of host cells. In principle, theevent may be coincidental with BCR‐ABL‐induced CML‐genesis, wherebyBCR‐ABL oncoprotein promotes the dysregulated expansion of HSCs by inter-acting with the intrinsic self‐renewal feature of HSCs (Huntly et al., 2004).According to a recent study, certain human squamous cell carcinoma cells showhigh levels of Rap1GTP. Rap1GAP transduction reportedly represses tumorigen-esis of such cancer cells in nude mice (Zhang et al., 2006b). These results suggestthat Rap1 may act as a ‘‘conditional’’ oncoprotein.Hitherto, the Rap1 signal has been documented to affect the invasiveness

and metastasis of tumors in vivo. Mutations of DOCK4 gene encoding a Rap1activator have been reported in certain human cancer cells (Yajnik et al., 2003).The mutant DOCK4 protein exhibits a dominant‐negative effect to incitedefective Rap1 activation in such cancer cells, resulting in the loss of intercel-lular adhesion among these abnormal cells. As a result, these cancers wouldmanifest highly invasive behavior in vivo. Intriguingly, introduction of a wild‐type DOCK4 gene restores the adherence junction among the cancer cells,and concomitantly represses the invasive tendency as the basal Rap1GTP isrestored (Yajnik et al., 2003). It has also been reported that the Rap1 signal incancer cells may play a critical role in metastasis. Employing the model ofspontaneous development of mammary tumors in transgenic mice of polyomamiddle T‐antigens under an MMTV promoter, Hunter and the colleagues haveindicated that genetic polymorphism of the SPA‐1 (also called SIPA‐1) gene inthe host is a major determinant for lung metastasis of primary mammarytumors (Park et al., 2005). Thus, mouse strains with SPA‐1/741A alleles displayextensive lung metastases, whereas far less lung metastases are observed inthose with SPA‐1/741T alleles. Interestingly, no significant differences ingrowth of the primary tumors are established between mice with a differentallele. The single amino acid polymorphism at position 741 in the PDZ domainof SPA‐1 protein affects Rap1GAP activity in cancer cells, with SPA‐1/741Abeing more active than SPA‐1/741T (Park et al., 2005). On the basis of thesefindings, the reduced Rap1 signal in cancer cells might favor metastasis inaddition to local invasiveness. In fact, these findings serve as the first directindication that the host genetic background can affect the metastatic behaviorof cancers (Threadgill, 2005). A report confirmed that certain SPA‐1 genehaplotypes are significantly associated with the presence of lymph nodemetastasi s and poo r prognosi s in huma n mamma ry cance rs (Crawford et al.,2006). Rec ently, we ha ve also confirme d that pro state cancer cells in primary


sites of patients with metastases exhibit expression of SPA‐1 protein signifi-cantly higher than those without metastasis (our unpublished observation). Inother words, the Rap1 signal in cancer cells may partially control malignantinvasiveness and remote metastasis by regulating intercellular adhesion amongthe cancer cells.

6. Conclusions and Perspectives

Extensive and intensive studies in recent years have unveiled unique biologicalactivities of Rap1. Twomajor activities of the Rap1 signal have been established:(1) control of cell–matrix and cell–cell adhesions via activation of integrins andother cell adhesion molecules and (2) regulation of the activation of variousMAPKs. Rap1 is activated by an extensive spectrum of extracellular stimuli viamany types of specific GEFs coupled with certain specific receptor systems, andthe activation status is tightly controlled by GAPs at different intracellularcompartments. Through such activities, Rap1 is involved in a range of diversecellular functions far more than originally anticipated. Unique and unanticipatedroles of the Rap1 signal in vivo have recently been uncovered by extensiveanalyses of gene‐targeted mice for Rap1 regulatory molecules. The Rap1 signalhas been demonstrated to play crucial roles in diverse aspects of the develop-ments and functions of immune and hematopoietic cells. Furthermore, Rap1dysregulation causes characteristically specific diseases, with highly resemblinghuman conditions. We anticipate that further analyses will reveal more as of yetundocumented important roles of the Rap1 signal in other biological systemssuch as the nervous and endocrine systems and malignant cells. While Rap1 isexpressed ubiquitously in most tissue cells in the body, predominant roles of theRap1 signal can be highly variable—depending on the contexts of specific celltypes and functions. This signaling molecule with multifaceted functional varia-bility provides a typical example, where a ubiquitous molecule may be cruciallyintegrated into the highly specified and sophisticated functions of manybiological events in a complex living system. Regulatory molecules of theRap1 signal may also serve as potentially reliable and rational molecular targetsfor controlling various human diseases including malignancy.

Acknowledgments

The authors are grateful to all personnel in the Department of Immunology and Cell Biology,Graduate School of Medicine and Graduate School of Biostudies, Kyoto University. In particular,we would like to thank Drs. D. Ishida, Li Su, Hailin Yang, Y. Hamazaki, Y. Shinozuka,M. Moriyama, M. Aoki, F. Wang, K. Shimatani, Y. Nakajima, and Y. Katayama for their kindcooperation in carrying out the study. This study was supported by Grants‐in‐Aid for ScientificResearch from the Ministry of Education, Science, Culture, Sport, and Technology of Japan.


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