Multiple roles of Rap1 in hematopoietic cells: complementary versus antagonistic functions

Rap1 (Ras-proximate-1) is a small G protein in the Ras superfamily. Rap1 is expressed in cells as one of 2 isoforms, Rap1a and Rap1b. It is most closely related to the 2 isoforms of Rap2 (Rap2a and Rap2b), small G proteins with overlapping but distinct functions.³  Like all G proteins, Rap1 exists in an inactive guanine nucleotide diphosphate (GDP)–bound state and is activated when GDP is exchanged for guanine nucleotide triphosphate (GTP). Rap1, like other small G proteins, is targeted to lipid membranes by the covalent attachment of lipid moieties to the carboxyl terminus via geranyl-geranyl lipids to the terminal cysteine during Rap1 synthesis and transport through the Golgi.⁴  This posttranslational modification anchors Rap1 to both endosomal and plasma membranes.⁴  Movement of Rap1 from endosomal membranes to the plasma membrane upon Rap1 activation has been reported in a number of cell types, including Jurkat T cells and megakaryocytes.^4-6  Upon activation by GTP binding, Rap1 undergoes a conformational change that exposes an effector-binding loop, allowing the recruitment of a variety of potential effectors. This recruitment triggers the signal pathways linking Rap1 to integrin signaling, ERK activation, and other effector pathways,⁷  some of which are shown in Figure 1. In the following sections, we will outline some of the mechanisms that regulate Rap1 activity in hematopoietic cells.

A diverse family of guanine nucleotide exchange factors (GEFs) stimulates the replacement of GDP for GTP to activate small G proteins. Several Rap1 GEFs have been identified (Table 1), enabling Rap1 to respond to a diverse set of stimuli.³  Three representative Rap1 GEFs are depicted in Figure 1.

C3G (RapGEF1), the first Rap1 GEF identified,⁸  is activated through its association via the Src homology 2 (SH2) domains of the Crk family of adaptor proteins, which include CrkI (CT10 regulator of kinase), CrkII, and CrkL.^20,21  In addition to Rap1, C3G may activate other small G proteins, including Rap2, R-Ras, TC21, and the Cdc42-related protein, TC10.³  The inducible association of Crk/C3G to sites of tyrosine phosphorylation at the plasma and intracellular membranes²²  occurs following ligand activation of a variety of receptors, including the receptor tyrosine kinases (RTKs) for epidermal growth factor (EGF), nerve growth factor (NGF), and insulin⁷ ; cytokine receptors for erythropoietin (EPO), interleukin 3 (IL-3),²⁰  interferon-γ,²³  as well as the T-cell receptor (TCR)²⁴  and the B-cell receptor.²⁵  For cytokine receptors, tyrosine phosphorylation is initiated by the Janus kinase (JAK) family kinases.

For both RTKs and cytokine receptors, the phospho-tyrosine residues that provide the docking sites for Crk/C3G can occur on specialized scaffolding molecules, including Cbl (Casitas B-lineage lymphoma proto-oncogene),²⁶  Cas,²⁷  and Gab1.²⁸  Phosphorylation of Crk binding sites on these molecules can be triggered by a variety of tyrosine kinases, including RTKs themselves as well as cytoplasmic tyrosine kinases, including c-src^29,30  and BCR/ABL.³¹ 

Calcium and diacylglycerol (CalDAG)–GEFs comprise another family of GEFs that respond to calcium and diacylglycerol, making them potential targets of phospholipase C (PLC) action (Figure 1). Four CalDAG-GEF gene products have been identified. CalDAG-GEFI, an alternatively spliced form of RasGRP2,¹⁵  is strongly expressed in megakaryocytes and platelets³²  and modestly expressed in neutrophils.³³  It shows preferential activation of Rap1, with some activity toward N-Ras, H-Ras, R-Ras, TC21, and Rap2.¹²  CalDAG-GEFII (RasGRP1) acts on Ras and R-Ras, but not Rap1, and is a critical mediator of TCR activation of Ras and ERKs in lymphocytes.³⁴  CalDAG-GEFIII (RasGRP3) can activate Rap1, Ras, and R-Ras³  and has been implicated in B-cell development.³⁵  RasGRP4 has been identified as a mast-cell–specific GEF that activates Ras.³⁶ 

Another class of Rap1GEFs includes cAMP (cyclic adenosine monophosphate)–GEFs or EPACs (exchange proteins directly activated by cAMP) that are activated by direct binding of cAMP. cAMP-GEFI (EPAC1) is widely expressed,¹¹  while cAMP-GEFII (EPAC2) is enriched in brain and adrenal¹²  and both are selective for Rap1.¹⁴  A role of EPACs has been proposed for Rap1-dependent cell adhesion to laminin in both epithelial cells³⁷  and red blood cells,³⁸  and in the regulation of vascular endothelial barrier function.^39,40 

Rap1 signaling is terminated by the hydrolysis of bound GTP to GDP. This is achieved through specific GTPase-activating proteins (GAPs). The specificity of Rap GAPs for Rap1 is dictated by unique sequences present in both the Rap GAPs and Rap1 that provides specificity for Rap1.⁴¹ 

The principal Rap1GAPs that have been identified in hematopoietic cells are Rap1GAP1 and SPA-1 (signal-induced proliferation-associated gene-1). Rap1GAP1 was originally identified on plasma membranes isolated from differentiated promyelocytic HL-60 cells.⁴²  The expression of SPA-1 is restricted to hematopoietic tissues.^43,44  Its expression is highest in lymphoid organs and regulated during differentiation. Immature bone marrow cells predominantly express SPA-1, whereas mature (lineage-positive) bone marrow cells predominantly express Rap1GAP.⁴⁵  This shift in expression of SPA-1 to Rap1GAP is also seen upon inducing granulocyte differentiation with granulocyte–colony-stimulating factor (G-CSF) of the IL-3–dependent promyeloblastic cell line 32D⁴⁵  and in HL-60 cells.^43,44  Recently, a novel Rap1GAP, RapGAP2, was identified in platelets and lymphocytes.⁴⁶ 

Figure 1 highlights some of the proposed effectors that bind selectively to Rap1GTP. For the purposes of this review, we have limited our discussion to the actions of Rap1 that regulate cell adhesion and the MAP kinase cascade. A more complete listing of Rap1 functions can be found in recent reviews.^1,3,7,47 

One of the best-studied effector pathways of Rap1 is the stimulation of cell adhesion. Activation of cell-surface receptors generate intracellular signals that increase integrin-mediated adhesion, termed “inside-out” signaling. A number of studies confirm a role for Rap1 in this process. It has been proposed that Rap1 activation initiates inside-out signaling to integrins, resulting in conformational changes that convert integrin to high-affinity states. In addition, integrins can undergo changes in lateral mobility that increase their avidity for ligand binding.^48,49  Rap2 has also been shown to play a role in the adhesion and migration of B cells.^50,51 

Inside-out signaling enhances the function of a wide range of integrins including αLβ2 (LFA-1), VLA-4, and VLA-5 on T cells,^49,52-54  αII_bβ₃ on megakaryocytes and platelets,^55,56  CD11b on monocytes and macrophages,⁵⁷  and others.¹  Integrins bind to a variety of ligands found in the extracellular matrix or on the surface of adhering cells.¹  In contrast, “outside-in” signaling refers to intracellular signals that are generated following the binding of integrins with their extracellular ligands.⁴⁸  Rap1 itself is a target of outside-in signaling,^58,59  suggesting the possibility of a positive feedback loop further enhancing integrin function.

A mediator of Rap1-dependent adhesion in T cells has recently been identified as RapL (also called Nore 1b).⁶⁰  RapL is an alternatively spliced product of RassF5 and is highly expressed in immune cells and binds Rap1 in a GTP-dependent manner. In T cells, this binding is stimulated by TCR and chemokine receptor activation. Following binding, it has been proposed to translocate to the plasma membrane, where it binds the T-cell integrin LFA-1 and enhances its avidity/affinity for its ligand intercellular adhesion molecule (ICAM).⁶¹  Expression of interfering mutants of RapL in T cells block Rap1-, TCR-, and chemokine-dependent adhesion to ICAM-1,⁶⁰  and RapL-null lymphocytes show reduced adhesion in both T and B cells as well as defects in homing of lymphocytes and dendritic cells.² 

RapL binds to the cytoplasmic tail of the α chain of LFA-1 to regulate LFA-1 affinity.⁶¹  Upon stimulation, both RapL and LFA-1 relocalize to the leading edge of T cells.⁶⁰  Mutants of LFA-1 that lack RapL binding do not relocalize, but, importantly, they do not prevent RapL from relocalizing, suggesting that the targeting of RapL to the plasma membrane may direct LFA-1 as well. These mutants abolish LFA-1 activation by Rap1 and TCR.⁶¹  Recent studies have also identified a role for RapL in the Rap1-dependent migration of vascular endothelial cells.⁶² 

Another effector of Rap1 implicated in integrin signaling is Rap1-GTP–interacting adaptor molecule (RIAM). RIAM induces β1 and β2 integrin–mediated adhesion in Jurkat T cells and is required for Rap1-dependent adhesion in these cells.⁶³  RIAM also binds both profillin and Ena/VASP (enabled/vasodilator-stimulated phosphoprotein), modulators of the actin cytoskeleton, and this action may participate in the recruitment of Rap1 and possibly LFA-1 to the lymphocyte plasma membrane upon activation.⁵  Rap1 enhancement of cell-cell interaction is not limited to its actions on integrins, and a wide range of adhesion molecules have been implicated in Rap1 action.⁴⁷ 

Rap1 was first identified as an antagonist of Ras-induced transformation in fibroblasts.⁶⁴  The recruitment of Raf-1 to GTP-bound Ras allows Raf-1 to activate the MAP/ERK kinase MEK, which subsequently activates the MAP kinase ERK. Active Rap1 can bind, but not activate Raf-1, and thereby may sequester Raf-1 from the Ras/ERK pathway.^65,66  The antagonism of Ras-dependent activation of Raf-1 has been proposed to mediate some of the antiproliferative actions of Rap1 in T lymphocytes⁶⁷  and other cell types,⁷  and may underlie the role of Rap1 in T-cell anergy,²⁴  although other mechanisms have been proposed.⁶⁸  The expression of Raf-1 is ubiquitous, yet the ability of Rap1 to inhibit the ERK cascade is not universal, even in cells where Raf-1 is the major Raf isoform expressed.⁶⁹ 

The ability of Rap1 to sequester effectors of Ras may also be seen with upstream activators of the p38 MAP kinase. For example, in thymocytes, IL-1 activates p38 in a Ras-dependent manner that is antagonized by Rap1. However, the direct target of Rap1 signaling is not known.⁷⁰  Another pathway in which Rap1 can antagonize Ras function in T cells is the suppression of Ras-dependent reactive oxygen species, an action that allows for T-cell proliferation.⁷¹ 

The effect of Rap1 on ERKs may be indirect. Adhesion of integrins can trigger signals back to the integrin-expressing cell through a variety of signaling cascades,⁷²  collectively referred to as outside-in signaling. Multiple mechanisms for this type of outside-in signaling from integrins to ERKs have been proposed.^73,74  Integrins can activate ERKs via both focal adhesion kinase (FAK)–dependent⁷⁵  and FAK-independent mechanisms.⁷⁴  Cross-talk with growth factor receptors also couples integrins to ERKs.⁷⁶  These pathways are often mediated via Ras, although Rap1-dependent activation of ERKs through integrins has been reported.^77,78 

A major target of the antagonism of Ras and Rap1 signaling is integrin adhesion itself. Ras and Rap1 have opposing actions on integrin-dependent adhesion^79,80  and cadherin-dependent adhesion.⁸¹  The antagonism may partially explain the initial observation that Rap1 antagonized Ras-dependent transformation.⁶⁴ 

Rap1 can also bind a second member of the Raf family, B-Raf.⁸²  Unlike Raf-1, the recruitment of B-Raf to Rap1 is capable of activating MEK and the ERK cascade. Although B-Raf was first identified in brain, it is expressed in a wide variety of hematopoietic cells and cell lines.^83-85 

The distinct and opposing actions of Rap1 on Raf-1 and B-Raf dictate that the outcome of Rap1 signaling to ERKs depends in part on the relative expression of these 2 Raf isoforms. The expression of B-Raf is critical for Rap1 to activate ERKs in megakaryocytes and myeloid precursors. In peripheral T cells that lack B-Raf, Rap1 antagonizes Ras-dependent activation of ERKs.

Given the diverse processes regulated by Rap1, it is likely that the choice of Rap1 effectors is highly regulated. In the following section, we will examine 3 hematopoietic cells that use multiple Rap1 effector pathways: megakaryocytes/platelets, leukocytes, and T lymphocytes. We will characterize Rap1 actions in these cells and speculate how the cell integrates these diverse actions to achieve the physiologic requirements of those cells.

The Rap1 isoform, Rap1b, is highly expressed in both mature megakaryocytes and platelets, and it is expressed at lower levels in immature megakaryocytes.⁶  Many studies support a role of Rap1 in megakaryocyte/platelet adhesion, which regulates critical aspects of platelet function, including aggregation, adhesion, and spreading. This occurs through direct interactions between platelet integrins and fibrin, fibrinogen, Von Willebrand factor, and collagen, as well as other ligands and receptors.⁸⁶ 

In murine megakaryocytes and human MEG-01 cells, the constitutively active mutant RapV12 dramatically increases the binding of the αII_bβ₃ integrin to fibrinogen in activated⁵⁶  and resting cells.⁵⁵  αIIβ₃ activation is negatively regulated by nitric oxide and cyclic guanosine monophosphate (cGMP) via the inhibition of Rap1,⁸⁷  possibly by protein kinase G (PKG)–dependent activation of Rap1GAP2.⁴⁶ 

Many stimuli trigger Rap1 activation in platelets,⁴⁸  including turbulence,⁵⁵  epinephrine, adenosine diphosphate (ADP), thrombin, thromboxane A₂, and platelet activating factor.^88,89  ADP, thrombin, and thromboxane A₂ are linked to PLC, protein kinase C (PKC) and rises in intracellular calcium via the G protein Gq. Indeed, platelets deficient in Gq show defects in platelet function.⁹⁰  However, evidence for calcium-independent activation of Rap1 in platelets also exists, particularly for epinephrine and ADP.⁹¹ 

One Rap1GEF, CalDAG-GEFI, has recently been implicated in mediating the activation of Rap1 and agonist-induced αII_bβ₃ binding to fibrinogen.³²  Since both calcium and DAG activate CalDAG-GEFI, it is a candidate to link calcium signaling to Rap1. Initially, CalDAG-GEFI was implicated by gene profiling for candidate integrin regulators.⁹²  The role for CalDAG-GEF in platelet aggregation and thrombus formation has emerged from studies in mice deficient for CalDAG-GEFI.³³  Platelets from these mice were deficient in aggregation in response to calcium, collagen, ADP, thromboxane A₂, and low but not high concentrations of thrombin or DAG analogs, suggesting that CalDAG-GEFI activation of Rap1 was a major mechanism for activation of αII_bβ₃ integrins but that other mechanisms also exist. RapL is also expressed in platelets and may mediate these effects.³³ 

The maturation of megakaryocytes in the bone marrow is tightly controlled by both extracellular ligands and interaction with stromal cells and a role for Rap1 regulation has been proposed for both processes (Figure 2). Differentiation of magakaryocytes by thrombopoietin (TPO), acting through the Mpl (myeloproliferative virus ligand) receptor, requires sustained ERK activation,^93,94  whereas erythropoietin (EPO) and granulocyte macrophage (GM)–CSF, which do not induce differentiation, induce a transient activation of ERKs.⁹⁵  A model has been proposed that sustained activation of ERKs during the lineage commitment phase of megakaryocytic differentiation results in the synthesis and secretion of autocrine factors that initiate terminal differentiation.⁹⁴ 

The requirement for Rap1 in megakaryocytic differentiation has been examined in the human leukemic cell line UT7. Using these cells, it was demonstrated that Ras/Raf-1 activation was responsible for transient, and Rap1/B-Raf for sustained ERK activation.⁹⁶  The participation of Rap1/B-Raf in sustained ERK signaling was shown using interfering mutants for both Rap1 and B-Raf, as wellas with a mutant Mpl receptor (Mpl-Δ3). TPO activation of Mpl-Δ3 was selectively deficient in sustained ERK activation,⁹⁶  and lacked activation of Rap1 and B-Raf but retained full activation of Ras and Raf-1. The ability of TPO to activate Ras to mediate transient, and Rap1 to mediate sustained ERK signals is similar to that seen in the neuronal differentiation of PC12 cells stimulated by NGF, which also requires the activation of Rap1 and B-Raf.⁹⁷  The specific Rap1GEFs downstream of TPO have not been identified; however, an association of a CrkL/C3G complex with Mpl has been described.^31,98 

Further support for the role of Rap1 in TPO-induced differentiation comes from coculture of megakaryocyte-precursor cell lines with bone marrow stromal cells, which blocks their differentiation. This block requires cell-cell contact and occurs via the inhibition of sustained ERK activation,⁹⁹  and can be overcome by expression of constitutive mutants of the MAP kinase cascade.¹⁰⁰  Stromal cells block sustained ERK activation by selectively interfering with the activation of Rap1, without affecting Ras.¹⁰⁰  Although the mechanism by which Rap1 is selectively targeted by stromal inhibition is not known, disruption of protein complexes involving the adaptor protein Cbl following stromal-cell interaction has been shown.¹⁰⁰  These studies strongly suggest that Rap1-dependent ERK activation participates in megakaryocytic maturation and is a target of stromal inhibition (Figure 2). It will be necessary to confirm these results using primary megakaryocyte precursors in culture.

For megakaryocytes, the consequence of Rap1 activation may be temporally regulated. In megakaryocyte precursors, Rap1 signaling through B-Raf enhances ERK activation. Signals from TPO to Rap1 promote the sustained activation of ERKs that triggers megakaryocyte differentiation. Since cell-cell contact with stromal cells inhibits Rap1 activation of ERKs in cocultured megakaryocyte precursors, it is possible that the ability of Rap1 activation to enhance adhesion to stromal cells during this critical period is limited by this potential negative feedback loop. It will be important to determine whether stromal-cell contact with megakaryocyte precursors can be regulated by Rap1, and whether TPO activation of Rap1 enhances adhesion of these precursor cells.

In platelets, the major function of Rap1 appears to be the activation of integrin-mediated adhesion; Rap1-dependent regulation of ERKs has not been reported. Are these 2 unique actions of Rap1 regulated at the different stages of development in these cells? The ability to channel Rap1 to effectors upstream of ERKs at the precursor stage, and to integrin activation at later stages, may be achieved by the selective activation of distinct pools of Rap1 that are preferentially coupled to each effector pathway. It is likely that distinct Rap1GEFs may be responsible for activation of B-Raf/ERK signaling and αIIβ₃ integrin enhancement. It is also possible that TPO activates selected pools of Rap1 in megakaryocyte precursors that can engage B-Raf, whereas in platelets, agonists may activate pools of Rap1 that are linked to RapL. Rap1 activation of ERK signaling has been shown to occur via C3G.³¹  Although C3G has been implicated in Rap1-dependent adhesion in many hematopoietic-cell types^58,101,102  in platelets, increases in adhesion by platelet agonists are thought to involve CalDAG exchangers.^32,33  Alternatively, effectors like B-Raf and RapL may also be differentially expressed during cell development. The megakaryocyte model provides the opportunity to address these general questions of Rap1 signaling.

Rap1 has been implicated indirectly in leukemic malignancies through the examination of genetic events resulting in leukemias. CalDAG-GEFI is recurrently activated by proviral insertion in the leukemia-prone BXH-2 mice.¹⁰³  The SPA-1 gene is located on chromosome 11g13.3, a hot spot in human hematologic malignancies.¹⁰⁴  However, the target of Rap1 activation relevant for the development of these leukemias has not been examined.

Human CML is characterized by the acquired translocations of the long arms of chromosomes 9 and 22 that generate the BCR/ABL fusion gene. The importance of dysregulated ABL kinase arising from this translocation is demonstrated by the efficacy of ABL kinase inhibitors in the treatment of this disease.¹⁰⁵  CrkL mediates mitogenic signals implicated in BCR/ABL-dependent transformation¹⁰⁶  and is highly phosphorylated on tyrosine in leukocytes from patients with CML, suggesting that it may be a substrate of BCR/ABL.³¹  Hematopoietic cells that have been transformed by expression of BCR/ABL show both constitutive phosphorylation of c-Cbl and its association with CrkL. C3G can also associate with BCR/ABL via CrkL.^106-108  However, some studies see a dissolution of the CrkL/C3G complex in BCR/ABL-expressing cells.¹⁰² 

The ability of BCR/ABL to activate Rap1 has recently been demonstrated in the murine hematopoietic cell line TonB210. This activation required ABL kinase activity and resulted in the activation of B-Raf and ERKs.⁸³  Inhibition of Rap1 activation by expression of SPA-1 or the interfering mutant Rap1N17 blocked ERK-dependent signaling,⁸³  demonstrating that in addition to activating Ras/Raf-1,¹⁰⁹  BCR/ABL can activate Rap1/B-Raf to activate ERKs. The mechanism of Rap1 activation by BCR/ABL was not examined, although the direct binding of CrkL/C3G to BCR/ABL has been reported¹⁰⁸  (Figure 3).

The most provocative study linking Rap1 activation to hematologic malignancies comes from the examination of Spa-1^–/– mice.⁴⁵  In Spa-1^–/– myeloid progenitor cells, basal levels of Rap1 activity were elevated. Nearly all mice developed overt leukemias, including those similar to CML in the chronic phase and chronic lymphocytic leukemia (CLL). The mechanism for leukemogenesis in these mice is unknown. However, preleukemic cells showed elevated basal Rap1 activation and constitutive ERK activation without changes in Ras activity. This suggests that in those cells the elevated Rap1 activity was contributing directly to the activation of ERKs, suggesting a possible role for Rap1/B-Raf signaling in the expansion of hematopoietic progenitor cells. However, examination of leukemic blast cell lines derived from Spa-1^–/– mice revealed a different pattern of high ERK activity and high Ras activity,⁴⁵  consistent with an indirect action of Rap1 on ERKs in these cells.

Hematopoietic cytokines including IL-3, EPO, TPO, GM-CSF, and stem-cell factors all activate adhesion of hematopoietic cells via β₁ integrins.²⁰  β₁ integrins are essential for fetal and adult hematopoiesis, in part by establishing proper homing and by increasing cell survival, and the function of leukocytes in trafficking, transendothelial migration, and targeting to sites of inflammation.

Rap1-dependent enhancement of β₁ integrin function in leukocyte cells has been examined in the promyelocyte 32D cell line. 32D cells are nonadherent but become adherent following treatment with G-CSF. Blockade of Rap1 activation by retroviral expression of SPA-1 inhibits adhesion by G-CSF.¹¹⁰  Rap1 is also required for β₁ integrin–mediated adhesion of 32D cells expressing the EPO receptor (EpoR)²⁰  following stimulation with IL-3, and with EPO. EPO- and IL-3–dependent cell lines respond to cytokine stimulation by inducing c-Cbl association with CrkL/C3G.²⁶  Integrin-mediated adhesion could be blocked by interfering mutants of Rap1, C3G, and CrkL and restored by constitutively active mutants of Rap1.²⁰  Additionally, conditional overexpression of either CrkL or C3G enhanced cytokine activation of Rap1 and adhesion.¹⁰¹  A modest role of PLC in both cytokine activation of Rap1 and adhesion has also been shown,²⁰  suggesting additional mechanisms for Rap1 activation in these cells. Direct evidence that CrkL is required for Rap1 activation by cytokines comes from studies in CrkL^–/– mouse embryo fibroblast that show loss of Rap1 activation in response to interferon-α.¹¹¹ 

As discussed in the section entitled “Rap1 and cell growth (leukemia),” the Spa-1^–/– mouse model implicated Rap1 activation of ERKs in the expansion of Spa-1^–/– progenitors.⁴⁵  Additionally, ERK-independent actions of Rap1 in blast cells have also been implicated by those studies.⁴⁵  Experiments performed in cell lines established from Spa-1^–/– leukemic cells in blast crisis, showed that Rap1 was not enhancing ERKs but that additional changes had occurred that constitutively activated Ras/ERK signals. Restoring SPA-1 expression in blast cells decreased leukemogenesis, suggesting that at this stage, constitutive Rap1 activity was leukemogenic without regulating ERKs.⁴⁵  It is possible that Rap1 activation of ERKs participates in the expansion of preleukemic cells but that other actions of Rap1 may enhance leukemogenesis at later stages. For example, Rap1 enhancement of adhesion may potentiate ERK activation via outside-in signals from integrins to Ras. Rap1 activation of adhesion may also contribute to the proliferative advantage of Spa-1^–/– cells by enabling blast cells to home to niches conducive to leukemogenesis, possibly through direct interaction with stromal cells. Indeed, proper interactions with stromal cells are essential for the immortalization of SPA-1^–/– blast cells.⁴⁵ 

Rap1 has recently been implicated in signaling downstream of BCR/ABL, and Rap1 activation of B-Raf and ERKs has been observed following expression of BCR/ABL in myelocyte cell lines.⁸³  However, Rap1 activation of ERKs may not be limited to BCR/ABL-expressing cells. The myeloproliferative disorders and elevated ERK activation seen within myelocytes in Spa-1^–/– mice suggest that the Rap1/B-Raf/ERK cascade can be activated in cells lacking BCR/ABL. These properties of Spa-1^–/– myelocytes suggest that in normal myelocytes, endogenous SPA-1 limits the activation of this pathway. Moreover, it is also possible that endogenous SPA-1 may regulate discrete pools of Rap1 by its recruitment to specific subcellular locales.

Rap1 activation of ERKs and enhancement of adhesion within the bone marrow may also synergize to augment proliferation of myelocyte progenitors. It will be important to evaluate myelocyte development in cells lacking either RapL (to limit Rap-1–dependent adhesion), or B-Raf (to limit Rap-1–dependent ERK activation) to distinguish the contributions of each arm of Rap1 signaling during leukocyte development.

Primary peripheral T cells are not thought to express B-Raf.¹¹²  In these cells Rap1 appears to antagonize Ras-dependent ERK activation,¹¹³  perhaps by sequestering Raf-1. A model of antagonism of T-cell function was first proposed for Rap1 when it was shown that Rap1 was constitutively active in nonfunctional or anergic T cells.²⁴  T cells activated through the TCR in the absence of CD28 costimulation become anergic,⁶⁸  which is characterized biochemically by constitutive Rap1 activity and decreased Ras, ERK, JNK, and AP-1 (activator protein-1) activation.⁶⁸  The anergic phenotype can be mimicked by overexpression of constitutively active mutants of Rap1.^24,54  Selected forms of anergy can be suppressed by transgenic expression of B-Raf, allowing Rap1 to couple to ERKs.¹¹³ 

Additional evidence for Rap1 antagonism of ERKs in T cells comes from studies using the Spa-1^–/– mice. These mice, which have provided a valuable model of CML, as described in the section entitled “Leukocyte cells,” provide insight into Rap1 signaling in T cells as well. The phenotype of Spa-1^–/– T cells is growth inhibition, in contrast to the hyperproliferative defect seen in Spa-1^–/– myelocytes. This difference in the actions of Rap1 correlates with the differences in ERK activation and B-Raf expression of the two cell types, but may also be due to other cell-type–specific actions of Rap1. Compared with wild-type T cells, Spa-1^–/– T cells were refractive to TCR stimulation, and were associated with constitutive Rap1 activation and diminished ERK activation despite normal or enhanced Ras activity.¹¹⁴  This suggests that the high levels of Rap1 activation achieved by eliminating SPA-1 can antagonize Ras signaling to ERKs in peripheral T cells. However, in that study these effects were age dependent. The age-dependent increase in anergic T cells at 9 to 12 months was attributed to the constitutive activation of Rap1.

Stimulation through the TCR activates Rap1.^113,115  Recent studies using Rap1GAP1-expressing T cells showed loss of Rap1 activation by TCR with Rap1GAP1 expression and enhanced TCR-mediated ERK activation.¹¹⁶  These and related studies⁶⁷  have demonstrated a role for Rap1 to uncouple Ras-mediated ERK activation in peripheral T cells.¹¹³  TCR function is enhanced by stimulation of the coreceptor CD28, which is characterized by enhanced ERK activation and IL-2 production^67,117  and the inhibition of Rap1^52,54  without effecting Ras.^113,116  It has been proposed that CD28 does this by inducing endogenous Rap1GAP activity.⁶⁷ 

In contrast to CD28, the coreceptor CTLA-4 (cytotoxic T lymphocyte associated protein-4) inhibits T-cell activation by limiting multiple signals downstream of TCR, including ERKs.^118,119  Recent studies have demonstrated that Rap1 mediates the inhibition of ERKs by CTLA-4.¹¹⁶  Rap1GAP1 expression in T cells blocks ERK inhibition by CTLA-4, and partially rescues IL-2 production in these cells. These studies suggest that TCR activation of Rap1 is inhibited by CD28 and activated by CTLA-4, to increase and decrease ERK activation, respectively (Figure 4).

In T cells, Rap1 has been implicated in the enhancement of the function of a variety of integrins, including α_Lβ₂ (LFA-1), α₄β₁ (VLA-4), and α₅β₁ (VLA-5).^49,52-54,120  Rap1-dependent adhesion via different integrin subsets facilitates T-cell migration and augments antigen-dependent T-cell activation.^49,54,121  In the human leukocyte adhesion deficiency, LAD III, a defect in Rap1 activation is responsible for the profound defect in integrin adhesiveness.¹²² 

Antigen-dependent activation of Rap1 causes T cells to form stable conjugates with antigen-presenting cells (APCs), which are inhibited by interfering mutants of Rap1, as well as SPA-1 and Rap1GAP1.^54,110,116  Transgenic expression of constitutively active mutants of Rap1 augments integrin-mediated T-cell adhesion by increasing the avidity of LFA-1.^49,54  Recent studies have shown that the expression of RapV12, a constitutively active mutant of Rap1, can potentiate T-cell responses to antigen, including enhanced thymocyte positive selection, increased proliferation, and IL-2 production.⁴⁹  In that study,⁴⁹  the expression of RapV12 had no inhibitory effect on ERK activation, in contrast to studies showing a negative regulatory role of Rap1 on ERK activation.^54,114,116  These differences may be due to differences in the level of Rap1-GTP achieved.¹¹⁴  It was proposed that the strong positive effect of Rap1 occurred via T-cell activation by enhancing adhesion to thymic epithelial cells and APCs. These actions on adhesion are thought to be mediated by the Rap1 effector RapL (see the section entitled “Rap1 effector functions”).^2,60  This study demonstrates that constitutive activation of Rap1 can induce Ras-independent effects on T-cell activation.

Rap1 antagonism of Ras-dependent ERK signaling provides a negative signal for T-cell activation, whereas enhancement of T-cell adhesion provides a positive signal for T-cell activation. These 2 actions of Rap1 are seemingly at odds with each other. T-cell adhesion to APCs is essential for T-cell activation, and constitutive activation of Rap increases T-cell adhesion and T-cell activation.^49,54  How can this action of Rap1 be coordinated with its ability to limit T-cell activation through its inhibition of ERKs?

One possibility is that the balance between these opposing functions may be influenced by the characteristics of the antigenic peptide that triggers the T-cell response. While LFA-1/ICAM interactions are essential for T-cell responses by low affinity peptides,^49,121  these interactions may not be necessary when high-affinity peptide is used.¹²¹  When interactions between APCs and T cells are of high affinity, T cells may become overstimulated by enhanced Rap1-mediated adhesion, resulting in cell death or anergy.^24,54  Therefore, in T lymphocytes the 2 functions of Rap1 may work together to enhance T-cell activation in response to weak stimuli, and depress T-cell activation in response to hyperstimulation. In this way, Rap1 activation may optimize T-cell responses to antigenic stimuli (Figure 4).

Early studies showing Rap1 antagonism of Ras/ERK signaling have relied on the use of constitutively active Rap1 mutants. However, subsequent studies in T cells and other cell types have used selective inhibition of Rap1 to establish this function of Rap1.^30,123-125  Additionally, Raf-1/ERK may not be the only Ras pathway antagonized by Rap1 in T cells. Rap1 inhibition of Ras-dependent signaling to p38 and reactive oxygen species has been described.^70,71  However, it is clear that the physiologic role of Rap1 is not limited to the antagonism of Ras signaling, but can activate T cells to proliferate via pathways that are independent of Ras.

The enhancement of T-cell activation via enhanced integrin/APC interactions forms the basis of Rap1's enhancement of T-cell function. It is generally thought that enhancing integrin adhesion can activate T-cell function by strengthening signals downstream of the TCR. However, the enhanced T-cell function seen in RapV12-overexpressing transgenic T cells is not associated with increased ERK signaling.⁴⁹  Therefore, it appears that Ras-independent signaling cascades are triggered by outside-in signaling to enhance IL-2 production in that model.

Both functions of Rap1 (enhancement of integrin function and antagonism of Ras-mediated signals) may also act in a temporal manner to optimize T-cell responses. The antagonism of Ras-mediated signals by Rap1 may set a threshold for activation of primed T cells that is regulated by coreceptors such as CD28 and CTLA-4. The enhancement of integrin function by Rap1 may also play a role in the initial TCR engagement and formation of the immunologic synapse by enhancing adhesion during early T-cell activation.

In the 3 examples included in this review, Rap1 displays dual actions: the regulation of ERK signaling and the augmentation of cell adhesion. The ability of Rap1 to activate or inhibit ERK signaling in each of these cells is dictated, in part, by the expression of B-Raf in megakaryocytes and myelocytes, and the absence of B-Raf in peripheral T cells (Figure 5). The activation of ERKs by Rap1/B-Raf participates in differentiation of megakaryocytes and, possibly, in the proliferation of myelocytes, whereas the inhibition of ERKs can limit the proliferation of a subset of peripheral T cells. Rap1 enhancement of cell adhesion also occurs within all of the 3 cell types via a distinct effector pathway possibly involving RapL. What is the consequence of this enhanced adhesion and how does the cell coordinate this action with that of ERK regulation to produce an integrated response to Rap1 activation? It is likely that each cell uses distinct strategies to ensure that Rap1 activation results in a coherent cellular response. Hematopoietic cells offer a unique opportunity to examine these potential distinct strategies by which the outcome of signaling pathways to Rap1 are restricted temporally and spatially to achieve cell-specific outcomes.

Note added in proof. Two recent reports support the ability of Rap1 to antagonize T cell function. According to one report, the transgenic expression of RapE63 in T lymphocytes blocked ERK activation, IL-2 production, and proliferation in peripheral T cells.¹²⁶  The other report confirms that CTLA-4 activates Rap1 and proposes that Rap1 activation contributes to decreased IL-2 production upon CTLA-4 engagement.¹²⁷  It is important to note that both studies demonstrate that the Rap1-dependent decrease in T-cell function was accompanied by increased LFA-1-mediated adhesion.