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

Abstract

Small G proteins serve as critical control points in signal transduction, integrating a wide range of stimuli to dictate discrete cellular outcomes. The outcomes of small G-protein signaling can both potentiate and antagonize one another. Studies in hematopoietic cells have uncovered multiple functions for the small G protein, Rap1 (Ras-proximate-1). Because Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms, it serves as a paradigm for the need for tight cellular control of small G-protein function. Rap1 has received recent attention for its role in enhancing integrin-dependent signals. This action of Rap1 augments a variety of processes that characterize hematopoietic-cell function, including aggregation, migration, extravasation, and homing to target tissues. Rap1 may also regulate cellular differentiation and proliferation via pathways that are distinct from those mediating adhesion, and involve regulation of the mitogen-activated protein (MAP) kinase or ERK (extracellular signal-regulated kinase) cascade. These actions of Rap1 occur in selected cell types to enhance or diminish ERK signaling, depending on the expression pattern of the MAP kinase kinase kinases of the Raf family: Raf-1 and B-Raf. This review will examine the functions of Rap1 in hematopoietic cells, and focus on 3 cellular scenarios where the multiple actions of Rap1 function have been proposed. Recent studies implicating Rap1 in the maturation of megakaryocytes, the pathogenesis of chronic myelogenous leukemia (CML), and activation of peripheral T cells will receive particular attention.

Figure 1.

Rap1 regulation and activation of effector proteins. Rap1 is activated (Rap1-GTP) by specific guanine nucleotide exchange factors (GEFs) and inhibited by specific GTPase activating proteins to regulated GTP-dependent binding to potential effector proteins. A diverse family of GEFs activates Rap1 by exchanging GDP for GTP (selected GEFs are shown). C3G, the first Rap1 GEF identified, is activated through its association with CrkL, a member of the Crk family of adaptor proteins, via a proline-rich (PPP) region on C3G and an SH3 region on CrkL. The inducible association of Crk/C3G to sites of tyrosine phosphorylation on receptor-associated adaptor/scaffold proteins occurs following ligand activation of a variety of receptors. CalDAG-GEFs respond to calcium (Ca²⁺) and diacylglycerol (DAG), making them targets of phospholipase C (PLC) action. cAMP-GEFs or EPACs (exchange proteins directly activated by cAMP; EPAC 1 shown here) are activated by directly binding cAMP to cyclic nucleotide binding domains (CNDB). Two Rap1GAPs are shown here: Rap1GAP1 and SPA-1. Rap1GAP1 is expressed widely and contains a GOLoCo motif that can associate with heterotrimeric G-protein alpha subunits. SPA-1 is highly expressed in hematopoietic cells and contains a PDZ (domain found in PSD-95, Discs large, and ZO-1) domain that can mediate interactions with membrane-associated proteins. Following GTP binding, Rap1-GTP associates with a number of potential effectors, some of which are shown here. One of the best-studied effector pathways of Rap1 is the stimulation of cell adhesion. RapL and RIAM (Rap1-GTP–interacting adaptor molecule) have been shown to mediate integrin-mediated adhesion in hematopoietic cells. Both contain a Ras association (RA) domain, and RIAM also contains a Pleckstrin-homology (PH) domain. B-Raf and Raf-1 are related kinases that contribute to the MAP kinase pathway upstream of ERK. Both are recruited to Rap1-GTP via their Ras binding domain (RBD). In cells expressing B-Raf, Rap1 activates B-Raf–dependent signals to ERKs. In B-Raf–negative cells, such as T cells, Rap1 cannot couple to ERK, but can antagonize Ras-dependent signals to ERK, in part by binding to Raf-1. Rap1 can also sequester other effectors of Ras, such as the upstream activators of the p38 MAP kinase, to antagonize the activation of p38 by IL-1. In addition, Rap1 has been proposed to inhibit the phosphatidylinositol 3-kinase–dependent phosphorylation of the survival factor Akt, in B cells. DEP indicates domain found in Disheveled, Eg-10, and pleckstrin; IP3, inositol 1,4,5 triphosphate.

Figure 2.

Differentiation of megakaryocyte precursors requires sustained ERK activation. (A) Thrombopoietin (TPO) activation of ERKs has 2 components, a Ras-dependent transient ERK activation and a Rap1-dependent sustained ERK activation. TPO binds its receptor (Mpl) to activate the JAK family kinases that promote tyrosine phosphorylation of the cytoplasmic tail. These phosphorylations serve as binding sites for signaling molecules, including Shc, which recruits Grb2 (growth factor receptor-bound protein-2)/SOS to activate Ras (Ras-GTP) resulting in a transient activation of ERKs (pERK). Sustained pERK requires the activation Rap1 (Rap1-GTP) and B-Raf. The recruitment of a Cbl/CrkL/C3G complex to Mpl has been proposed to trigger the activation of Rap1. (B) Stimulation of megakaryocytes with TPO induces sustained activation/phosphorylation of ERKs (pERK), which is required for megakaryocyte precursor differentiation (+). The sustained ERK activation by TPO has 2 components mediated by Ras and Rap1, as described in panel A. In contrast, stimulation of megakaryocytes with GM-CSF or EPO induces a Ras-dependent transient pERK, which does not lead to cellular differentiation (–). Direct contact between stromal cells and megakaryocytes inhibits the sustained pERK to prevent differentiation (–) by specifically disrupting Rap1/B-Raf signals. Shc indicates SH2-containing protein; STAT, signal transducer and activator of transcription.

Figure 3.

BCR/ABL can activate ERKs via Ras and Rap1. BCR/ABL is a fusion protein between the BCR and the kinase ABL. The ABL kinase contains Src homology domains 1, 2, and 3 (SH1, SH2, and SH3) and a proline-rich domain (PRD). BCR/ABL binds multiple proteins to trigger intracellular signaling cascades. The binding of Grb2, Cbl, and CrkL are shown here. Grb2 associates with BCR/ABL as a complex with the RasGEF SOS (son of sevenless). The SH2 domain of Grb2 binds to phosphotyrosine 177 in the BCR region of BCR-ABL to allow SOS to activate Ras (Ras-GTP), which in turn recruits Raf-1 (and possibly B-Raf) to activate ERK. CrkL exists as a complex with the Rap1 GEF C3G. Recruitment of CrkL/C3G to the PRD of BCR/ABL (via a CrkL SH3 domain) allows C3G to activate Rap1 (Rap1-GTP), which recruits B-Raf to activate ERK. The scaffold molecule Cbl binds the SH2 domain of BCR/ABL and may also participate in the complex with CrkL/C3G.

Figure 4.

In peripheral T cells Rap1 has 2 opposing functions: one is to enhance integrin-mediated adhesion and the other is the regulation of Ras-dependent ERK activation. (A) T-cell activation requires multiple interactions with antigen-presenting cells (APCs). Antigen-dependent interactions are mediated by TCR recognition of antigen bound by the major histocompatibility complex (MHC) on the surface of APCs. Antigen-dependent signals downstream of TCR activate both Rap1 and Ras. Rap1 can enhance T-cell adhesion by stimulating the association of RapL with the α-chain of the integrin LFA-1, enhancing its affinity/avidity for ICAM. TCR activation of Ras and ERKs leads to the activation of multiple transcription factors, including the induction of the transcription factor Fos, which, in conjunction with other transcription factors (NFAT [nuclear factor of activated T cells] and nuclear factor κB [NFκB]), stimulates the transcription and production of IL-2, the major proliferative cytokine of activated T cells. TCR-dependent Rap1 activation antagonizes Ras signaling to ERKs, limiting IL-2 production. APCs express a transmembrane ligand B7 that binds 2 coreceptors, CD28 and CTLA-4, on T cells. Both CD28 and CTLA-4 regulate Rap1. CD28 augments signals from the TCR to IL-2 via multiple pathways, including ERKs shown here. CD28 inhibits TCR-dependent Rap1 activation, providing a mechanism for enhancing ERK-dependent IL-2 production. CTLA-4 antagonizes multiple stimulatory signals from the TCR to IL-2, including ERKs. In contrast to CD28, CTLA-4 activates Rap1, leading to down-regulation of ERK activity, providing a mechanism for the CTLA-4–dependent inhibition of IL-2 production. (B) The balance between the inhibition of ERK-dependent T-cell activation and the enhancement of LFA-dependent T-cell activation optimizes the T-cell response to antigen.

Figure 5.

Rap1 has multiple functions in hematopoietic cells. Rap1 can enhance integrin-mediated adhesion in all hematopoietic cells examined to date. Rap1 can also regulate ERK activation in the 3 types of cells shown here (megakaryocytes, myelocytes, and T lymphocytes). Rap1 activates ERK via B-Raf in megakaryocytes and myelocytes that express B-Raf. In T cells that do not express B-Raf, Rap1 antagonizes Ras-dependent ERK activation. The proposed actions of Rap1 on ERK and adhesion and the overall cellular responses to Rap1 are listed below each cell type.