Integrins on the surface of resting lymphocytes normally have low affinity and low avidity for their ligands. However, external stimuli such as chemokines and antigens generate intracellular signals that rapidly activate integrins, converting them to a clustered high affinity/high avidity state that can bind adhesion molecules on other cells or ECM components (1, 12). The nature of this "inside-out" signaling that promotes integrin activation is not completely understood. Many signaling pathways contribute to integrin activation, including the pathways that involve phosphatidylinositol 3-kinase, protein kinase C, Fyb/ADAP (adhesion and degranulation adaptor protein), and the Rho GTPase (12-15).Once activated, integrins can bind their ligands and initiate intracellular signaling pathways that regulate many aspects of cell behavior, including cell survival, proliferation, and differentiation and reorganization of the actin cytoskeleton (16). The reorganization of a cell's actin cytoskeleton and the resulting * This work was supported by a grant from the Cancer Research Society of Canada (to M. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for B cells and B cell progenitors. Although the binding of SDF-1 to its receptor, CXCR4, activates multiple signaling pathways, the mechanism by which SDF-1 regulates cell migration is not completely understood. In this report we show that activation of the Rap GTPases is important for B cells to migrate toward SDF-1. We found that treating B cells with SDF-1 resulted in the rapid activation of both Rap1 and Rap2. Moreover, blocking the activation of Rap1 and Rap2 via the expression of a Rap-specific GTPase-activating protein significantly reduced the ability of B cells to migrate toward SDF-1. Conversely, expressing a constitutively active form of Rap2 increased SDF-1-induced B cell migration. Thus, the Rap GTPases control cellular processes that are important for B cells to migrate toward SDF-1.

The B cell antigen receptor (BCR) activates Ras, a GTPase that promotes cell proliferation by activating the Raf-1/MEK/ERK signaling module and other signaling enzymes. In its active GTP-bound form, the Rap1 GTPase may act as a negative regulator of Ras-mediated signaling by sequestering Ras effectors (e.g., Raf-1) and preventing their activation. In this report, we show that BCR engagement activates Rap1 and that this is depend-

Signaling by the B cell antigen receptor (BCR) activates the Rap1 and Rap2 GTPases, putative antagonists of Ras-mediated signaling. Because Ras can activate the Raf-1/ERK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we asked whether Rap activation limits the ability of the BCR to signal via these pathways. To do this, we blocked the activation of endogenous Rap1 and Rap2 by expressing the Rap-specific GTPase-activating protein RapGAPII. Preventing Rap activation had no effect on BCR-induced activation of ERK. In contrast, BCR-induced phosphorylation of Akt on critical activating sites was increased 2-to 3-fold when Rap activation was blocked. Preventing Rap activation also increased the ability of the BCR to stimulate Akt-dependent phosphorylation of the FKHR transcription factor on negative regulatory sites and decreased the levels of p27 Kip1 , a pro-apoptotic factor whose transcription is enhanced by FKHR. Moreover, preventing Rap activation reduced BCR-induced cell death in the WEHI-231 B cell line. Thus activation of endogenous Rap by the BCR limits BCR-induced activation of the PI3K/ Akt pathway, opposes the subsequent inhibition of the FKHR/p27Kip1 pro-apoptotic module, and enhances BCR-induced cell death. Consistent with the idea that Rap-GTP is a negative regulator of the PI3K/Akt pathway, expressing constitutively active Rap2 (Rap2V12) reduced BCR-induced phosphorylation of Akt and FKHR. Finally, our finding that Rap2V12 can bind PI3K and inhibit its activity in a manner that depends upon BCR engagement provides a potential mechanism by which Rap-GTP limits activation of the PI3K/Akt pathway, a central regulator of B cell growth and survival.Signaling by the B cell antigen receptor (BCR) 1 is required for B cell development and survival, for the elimination or silencing of self-reactive B cells, and for the activation of B cells that recognize foreign antigens (1). The BCR activates multiple signaling pathways, including the phospholipase C pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the kinase cascades that lead to activation of the mitogen-activated protein kinases (MAPKs) (1-3). Downstream targets of the phospholipase C pathway include protein kinase C (PKC) enzymes as well as the NF-AT and NF-B transcription factors (4). PI3K produces lipid second messengers that regulate a network of protein kinases, including 3-phosphoinositide-dependent kinase-1, PKC-, PKC-⑀, p70 S6 kinase, the Btk tyrosine kinase (1, 5), and Akt/protein kinase B, a kinase that plays a key role in B cell survival (6). The three major classes of MAPKs, extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), and p38 MAPK, phosphorylate a variety of transcription factors, increasing their ability to promote transcription. The activation of distinct combinations of these signaling pathways may account for the ability of the BCR to promote survival, apoptosis, proliferation, or differentiation depending on the maturation state of the B cell and the nature of the antigen (2)...

The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM. Cancer Res; 70(11); 4590-601. ©2010 AACR.