Activation of the Rap GTPases in B Lymphocytes Modulates B Cell Antigen Receptor-induced Activation of Akt but Has No Effect on MAPK Activation

Abstract

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)...

Characterization of Interactions of Adapter Protein RAPL/Nore1B with RAP GTPases and Their Role in T Cell Migration

“…2) are consistent with several previous observations in B cells, where supporting evidence was mainly obtained from overexpression of activated Rap2 and the use of Rap GAPII that blocks the activation of both Rap1 and Rap2 (42,43,45). We also show that expression of constitutively active Rap2 enhances migration and, more importantly, that partial depletion of endogenous Rap2 by specific siRNA reduces migration of HSB2 T cells.…”

Role of Rap1B and Tumor Suppressor PTEN in the Negative Regulation of Lysophosphatidic Acid—induced Migration by Isoproterenol in Glioma Cells

“…We observed the opposite suppressive effect on Akt activation by treatment of cells with ISO, which stimulated Rap1 in an Epac-dependent manner (Figures 3 and 4C). These findings were supported by notable studies in B-lymphocytes, in which the activation of Rap GTPases modulated the B-cell antigen receptor-mediated activation of Akt (Christian et al, 2003). A controversy in the Rap1 effect on Akt from the studies could be explained by the difference in the type of Rap1 protein expressed in one or another cell type.…”

BCR/ABL and IL-3 activate Rap1 to stimulate the B-Raf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion

“…Previous reports have implicated Rap1 as a positive or a negative regulator of Akt activation in various types of cells stimulated with different stimuli (Tsygankova et al, 2001;Wang et al, 2001;Lou et al, 2002;Christian et al, 2003). Although the molecular mechanisms underling these effects have not been elucidated, Rap1 has been proposed to bind PI3K through its effector-binding region, which is identical with that of Ras (Downward, 1997;Bos et al, 2001).…”

“…Thus, depending on cell types, Rap1 may activate the MEK/Erk signaling pathway through B-Raf or may reduce its activation through its inhibitory effect on Raf-1. Similarly, recent studies have shown that Rap1 may also interact with the Ras effector PI3K and show a negative or positive effect on activation of the PI3K/Akt pathway depending on cell types and stimuli (Tsygankova et al, 2001;Wang et al, 2001;Lou et al, 2002;Christian et al, 2003). In our previous study, we found that IL-3 as well as Epo activates b1 integrin through Rap1 and induces hematopoietic cell adhesion, which agrees with recent studies showing that Rap1 plays important roles in regulation of cell adhesion through various integrins (Arai et al, 2001;Bos et al, 2001;Stork and Dillon, 2005).…”