Rap1 Is Activated by Erythropoietin or Interleukin-3 and Is Involved in Regulation of β1 Integrin-mediated Hematopoietic Cell Adhesion

Abstract

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates ␤ 1 integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated ␤ 1 integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas U73122, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase C␥ and strongly implicate Rap1 in regulation of ␤ 1 integrin-mediated hematopoietic cell adhesion.

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

“…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). However, the roles Rap1 plays in activation of downstream signaling pathways, including those involving B-Raf, Raf-1, MEK, Erk, and Akt1, in cells stimulated by IL-3 or BCR/ABL have largely remained to be examined.…”

“…The ability of TPO to activate Rap1 to mediate sustained Erk signaling agrees with the observation that several hours of stimulation with TPO are required for activation of Rap1 and is similar to that seen in the neuronal differentiation of PC12 cells stimulated by NGF (Garcia et al, 2001;Stork, 2003;Stork and Dillon, 2005). On the other hand, IL-3 stimulation rapidly induces activation of Rap1 (Arai et al, 2001), and Rap1 inactivation by SPA-1 inhibited the transient early Erk activation (Figure 2b). The difference in time course of activation of Rap1-mediated signaling between IL-3 and TPO may respond to the difference in signaling for proliferation and differentiation, although the molecular mechanisms responsible remain to be examined.…”

“…We first examined whether SPA-1 expressed in Ton.B210/SPA-1 cells inhibits Rap1 activation by IL-3 stimulation or by BCR/ABL expression. In accordance with our previous reports, IL-3 stimulation as well as the induction of BCR/ABL expression by Dox treatment activated Rap1 in Ton.B210 cells (Figure 1b) (Arai et al, 2001;Mizuchi et al, 2005). On the other hand, the SPA-1 expression in Ton.B210/SPA-1 cells not only reduced the basal level of Rap1-GTP significantly but also inhibited Rap1 activation by IL-3 and BCR/ABL almost completely (Figure 1b).…”

“…We have previously shown that Rap1 plays an important role in activation of integrin-mediated hematopoietic cell adhesion in response to cytokine stimulation (Arai et al, 2001). Although it still remains controversial, BCR/ABL has also been reported to activate integrin-mediated hematopoietic cell adhesion (Bazzoni et al, 1996;Kramer et al, 1999;Wertheim et al, 2002).…”

“…We previously found that hematopoietic cytokines, IL-3 and Epo, as well as BCR/ABL activate Rap1 in hematopoietic cells (Arai et al, 2001;Mizuchi et al, 2005). Rap1 is a small GTPase of the Ras family, which was initially identified as a homolog of Ras (Pizon et al, 1988) and was also identified independently by its ability to induce a flat revertant phenotype in K-Ras-transformed cells (Kitayama et al, 1989).…”