Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane

Abstract

RasGRP1 is a Ras-activating exchange factor that is positively regulated by translocation to membranes. RasGRP1 contains a diacylglycerol-binding C1 domain, and it has been assumed that this domain is entirely responsible for RasGRP1 translocation. We found that the C1 domain can contribute to plasma membrane-targeted translocation of RasGRP1 induced by ligation of the B cell antigen receptor (BCR). However, this reflects cooperativity of the C1 domain with the previously unrecognized Plasma membrane Targeter (PT) domain, which is sufficient and essential for plasma membrane targeting of RasGRP1. The adjacent suppressor of PT (SuPT) domain attenuates the plasma membranetargeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase C␥2, the C1 domain counteracts the SuPT domain and enables efficient RasGRP1 translocation to the plasma membrane. In fibroblasts, the PT domain is inactive as a plasma membrane targeter, and the C1 domain specifies constitutive targeting of RasGRP1 to internal membranes where it can be activated and trigger oncogenic transformation. Selective use of the C1, PT, and SuPT domains may contribute to the differential targeting of RasGRP1 to the plasma membrane versus internal membranes, which has been observed in lymphocytes and other cell types. INTRODUCTIONRasGRP1 is a guanine nucleotide exchange factor that couples antigen receptors to the activation of Ras GTPases (Dower et al., 2000;Ebinu et al., 2000;Priatel et al., 2002;Bivona et al., 2003;Caloca et al., 2003b;Layer et al., 2003;Norment et al., 2003;Oh-hora et al., 2003;Guilbault and Kay, 2004;Perez de Castro et al., 2004;Reynolds et al., 2004;Zugaza et al., 2004;Coughlin et al., 2005;Roose et al., 2005). Deletion of the RasGRP1 gene perturbs the immunological selection and activation of lymphocytes (Dower et al., 2000;Priatel et al., 2002Priatel et al., , 2006Layer et al., 2003) and mast cells (Liu et al., 2007), whereas aberrant expression of RasGRP1 initiates oncogenic transformation of lymphocytes (Li et al., 1999;Mikkers et al., 2002;Suzuki et al., 2002;Kim et al., 2003;Dupuy et al., 2005;Klinger et al., 2005), fibroblasts (Ebinu et al., 1998;Tognon et al., 1998) and keratinocytes (Oki-Idouchi and Lorenzo, 2007).To be active as an exchange factor, RasGRP1 must be localized to cell membranes where Ras GTPases reside. This requirement provides an opportunity for positive or negative regulation. RasGRP1 contains a C1 domain that binds the lipid second messenger diacylglycerol (DAG), or DAGmimetic phorbol esters (Ebinu et al., 1998;Lorenzo et al., 2000;Shao et al., 2001;Rong et al., 2002;Carrasco and Merida, 2004;Madani et al., 2004). Treatment of cells with DAG or phorbol esters results in the translocation of RasGRP1 to membranes (Ebinu et al., 1998;Tognon et al., 1998;Bivona et al., 2003;Rambaratsingh et al., 2003;Caloca et al., 2004;Stone et al., 2004), and it stimulates Ras activation via RasGRP1 (Ebinu et al., 1998;...

PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

“…We next tried to identify the domain(s) necessary for the interaction of RasGRP1 with PAQR10 using different deletion or truncation forms of RasGRP1 [15,41] ( Figure 6A). PAQR10 could significantly increase Golgi localization of the full-length RasGRP1, as well as its mutant RasGRP1(1-606) that had a deletion of the Cterminus containing SuPT and PT domains ( Figure 6B).…”

Phosphoinositide 3-Kinase Regulates Plasma Membrane Targeting of the Ras-specific Exchange Factor RasGRP1

“…In lymphocytes, initiation of Ras signaling by RasGRP1 controls progression through developmental and immunoselection checkpoints, whereas misregulation of RasGRP1 can trigger autoimmunity or oncogenic transformation (5-10). Depending on the quality and intensity of signaling from receptors, RasGRP1 can be specifically targeted to the plasma membrane (11)(12)(13)(14)(15) or to endomembranes such as Golgi or the endoplasmic reticulum (11, 13, 16 -19). The site of RasGRP1 localization can have important consequences for how cells respond to the resulting activation of Ras GTPases.…”

“…1A), and remarkably, each of these can affect the cellular membranes to which RasGRP1 localizes (11,17,19,(21)(22)(23). The C1 domain was the first membranelocalizing domain identified in RasGRP1, making it the focus of the initial models of membrane-selective targeting of RasGRP1.…”

“…The C1 domain was the first membranelocalizing domain identified in RasGRP1, making it the focus of the initial models of membrane-selective targeting of RasGRP1. The C1 domain binds to the lipid second messenger diacylglycerol (DAG) 3 (24, 25) and thus has the potential to mediate translocation of RasGRP1 to membranes when DAG-generating phospholipase Cs (PLC) or phosphatidic acid phosphatases are activated (11,13,21,26). By this mechanism, membraneselective targeting of RasGRP1 could be attained by generation * This work was supported by grants from the Canadian Institutes of Health Research (to R. B. C. and R. J. K.).…”

Rac1/p21‐activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes

“…IP 3 binds to IP 3 receptors in specialized areas of the endoplasmic reticulum triggering Ca 2+ release [15], while DAG remains attached to the inner region of the plasma membrane and serves to recruit and contribute to activating the cytosolic protein kinase C (PKC) [16]. Additionally, DAG cooperates with the Vav/Rac pathway in order to facilitate the localization of RAS guanyl releasing protein (RasGRP) (guanine nucleotide exchange factors (GEFs) for Ras) family members into actin juxtamembrane structures, thereby facilitating Ras activation [2,17].…”