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

Zahedi, Bari; Goo, Hyun-jung; Beaulieu, Nadine; Tazmini, Ghazaleh; Kay, R.; Cornell, Rosemary B.

doi:10.1074/jbc.m110.189605

Cited by 19 publications

(33 citation statements)

References 71 publications

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“…These results suggest that RasGRP1/3 are required for ERK activation in response to SDF1α/CXCR4 signaling, but RasGRP1/3 deficiency does not impair PI3K/AKT signaling in response to SDF1α. Interestingly, RasGRP1 contains a C-terminal plasma membrane targeting (PT) domain that includes a basic/hydrophobic cluster of amino acids shown to interact with phosphoinositides [32], [33]. Therefore, targeting of RasGRP1/3 to the plasma membrane and subsequent Ras activation may be dependent on PI3K activity.…”

Section: Resultsmentioning

confidence: 99%

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

et al. 2013

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T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4−CD8− ‘double negative’ (DN) thymocytes, pass through a checkpoint termed “β-selection” before maturing into CD4+CD8+ ‘double positive’ (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

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Section: Resultsmentioning

confidence: 99%

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

et al. 2013

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“…In the plasma membrane targeting (PT) domain of RasGRP1, a small segment enriched in basic hydrophobic residues, termed the BHC motif, has been identified that binds directly to the plasma membrane enriched in anionic phospholipids [48]. This unstructured basic/hydrophobic cluster can mediate membrane binding by presenting positive charges that electrostatically interact with the strongly negatively charged phosphoinositide headgroups; additionally, it has aromatic and long aliphatic side chains that insert into the lipid bilayer [49, 50, 51, 52].…”

Section: Resultsmentioning

confidence: 99%

“…It is elevated in many cancers either through constitutively active phosphatidylinositol 3-kinase or through inactivation of its degradative pathway as a result of PTEN mutation. The report that RasGRP1 bound PIP 3 through its PT domain [48], which is divergent from that of RasGRP3, suggested that RasGRP3 might show markedly different behavior depending on the membrane environment.…”

Section: Discussionmentioning

confidence: 99%

“…The plasma membrane targeting domain is directly responsible for targeting RasGRP1 to the plasma membrane in some cell types [47]. In the PT domain, a small segment enriched in basic and hydrophobic residues, the BHC motif has been identified [48]. A similar motif has been found in other proteins that bind directly to plasma membranes enriched in anionic phospholipids, particularly phosphoinositides [49, 50, 51, 52].…”

Section: Introductionmentioning

confidence: 99%

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Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3

Czikora¹,

Kedei²,

Kalish³

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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RasGRP comprises a family of guanine nucleotide exchange factors, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP1 possesses REM (Ras exchange), GEF (catalytic), EF-hand, C1, SuPT (suppressor of PT), and PT (plasma membrane-targeting) domains, among which the C1 domain drives membrane localization in response to diacylglycerol or phorbol ester and the PT domain recognizes phosphoinositides. The homologous family member RasGRP3 shows less plasma membrane localization. The objective of this study was to explore the role of the different domains of RasGRP3 in membrane translocation in response to phorbol esters. The full-length RasGRP3 shows limited translocation to the plasma membrane in response to PMA, even when the basic hydrophobic cluster in the PT domain, reported to be critical for RasGRP1 translocation to endogenous activators, is mutated to resemble that of RasGRP1. Moreover, exchange of the C-termini (SuPT-PT domain) of the two proteins had little effect on their plasma membrane translocation. On the other hand, while the C1 domain of RasGRP3 alone showed partial plasma membrane translocation, truncated RasGRP3 constructs, which contain the PT domain and are missing the REM, showed stronger translocation, indicating that the REM of RasGRP3 was a suppressor of its membrane interaction. The REM of RasGRP1 failed to show comparable suppression of RasGRP3 translocation. The marked differences between RasGRP3 and RasGRP1 in membrane interaction necessarily will contribute to their different behavior in cells and are relevant to the design of selective ligands as potential therapeutic agents.

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“…The coordination of multiple lipid binding domains to enhance sensitivity to regulation is well recognized for PKC and AKT (49). In Ras-GRP1, for example, the plasma membrane targeter (PT) domain has been described as a key contributor along with the C1 domain (63).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity

Czikora

Lundberg

Abramovitz

et al. 2016

Journal of Biological Chemistry

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The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. 3 H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (K d ‫؍‬ 8.2 ؎ 1.1 nM for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length Ras-GRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. RasGRP2 is exceptional in that its

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

Cited by 19 publications

References 71 publications

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

Importance of the REM (Ras exchange) domain for membrane interactions by RasGRP3

Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity

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