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

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 membrane-targeting activity of the PT domain, thus preventing constitutive plasma membrane localization of RasGRP1. By binding to diacylglycerol generated by BCR-coupled phospholipase Cγ2, 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.

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TAPP1 and TAPP2 Are Targets of Phosphatidylinositol 3-Kinase Signaling in B Cells: Sustained Plasma Membrane Recruitment Triggered by the B-Cell Antigen Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5479-5491.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5479-5491 ◽

Cited By ~ 72

Author(s):

Aaron J. Marshall ◽

Allyson K. Krahn ◽

Kewei Ma ◽

Vincent Duronio ◽

Sen Hou

Keyword(s):

Plasma Membrane ◽

B Cell ◽

Antigen Receptor ◽

B Cell Antigen Receptor ◽

Ph Domain ◽

Phosphatidylinositol 3 Kinase ◽

Cell Antigen Receptor ◽

Membrane Recruitment ◽

Cell Antigen ◽

Phosphatidylinositol 3

ABSTRACT We report the characterization of two signal transduction proteins related to Bam32, known as TAPP1 and TAPP2. Bam32, TAPP1, and TAPP2 share several characteristics, including small size (32 to 47 kDa), lack of enzymatic domains, high conservation between humans and mice, and the presence of pleckstrin homology (PH) domains near their C termini which contain the 3-phosphoinositide-binding motif. Unlike Bam32, the N-terminal regions of TAPP1 and TAPP2 contain a second PH domain. TAPP1 and TAPP2 transcripts are expressed in a variety of tissues including lymphoid tissues. Using live-cell imaging, we demonstrate that TAPP1 and TAPP2 are recruited to the plasma membrane of BJAB human B-lymphoma cells upon activation through the B-cell antigen receptor (BCR). The C-terminal PH domain is necessary and sufficient for BCR-induced membrane recruitment of both TAPP1 and TAPP2. Blockade of phosphatidylinositol 3-kinase (PI3K) activity completely abolished BCR-induced recruitment of TAPP1 and TAPP2, while expression of active PI3K is sufficient to drive constitutive membrane localization of TAPP1 and TAPP2. TAPP1 and TAPP2 preferentially accumulate within ruffled, F-actin-rich areas of plasma membrane, suggesting a potential role in PI3K-driven cytoskeletal reorganization. Like Bam32, BCR-driven TAPP1 and TAPP2 recruitment is a relatively slow and sustained response, in contrast to Btk recruitment and Ca2+ mobilization responses, which are rapid and transient. Consistent with recent studies indicating that Bam32, TAPP1, and TAPP2 can bind to PI(3,4)P2, we find that membrane recruitment correlates well with production of PI(3,4)P2 but not with that of PI(3,4,5)P3. Our results indicate that TAPP1 and TAPP2 are direct targets of PI3K signaling that are recruited into plasma membranes with distinctive delayed kinetics and accumulate within F-actin-rich membrane ruffles. We postulate that the TAPPs function to orchestrate cellular responses during the sustained phase of signaling.

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