DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation

Anderson, Karen E.; Lipp, Peter; Bootman, Martin D.; Ridley, S.; Coadwell, John; Rönnstrand, Lars; Lennartsson, Johan; Holmes, Alexander J.; Painter, GF; Thuring, Jan Willem; Lim, Ze-Yi; Erdjument‐Bromage, Hediye; Grewal, Anita; Tempst, Paul; Stephens, Len R.; Hawkins, Phillip T.

doi:10.1016/s0960-9822(00)00794-6

Cited by 40 publications

(58 citation statements)

References 33 publications

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“…Previous studies, as well our own unpublished data, indicate that Bam32 phosphorylation requires PI3K activation and PH domain-mediated membrane recruitment (30,31). 3 Phosphorylation of Bam32 in response to EGF or BCR stimulation is blocked by inhibitors of Src family kinases (31), 4 and SH2 mutant Bam32 fails to be phosphorylated (43), suggesting that this domain plays a role in bringing Bam32 together with the Src family kinases that act upon Tyr-139. Together, these results suggest a multi-step model whereby PH domain-mediated membrane recruitment of Bam32 facilitates SH2 domain-mediated interactions with other membrane-proximal proteins, allowing access to Src family kinases that phosphorylate Tyr-139.…”

Section: Discussionmentioning

confidence: 80%

The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

Allam

Niiro

Clark

et al. 2004

Journal of Biological Chemistry

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The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actinrich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.Signaling through antigen receptors expressed by T and B lymphocytes is critical for the induction and regulation of immune responses. These multi-subunit protein tyrosine kinaselinked receptors share many common signaling mechanisms with other protein tyrosine kinase-linked receptors, including the recruitment of the activated receptor to lipid rafts, the activation of several protein tyrosine kinases, and the use of adaptor proteins to link the kinase cascade to downstream amplification and effector pathways. The B cell antigen receptor (BCR)

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Section: Discussionmentioning

confidence: 80%

The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

Allam

Niiro

Clark

et al. 2004

Journal of Biological Chemistry

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“…Transient Transfection-PAE and COS-7 cells were transfected by electroporation with 20 g of total plasmid DNA as described previously (10). Transfected PAE cells were plated onto glass coverslips (ϳ8 ϫ 10 4 cells per coverslip) in Ham's F-12 media containing 10% HI-FBS for 12 h, then serum starved for 6 -8 h in F-12 media supplemented with 0.5% fatty acid-free BSA, in the presence of 1 units/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

Paranavitane¹,

Coadwell²,

Eguinoa³

et al. 2003

Journal of Biological Chemistry

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We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5␤). The protein bound PtdIns(3,4,5)P 3 selectively in vitro. Strikingly, a substantial proportion of LL5␤ became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P 3 levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5␤ to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5␤ that could not bind PtdIns(3,4,5)P 3 were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P 3 levels, LL5␤ was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns-(3,4,5)P 3 levels, it became significantly plasma-membrane localized. The distribution of LL5␤ is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P 3 indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P 3 . In addition, LL5␤ bound to the cytoskeletal adaptor, ␥-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed ␥-filamin with GFP-LL5␤ in the unidentified vesicles.Phosphoinositide 3-kinases (PI3Ks) 1 3-phosphorylate phosphoinositides. There are three classes of PI3Ks. The type I enzymes seem to act as PtdIns(4,5)P 2 3-kinases in vivo; they can be activated by a variety of close-to-receptor transduction events and drive accumulation of PtdIns(3,4,5)P 3 in the inner leaflet of the plasma membrane. This PtdIns(3,4,5)P 3 serves as signal recruiting proteins from the cytosol that possess modules, typically PH domains, capable of binding its head group (1).There are a variety of reagents that can be used to inhibit PI3K activity. Most widely used are wortmannin (2) and LY294002 (3); both of which potently inhibit nearly all classes of PI3K and hence cannot generally be used to implicate a particular PI3K in a process. More specific are receptor tyrosine kinase Tyr 3 Phe mutants. A number of receptor tyrosine kinases (relevant here, the PDGF ␤-receptor) are capable of binding type IA PI3Ks at specific tyrosine residues that become phosphorylated following ligand binding (4). Mutation of these tyrosines to phenylalanine blocks type IA PI3K binding and activation but does not affect association of other effectors (1, 5). Stable, clonal cell lines have been created overexpressing wild-type PDGF-␤ receptors or (Y740F/Y751F) PDGF-␤ receptors (6) that have allowed the impact of selectivity blocking type IA PI3K activation to be assessed in vivo (7).There is now a substantial famil...

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“…The B cell adaptor molecule of 32 kDa (Bam32), also known as DAPP1, was identified during a screen for genes highly expressed in human GC cells (14) and through screens for proteins binding to phosphoinositide products of phosphoinositide 3-kinase (PI3K) (15)(16)(17). PI3K enzymes are strongly activated by BCR ligation (18,19), and the importance of PI3K signaling in B cell development and activation is well established (20,21).…”

mentioning

confidence: 99%

“…Bam32 binds PI3K lipid products, PI(3,4)P 2 and PI(3,4,5)P 3 , in vitro (15) and is recruited to the plasma membrane in a PI3K-dependent manner upon BCR ligation (14). Bam32 phosphorylation by Src family kinases is also PI3K-dependent (17,22). Bam32 has been implicated in BCR signaling processes, including activation of the GTPase Rac1, and MAPKs ERK and JNK (23)(24)(25).…”

mentioning

confidence: 99%

The Pleckstrin Homology Domain Adaptor Protein Bam32/DAPP1 Is Required for Germinal Center Progression

Zhang

Al‐Alwan²,

Marshall

2009

The Journal of Immunology

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Ab affinity maturation within germinal centers (GCs) requires weeks to complete. Several signaling pathways in B cells have been shown to be required for initiation of the GC response; however, the signaling checkpoints controlling progression and eventual dissolution of the GC reaction are poorly understood. The adaptor protein Bam32/DAPP1 was originally isolated from human GCs and functions downstream of phosphoinositide 3-kinase enzymes, which are known to have critical roles in B cell activation and GC responses. In this study we identify a unique role of Bam32/DAPP1 in promoting GC progression. Bam32-deficient mice show normal GC initiation, but premature GC dissolution after immunization with protein Ag in alum or low doses of sheep red blood cells. Adoptive transfer studies confirmed that Bam32-deficient B cells have an intrinsic impairment in the ability to mount sustained GC responses. Bam32 deficiency was also associated with impaired Ab affinity maturation. Proliferation of Bam32-deficient GC B cells was not compromised; however, these cells show impaired switch to IgG1 and increased apoptosis in situ. GCs formed by Bam32-deficient B cells contain fewer T cells, indicating that Bam32 is required for B cell–dependent T cell accumulation within established GCs. Exogenous CD40 ligand restored GC B cell numbers and switch to IgG1, indicating that Bam32-deficient B cells are competent to respond to CD40 stimulation when ligand is available. These data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival.

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DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation

Abstract: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

Cited by 40 publications

References 33 publications

The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

The Pleckstrin Homology Domain Adaptor Protein Bam32/DAPP1 Is Required for Germinal Center Progression

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