Characterization of the Pleckstrin Homology Domain of Btk as an Inositol Polyphosphate and Phosphoinositide Binding Domain

Kojima, Toshio; Fukuda, Mitsunori; Watanabe, Yutaka; Hamazato, Fumiaki; Mikoshiba, Katsuhiko

doi:10.1006/bbrc.1997.6947

Cited by 80 publications

(53 citation statements)

References 35 publications

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“…The activation of Btk was observed at concentrations of 10 nM to 10 M, thus yielding a 50% maximal activation (EC 50 ) of ϳ100 nM. This result closely corresponds to the value reported by others for the affinity of the Btk PH domain for PtdIns-3,4,5-P 3 (100 -700 nM) and is consistent with a direct interaction between PtdIns-3,4,5-P 3 and the Btk PH domain being involved in the kinase activation effect (16,22).…”

Section: Ptdins-345-psupporting

confidence: 89%

Interaction between the Btk PH Domain and Phosphatidylinositol-3,4,5-trisphosphate Directly Regulates Btk

Saito

Scharenberg²,

Kinét

2001

Journal of Biological Chemistry

124

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Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) through the Btk pleckstrin homology (PH) domain, an interaction thought to be required for Btk membrane translocation during B cell receptor signaling. Here, we report that interaction of PtdIns-3,4,5-P 3 with the PH domain of Btk directly induces Btk enzymatic activation in an in vitro kinase assay. A point mutation that reduces interaction of PtdIns-3,4,5-P 3 with the Btk PH domain blocks in vitro PtdIns-3,4,5-P 3 -dependent Btk activation, whereas the PH domain deletion enhances Btk basal activity but eliminates the PtdIns-3,4,5-P 3 -dependent stimulation. Btk kinase activity and the Btk activation loop phosphorylation site are both required for the PtdIns-3,4,5-P 3 -mediated stimulation of Btk kinase activity. Together, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; when interacting with PtdIns-3,4,5-P 3 , this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors and Btk catalytically inactive mutants, we demonstrate that in vivo, the activation of Btk is due to both Lyn phosphorylation and PtdIns-3,4,5-P 3 -mediated direct activation. Thus, the Btk-PtdIns-3,4,5-P 3 interaction serves to translocate Btk to the membrane and directly regulate its signaling function. The B cell antigen receptor (BCR)1 produces an inositol-1,4,5-trisphosphate (IP 3 )-mediated calcium signal that is required for antigen-driven B cell development (1, 2). One of the most important components of this BCR calcium signaling pathway is mediated by Bruton's tyrosine kinase (Btk), the predominant member of the Tec kinase family that is expressed in B cells (3). Btk is required for the majority of BCR-stimulated IP 3 production, and its function in this process is tightly linked to the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) (4 -6). The critical role of Btk/ PtdIns-3,4,5-P 3 -dependent IP 3 production in the B cell receptor signal is demonstrated by the ability of targeted deletions of Btk, the p85 subunit of phosphatidylinositol 3-kinase or phospholipase C␥2, to produce highly similar B cell immunodeficiencies in the mouse (7-11) and by the association of Btk mutations that abrogates phospholipase C␥2 activation with X-linked agammaglobulinemia in humans (12, 13). Furthermore, Btk/PtdIns-3,4,5-P 3 -dependent IP 3 signaling is blocked by the Fc␥RIIb1 inhibitory receptor, whose coengagement with the BCR has been shown to inhibit BCR-mediated IP 3 production, BCR-mediated calcium signaling, BCR-dependent antibody production, and B cell proliferation (14).The Btk recruitment into activated BCR signaling complexes is thought to be a direct result of its interaction with PtdIns-3,4,5-P 3 in the plasma membrane following local accumulation after BCR engagement (4, 5). This model of Btk activation is based on evidence from direct binding of the Btk PH domain and...

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Section: Ptdins-345-psupporting

confidence: 89%

Interaction between the Btk PH Domain and Phosphatidylinositol-3,4,5-trisphosphate Directly Regulates Btk

Saito

Scharenberg²,

Kinét

2001

Journal of Biological Chemistry

124

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“…As has previously been reported, substitution of R28C in the Btk PH domain reduces binding to PtdInsP 3 (8), whereas substitution of E41K dramatically increases binding. The wild-type Tec PH domain binds PtdInsP 3 but to a lesser degree than the wild-type Btk PH domain, as was previously reported (6). Interestingly, both the R29C and E42K substitutions in the Tec PH domain substantially reduce binding to PtdInsP 3 .…”

Section: Figuresupporting

confidence: 77%

Tec Kinase Signaling in T Cells Is Regulated by Phosphatidylinositol 3-Kinase and the Tec Pleckstrin Homology Domain

Yang

Ching

Tsoukas

et al. 2001

The Journal of Immunology

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Tec, the prototypical member of the Tec family of tyrosine kinases, is abundantly expressed in T cells and other hemopoietic cell types. Although the functions of Itk and Txk have recently been investigated, little is known about the role of Tec in T cells. Using antisense oligonucleotide treatment to deplete Tec protein from primary T cells, we demonstrate that Tec plays a role in TCR signaling leading to IL-2 gene induction. Interestingly, Tec kinases are the only known family of tyrosine kinases containing a pleckstrin homology (PH) domain. Using several PH domain mutants overexpressed in Jurkat T cells, we show that the Tec PH domain is required for Tec-mediated IL-2 gene induction and TCR-mediated Tec tyrosine phosphorylation. Furthermore, we show that Tec colocalizes with the TCR after TCR cross-linking, and that both the Tec PH and Src homology (SH) 2 domains play a role in this association. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, abolishes Tec-mediated IL-2 gene induction and Tec tyrosine phosphorylation, and partially suppresses Tec colocalization with the activated TCR. Thus, our data implicate the Tec kinase PH domain and phosphatidylinositol 3-kinase in Tec signaling downstream of the TCR.

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“…The diversity of primary sequence appears to lend to the ability to bind other molecules with variable a nities, when comparing di erent PH domains. For example, phospholipid binding a nities vary greatly between individual PH domains with the PH domain from PLC-d1 preferring Ins-(1,4,5)P3 and PtdIns(4,5)P2, while others, like the PH domains from Btk and PKB, prefer PtdIns(3,4,5)P3 (Gray et al, 1999;Kojima et al, 1997;Lemmon et al, 1995). Similar disparities for protein binding are seen for protein binding partners for PH domains (Rodriguez et al, 1999).…”

Section: Ph Domain-directed Interactions Between Afap-110 and Signalimentioning

confidence: 88%

The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity

et al. 2001

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Characterization of the Pleckstrin Homology Domain of Btk as an Inositol Polyphosphate and Phosphoinositide Binding Domain

Cited by 80 publications

References 35 publications

Interaction between the Btk PH Domain and Phosphatidylinositol-3,4,5-trisphosphate Directly Regulates Btk

Interaction between the Btk PH Domain and Phosphatidylinositol-3,4,5-trisphosphate Directly Regulates Btk

Tec Kinase Signaling in T Cells Is Regulated by Phosphatidylinositol 3-Kinase and the Tec Pleckstrin Homology Domain

The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity

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