Abstract:
The members of the Tec family of tyrosine kinases contain Src homology 2 (SH2), SH3, and pleckstrin homology (PH) domains, in addition to their kinase domains. Unlike the Src family of tyrosine kinases, which have a negative regulatory tyrosine located at their COOH-termini, members of the Tec family do not have an obvious negative regulatory site. Liu et al. (News and Views by Koyasu) have now identified a protein, termed IBtk, that interacts with and inhibits Bruton's tyrosine kinase (Btk) activity. IBtk and Btk associated in vitro and in vivo; however, IBtk did not interact with the related Tec family kinase Itk, suggesting that IBtk might specifically interact with Btk. Chicken B cells transfected with IBtk exhibited reduced changes of calcium concentrations and inhibition of NF-κB activation, in response to B cell-receptor stimulation. Other experiments revealed that the COOH-terminal portion of IBtk bound the the PH domain of Btk, and was responsible for mediating the inhibtion of Btk activity. Activation of B cells led to the localization of Btk and IBtk to the plasma membrane, which suggests that IBtk might inhibit Btk activity soon after B cell receptor-dependent activation to tightly control Btk-mediated signaling. These data are likely to spur a search for specific inhibitors of other Tec family members. W. Liu, I. Quinto, X. Chen, C. Palmieri, R. L. Rabin, O. M. Schwartz, D. L. Nelson, G. Scala, Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nature Immunol. 2 , 939-946 (2001). [Online Journal] S. Koyasu, Beating a kinase? Nature Immunol. 2 , 897-898 (2001). [Online Journal]Keywords:
Pleckstrin homology domain
Src family kinase
Pleckstrin homology domain
X-linked agammaglobulinemia
Protein kinase domain
Inositol phosphate
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Bruton's tyrosine kinase (Btk) is required for normal B cell development and signal transduction through cell surface molecules, and its defects lead to X-linked immune deficiency in mice and X-linked agammaglobulinemia in humans. In this report, we will describe the identification and characterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk. A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, to human LTG19/ENL, that was originally identified as one of the fusion partners involved in chromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutants demonstrated that the region of BAM11 required for binding to Btk was localized between amino acid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246–368) inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16 cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246–368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btk plays a regulatory role in the Btk signal transduction pathway. A cell fractionation study and analysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within the nucleus.
Pleckstrin homology domain
Identification
Characterization
Homology
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Tyrosine phosphorylation of CAS (Crk-associated substrate, p130Cas) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site.
Adapter molecule crk
Tyrosine-protein kinase CSK
Phosphorylation cascade
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Pleckstrin homology domain
X-linked agammaglobulinemia
Protein kinase domain
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Citations (192)
Bruton's tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP6) binding domain (Kd of approximately 40 nM for IP4), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP4 binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP4 binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP6 with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP4 binding capacity of the PH domain of Btk.
Pleckstrin homology domain
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Pleckstrin homology domain
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Roles of Src-family kinase isoforms, Lyn, Fyn, Fgr, and c-Src on degranulation in RBL-2H3 mast cells
흰쥐유래의 비만세포인 RBL-2H3 세포는 다양한 Src-family kinase를 발현한다. 현재까지의 연구결과에 의하면 비만세포의 초기 활성화에 Lyn kinase가 중요한 역할을 한다고 알려져 왔다. 그러나 그 세포에서 발현되는 다양한 다른 Src-family kinase의 역할은 불분명하다. 본 연구에서는 비만세포에서 다양한 Src-family kinase가 세포내 다른 곳에서 다양하게 발현되고 있다는 사실을 RT-PCR, immunoblotting 그리고 confocal microscopy 기법을 이용하여 증명하였다. 그 결과 Lyn 및 Fgr kinase는 세포막에 위치하고 c-Src 및 Yes kinase는 세포 내 과립에 존재하는 것을 알 수 있었다. 모든 Src-family kinase를 클로닝하고 과발현하여 탈과립 대한 영향을 평가하였다. 그 결과 fyn과 Fgr kinase는 비만세포에서 항원 유도의 탈과립을 증가시켰으며 반면 Lyn kinsae는 탈과립을 억제시키는 것을 확인할 수 있었다. 이러한 결과는 비만세포 초기 신호전달계에서 Fgr가 중요한 역할을 할 가능성을 제시한다. The rat RBL-2H3 mast cells contain various Src-family kinases. Previous reports with this cell line indicated that Lyn activation is an important initial signaling for the activation of the cells. However, the role and location of other Src-family kinase isoforms which are expressed in the cells are not clear. In this study, we now show that isoforms of Src-family kinases, Lyn, fyn, Fgr, c-Src, and Yes are differentially expressed and located differently in the cells as indicated by RT-PCR, immunoblotting analysis, and confocal microscopy. Lyn and Fgr were located on plasma membrane but on the other hand c-Src and Yes were located on intracellular organelle. All of Src-family kinases were cloned and overexpressed for investigating the roles of the isoforms. Overexpression of Fyn and Fgr, not Lyn and c-Src, stimulated Ag-induced degranulation in the cells. Our findings strongly suggest for the first time that each of Src-family kinase isoform can regulate differentially $Fc{\varepsilon}RI$ -mediated signaling in RBL-2H3 mast cells.
LYN
Src family kinase
FYN
Tyrosine-protein kinase CSK
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Significance Bruton’s tyrosine kinase (BTK) is targeted in treatment of immune cancers. As patients experience drug resistance, there is a need for alternative approaches to inhibit BTK. Other recently published findings clarify the role of the BTK pleckstrin homology (PH) domain in mediating activation via dimerization and sensing of ligand concentration at the membrane. Work presented here provides insight into the autoinhibitory BTK structure that has so far been elusive via crystallographic methods. In the resting state, the BTK PH domain binds to the activation loop face of the kinase domain and allosterically alters key sites within the kinase domain. The findings define a new regulatory site, the PH/kinase interface, that can be exploited in drug discovery efforts.
Pleckstrin homology domain
Protein kinase domain
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The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on protein-ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton's tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a phosphatidylinositol (3-5)-trisphosphate (PIP
Pleckstrin homology domain
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Pleckstrin homology domain
Protein kinase domain
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