Regulation of Btk by Src Family Tyrosine Kinases
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Abstract:
Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.Keywords:
Src family kinase
Tyrosine-protein kinase CSK
Diversity in Src-family kinase activation mechanisms: Implications for selective inhibitor discovery
The Src kinase family encompasses eight non-receptor protein tyrosine kinases in mammals that regulate signaling pathways in virtually every cell type. Src-family kinases (SFKs) share a common regulatory mechanism that requires two intramolecular interactions to maintain the inactive state. These involve binding of the SH3 domain and a PPII helix in the SH2-kinase linker and interaction of the SH2 domain and a phosphotyrosine residue in the C-terminal tail. To compare the activation dynamics of the individual SFKs, a synthetic SFK SH3 domain-binding peptide (VSL12) was used to probe the sensitivity of SFKs to SH3-based activation. Surface plasmon resonance was used to confirm equivalent binding of the VSL12 peptide to the SH3 domains and near-full-length kinases. SFKs were tested with VSL12 in a kinetic kinase assay to measure the changes in the rate of activity induced by SH3:linker displacement. All SFKs tested were susceptible to activation, but to varying degrees. Further, autophosphorylation of the c-Src and Hck activation loops prior to VSL12-induced activation revealed that c-Src can achieve a higher level of activation if primed before SH3 domain displacement. These results suggest that distinct activation thresholds may exist for individual SFKs. To apply these findings in an inhibitor discovery setting, the interaction of c-Src with focal adhesion kinase (FAK) was studied. FAK activates c-Src by binding to its SH2 and SH3 domains and disrupting their regulatory influence on the kinase domain. The c-Src:FAK complex plays a major role in the migration and invasion of both normal and cancer cells, making it an attractive target for drug discovery. To test the idea that FAK binding induces allosteric changes in the kinase domain active site, a screening assay was developed to target these changes with small molecule inhibitors. Assay conditions were identified where c-Src activity was dependent on a phosphopeptide encompassing the FAK SH3- and SH2-binding motifs for c-Src. A focused library of kinase-biased inhibitors was screened to identify compounds displaying selectivity for the c-Src:pFAK peptide complex. An aminopyrimidinyl carbamate, WH-4-124-2, was discovered that showed five-fold selectivity for the complex. Molecular docking studies of this inhibitor on the kinase domain of Lck bound to imatinib suggest that WH-4-124-2 is a “Type II” kinase inhibitor that prefers the unphosphorylated, “DFG-out” conformation of the kinase. Selective inhibitors of a specific FAK-induced c-Src conformation may provide a unique approach to selective targeting of this key cancer cell signaling pathway.
Tyrosine-protein kinase CSK
Src family kinase
Protein kinase domain
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Src is a nonreceptor type protein tyrosine kinase and a prototype of a family consisting of eight members in vertebrates: Src, Yes, Fyn, Fgr, Lyn, Hck, Lck, Blk. Src is an evolutionary well-conserved gene in vertebrates with homologs expressed in invertebrates. When compared with other nonreceptor type 1 tyrosine kinases in the human genome, the Src family is the largest, with its members very closely related to each other, particularly those within two Src family subgroups (Fig. 1). Such a high degree of similarity is also present in receptor tyrosine kinase families, e.g., the epidermal growth factor receptor (EGFR) family, which is the most similar to nonreceptor tyrosine kinases (Fig. 1). Interestingly, the presence of many close family members within the Src family corresponds to their biological activity, which is linked to physical and functional association with receptor tyrosine kinases at the plasma membrane (1).
Tyrosine-protein kinase CSK
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FYN
Src family kinase
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Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.
FYN
Tyrosine-protein kinase CSK
Src family kinase
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Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.
Tyrosine-protein kinase CSK
Src family kinase
Protein kinase domain
LYN
Histidine kinase
HAMP domain
Linker
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Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type.
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Tyrosine-protein kinase CSK
Src family kinase
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Protein kinase domain
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We previously identified a gene, LckBP1, which encodes a protein that binds to the Lck SH3 domain and is identical to murine HS1. Using unstimulated T lymphocytes, we further demonstrated that Lck binds to HS1 in vivo and that HS1 is tyrosine phosphorylated upon TCR stimulation. In the present report, we analyzed the binding pattern of several src kinases and HS1 in greater detail. The Lck SH3 domain binds to HS1 constitutively, while the Lck SH2 domain associates with HS1 only upon TCR stimulation. A similar binding pattern was observed with Lyn and HS1, but not with Fyn and HS1, in which the Fyn SH3 region associates with HS1 upon TCR stimulation but the Fyn SH3 region does not associate with HS1 regardless of TCR stimulation. Such distinct binding patterns of the src kinase SH2 and SH3 domains to HS1 may represent a mechanism by which src family kinases select substrates and activate particular downstream signaling pathways.
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FYN
Tyrosine-protein kinase CSK
Src family kinase
Protein kinase domain
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Src family kinase
Tyrosine-protein kinase CSK
Protein kinase domain
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Bruton's tyrosine kinase (Btk) is a recently described B-cell-specific tyrosine kinase. Mutations in this gene lead to human X chromosome-linked agammaglobulinemia and murine X-linked immunodeficiency. Although genetic evidence strongly suggests that Btk plays a crucial role in B-lymphocyte differentiation and activation, its precise mechanism of action remains unknown, primarily because the proteins that it interacts with have not yet been identified. Here, we show that Btk interacts with Src homology 3 domains of Fyn, Lyn, and Hck, protein-tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-aa motifs in Btk. An analogous site with the same specificity is also present in Itk, the T-cell-specific homologue of Btk. Our data extend the range of interactions mediated by Src homology 3 domains and provide an indication of a link between Btk and established signaling pathways in B lymphocytes.
LYN
FYN
Src family kinase
Tyrosine-protein kinase CSK
X-linked agammaglobulinemia
Protein kinase domain
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Tyrosine-protein kinase CSK
LYN
Src family kinase
Rous sarcoma virus
Protein kinase domain
HAMP domain
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FYN
Tyrosine-protein kinase CSK
Src family kinase
Jurkat cells
Protein kinase domain
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