Dataset Information

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec.

ABSTRACT: IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone ?-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk ?-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.

SUBMITTER: Boyken SE

PROVIDER: S-EPMC3631358 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec.

Boyken Scott E SE Fulton D Bruce DB Andreotti Amy H AH

Protein science : a publication of the Protein Society 20120716 9

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand bindi ...[more]

PMID: 22761113

Similar Datasets

Project description:BackgroundMast cells are indispensable for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergens, but their roles in mast cell responses in LPS-induced sepsis are unclear.ObjectiveWe sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation.MethodsMice (genetically modified or bone marrow-derived mast cell-reconstituted Sash) were given LPS to induce septic hypothermia in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-α production in peritoneal cells. Microarray was used for genomewide gene expression analysis on bone marrow-derived mast cells. Quantitative PCR and multiplex were used to determine transcribed and secreted proinflammatory cytokines. Microscopy and Western blotting were used to determine activation of signal transduction pathways.ResultsThe absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk(-/-)Btk(-/-) mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell-deficient Sash mice. LPS-induced nuclear factor kappa B, Akt, and p38 activation is enhanced in Itk(-/-)Btk(-/-) mast cells, and blockage of phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, or p38 downstream mitogen-activated protein kinase interacting serine/threonine kinase 1 activation significantly suppresses TNF-α hyperproduction and attenuates septic hypothermia.ConclusionsITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of Toll-like receptor 4/LPS in mast cells, through regulating the activation of canonical nuclear factor kappa B, phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt, and p38 signaling pathways.

Dataset Information

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec.

Publications

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets