title: 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 α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-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.
authors: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, USA.
authors: Scott E
authors: SE
authors: Boyken
authors: D Bruce
authors: DB
authors: Fulton
authors: Amy H
authors: AH
authors: Andreotti
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D000077329:Agammaglobulinaemia Tyrosine Kinase; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins; C089848:Tec protein-tyrosine kinase; C080813:emt protein-tyrosine kinase