title: Characterization of Itk tyrosine kinase: contribution of noncatalytic domains to enzymatic activity.
abstract: Itk is a Tec family tyrosine kinase found in T cells that is activated upon ligation of the T cell receptor (TCR/CD3), CD2, or CD28. Itk contains five domains in addition to the catalytic domain: pleckstrin homology, Tec homology which contains a proline-rich region, Src homology 3, and Src homology 2. To provide a basis for understanding the contribution of these various domains to catalysis, recombinant Itk was purified and its substrate specificity determined by steady-state kinetic methods. Measurements of the rates of phosphorylation of various protein substrates, including Src associated in mitosis 68K protein (SAM68), CD28, linker for activation of T cells, and CD3 zeta, at a fixed concentration indicated that SAM68 was phosphorylated most rapidly. Wild-type Itk and three Itk mutants were characterized by comparing their activity (k(cat)) using the SAM68 substrate. A deletion mutant removing the pleckstrin homology domain and part of the Tec homology domain (Itk(Delta152)) had approximately 10-fold less activity than wild type, a mutant with an altered proline-rich domain (P158A,P159A) had a more dramatic 100-fold loss of activity, and the catalytic domain alone was essentially inactive. Itk(Delta152) had K(m) values for ATP and SAM68 nearly identical to those of the wild-type enzyme, while Itk(P158A,P159A) had approximately 3-fold higher K(m) values for each substrate. SAM68 phosphorylation by the wild-type and mutant enzymes in the presence of several tyrosine kinase inhibitors were compared using a homogeneous time-resolved fluorescence assay. Both the Itk(Delta152) deletion mutant and the Itk(P158A,P159A) mutant had IC(50) values similar to those of the wild-type enzyme for staurosporine, PP1, and damnacanthal. These comparisons, taken together with the similar K(m) values for ATP and SAM68 substrate between the wild-type and the mutant enzymes, indicate that the amino acids in the N-terminal 152 residues and proline-rich domains enhance catalysis by affecting turnover rate rather than substrate binding.
authors: Department of High Throughput Screening and Automation, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
authors: J
authors: J
authors: Hawkins
authors: A
authors: A
authors: Marcy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D002384:Catalysis; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000880:Anthraquinones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C489559:KHDRBS1 protein, human; C513253:Khdrbs1 protein, mouse; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C073882:phosphoprotein phosphatase inhibitor 1; C079170:damnacanthal; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases; D019311:Staurosporine