Characterization of Itk tyrosine kinase: Contribution of noncatalytic domains to enzymatic activity

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 fiv e domains in addition to the catalytic domain: pleckstrin homology, Tec hom ology which contains a proline-rich region, Src homology 3, and Src homolog y 2. To provide a basis for understanding the contribution of these various domains to catalysis, recombinant Itk was purified and its substrate speci ficity determined by steady-state kinetic methods. Measurements of the rate s of phosphorylation of various protein substrates, including Src associate d in mitosis 68K protein (SAM68), CD28, linker for activation of T cells, a nd CD3 zeta, at a fixed concentration indicated that SAM68 was phosphorylat ed most rapidly. Wild-type Itk and three Itk mutants were characterized by comparing their activity (k(cat)) using the SAM68 substrate. A deletion mut ant removing the pleckstrin homology domain and part of the Tec homology do main (Itk(Delta 152)) had approximately 10-fold less activity than wild typ e, a mutant with an altered proline-rich domain (P158A,P159A) had a more dr amatic 100-fold loss of activity, and the catalytic domain alone was essent ially inactive. Itk(Delta 152) had K-m values for ATP and SAM68 nearly iden tical to those of the wild-type enzyme, while Itk(P158A,P159A) had approxim ately 3-fold higher K-m values for each substrate. SAM68 phosphorylation by the wild-type and mutant enzymes in the presence of several tyrosine kinas e inhibitors were compared using a homogeneous time-resolved fluorescence a ssay. Both the Itk(Delta 152) deletion mutant and the Itk(P158A,P159A) muta nt had IC50 values similar to those of the wild-type enzyme for staurospori ne, PP1, and damnacanthal. These comparisons, taken together with the simil ar K-m values for ATP and SAM68 substrate between the wild-type and the mut ant enzymes, indicate that the amino acids in the N-terminal 152 residues a nd proline-rich domains enhance catalysis by affecting turnover rate rather than substrate binding. (C) 2001 Academic Press.