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Article
Mechanism and Functional Significance of Itk Autophosphorylation
Journal of Molecular Biology
  • Raji E. Joseph, Iowa State University
  • D. Bruce Fulton, Iowa State University
  • Amy H. Andreotti, Iowa State University
Document Type
Article
Publication Version
Accepted Manuscript
Publication Date
11-9-2007
DOI
10.1016/j.jmb.2007.08.060
Abstract

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full length Itk mutant containing a phenylalanine in place of the autophosphorylated tyrosine has been previously studied in Itk deficient primary T cells. These studies revealed that the nonphosphorylated enzyme only partially restores Itk mediated signaling. In spite of these insights, the precise role of the Tec kinase autophosphorylation site remains unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay we also show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand binding capability of the Itk SH3 domain in a ligand dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.

Comments

This is a manuscript of an article published as Joseph, Raji E., D. Bruce Fulton, and Amy H. Andreotti. "Mechanism and functional significance of Itk autophosphorylation." Journal of molecular biology 373, no. 5 (2007): 1281-1292. doi: 10.1016/j.jmb.2007.08.060. Posted with permission.

Creative Commons License
Creative Commons Attribution-Noncommercial-No Derivative Works 4.0
Copyright Owner
Elsevier Ltd
Language
en
File Format
application/pdf
Citation Information
Raji E. Joseph, D. Bruce Fulton and Amy H. Andreotti. "Mechanism and Functional Significance of Itk Autophosphorylation" Journal of Molecular Biology Vol. 373 Iss. 5 (2007) p. 1281 - 1292
Available at: http://works.bepress.com/amy_andreotti/24/