Identification of Itk/Tsk Src Homology 3 Domain Ligands

doi:10.1074/jbc.271.41.25646

Identification of Itk/Tsk Src Homology 3 Domain Ligands*

From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138,
^‡ ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, the
^¶ Department of Cell Biology and
^§ Department of Pediatrics, Harvard Medical School, and The Center for Blood Research, Boston, Massachusetts 02115

^∥ To whom correspondence should be addressed. Tel.: 617-496-8121; Fax: 617-496-8123; E-mail, berg{at}biosun.harvard.edu

Abstract

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated Jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.

Footnotes

↵* This work was supported by grants from the Arthritis Foundation, the Council for Tobacco Research, Inc., and National Institutes of Health Grant AI37584 (to L. J. B.), a National Institutes of Health grant (to T. K.), and by the WAS Fund of the Center for Blood Research (to T. K. and R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Btk

Bruton's tyrosine kinase

SH3

Src homology 3

SH2

Src homology 2

PH

pleckstrin homology

TH

Tec homology

GST

glutathione S-transferase

WASP

Wiskott-Aldrich Syndrome protein

PAGE

polyacrylamide gel electrophroresis

hnRNP

heterogeneous nuclear ribonucleoprotein.
↵² S. C. Bunnell and L. J. Berg, unpublished data.
↵³ S. C. Bunnell and L. J. Berg, manuscript in preparation.
- Received May 9, 1996.
- Revision received July 23, 1996.