Syk, Activated by Cross-linking the B-cell Antigen Receptor, Localizes to the Cytosol Where It Interacts with and Phosphorylates -Tubulin on Tyrosine

doi:10.1074/jbc.271.9.4755

Syk, Activated by Cross-linking the B-cell Antigen Receptor, Localizes to the Cytosol Where It Interacts with and Phosphorylates -Tubulin on Tyrosine (*)

From the (1)Departments of Medicinal Chemistry and Molecular Pharmacology and
(2)Biological Sciences, Purdue University, West Lafayette, Indiana 47907

** To whom correspondence should be addressed:
Dept. of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Bldg., Purdue University, West Lafayette, IN 47907.
Tel.: 317-494-1457; Fax: 317-494-9193.

Abstract

Syk (p72) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as α-tubulin. α-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. α-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including α-tubulin.

Footnotes

↵^§ Supported by United States Public Health Service Training Grant GM08298.
↵^¶ Supported by a fellowship from the American Heart Association, Indiana Affiliate, Inc.
↵* This research was supported in part by United States Public Health Service, National Institutes of Health Grants CA37372 (to R. L. G. and M. L. H.) and GM49889 (to D. L. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

BCR

B-cell antigen receptor

TCR

T-cell antigen receptor

ITAM

immunoreceptor tyrosine activation motif

GST

glutathione S-transferase

cfb3

cytoplasmic fragment of erythrocyte band 3

PAGE

polyacrylamide gel electrophoresis

MAP

microtubule-associated protein.
↵²M. T. Furlong and R. L. Geahlen, manuscript in preparation.
↵³M. T. Samson and R. L. Geahlen, manuscript in preparation.
↵⁴J. D. Peters and R. L. Geahlen, unpublished observations.
- Received September 11, 1995.
- Revision received December 20, 1995.