Phosphorylation- and Activation-independent Association of the Tyrosine Kinase Syk and the Tyrosine Kinase Substrates Cbl and Vav with Tubulin in B-Cells

doi:10.1074/jbc.274.3.1401

Phosphorylation- and Activation-independent Association of the Tyrosine Kinase Syk and the Tyrosine Kinase Substrates Cbl and Vav with Tubulin in B-Cells*

From the Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907

Abstract

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk⁻ B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Footnotes

↵* This research was supported by Public Health Service Grant CA37372 awarded by the National Institutes of Health.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.
↵‡ Supported by Public Health Service Predoctoral Fellowship 3 F31 GM18062-01S1 awarded by the National Institutes of Health.
↵§ To whom correspondence should be addressed: Dept. of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Science Research Bldg., Purdue University, West Lafayette, IN 47907. Tel.: 765-494-1457; Fax: 765-494-9193; E-mail:geahlen{at}pharmacy.purdue.edu.
Abbreviations:

BCR

B-cell antigen receptor

SH2

Src homology 2

ITAM

immunoreceptor tyrosine-based activation motif

PAGE

polyacrylamide gel electrophoresis

WT

wild-type.
- Received August 31, 1998.
- Revision received October 20, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.