Tyrosine Phosphorylation of Syndecan-1 and -4 Cytoplasmic Domains in Adherent B82 Fibroblasts

doi:10.1074/jbc.273.52.35291

Tyrosine Phosphorylation of Syndecan-1 and -4 Cytoplasmic Domains in Adherent B82 Fibroblasts*

Vanessa L. Ott and
Alan C. Rapraeger‡

From the Program in Cellular and Molecular Biology and Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706

Abstract

The syndecans, a family of cell surface proteoglycans, have highly conserved cytoplasmic domains that bind proteins containing PDZ domains and co-localize with the actin cytoskeleton. The syndecan cytoplasmic domains contain four conserved tyrosine residues, two of which are located within favorable sequences for phosphorylation. Endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected in adherent B82 fibroblasts. Approximately 1.5% of total syndecan is endogenously phosphorylated, while most, if not all, cell surface syndecan is phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate. Syndecan phosphorylation is also detected in Raji-S1 and NMuMG cells, but only following treatment with vanadate or pervanadate, suggesting that endogenous phosphorylation is maintained in an “off” state in these cells. Endogenous syndecan phosphorylation in B82 cells is rapidly blocked by genistein (IC₅₀ < 10 μm) confirming the presence of a constitutively active kinase and a corresponding tyrosine phosphatase. Phosphorylation is also inhibited by herbimycin A (IC₅₀ < 1.0 μm) and staurosporine (IC₅₀ < 1.0 nm), suggesting a role for Src family kinases in regulating syndecan phosphorylation. Together, these data suggest an important role for tyrosine phosphorylation of the syndecan cytoplasmic domains in regulating downstream signaling events in response to cell adhesion and/or growth factor activity.

Footnotes

↵* This work was supported by Grants HD21881 and HD07477 from 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.
↵‡ To whom correspondence should be addressed. Tel.: 608-262-7577; Fax: 608-265-3301; E-mail: acraprae{at}facstaff.wisc.edu.
Abbreviations:

GAG

glycosaminoglycan

FAK

focal adhesion kinase

TES

Tris-EDTA saline

TBS

Tris-buffered saline

TBS/EDTA

Tris-buffered saline containing EDTA

MES

2-(N-morpholino)ethanesulfonic acid

TBST

Tris-buffered saline containing Tween 20

PAGE

polyacrylamide gel electrophoresis

ITAM

immunoreceptor tyrosine-based activation motif

PBS

phosphate-buffered saline

mAb

monoclonal antibody

SH2

Src homology domain 2.
- Received May 7, 1998.
- Revision received October 6, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.