Shift syndecan-2 from RACK1 to caveolin-2 upon transformation with oncogenic ras

doi:10.1016/j.bbrc.2006.09.035

Biochemical and Biophysical Research Communications

Volume 350, Issue 1, 10 November 2006, Pages 227-232

https://doi.org/10.1016/j.bbrc.2006.09.035 Get rights and content

Abstract

Syndecan-2 was found to detach from RACK1 and associate with caveolin-2 and Ras in cells transformed with oncogenic ras. Most of syndecan-2 from transformed cells was revealed with negligible phosphorylations at tyrosine residues. We experimented with HeLa cells transfected with plasmids encoding syndecan-2 and its mutants (syndecan-2^Y180F, syndecan-2^Y192F, and syndecan-2^Y180,192F) to provide evidences that PY¹⁸⁰ of syndecan-2 is a binding site for RACK1 and is deprived in cells transfected with oncogenic ras. However, in HeLa cells transfected with syndecan-2^Y180F, RACK1 was found to sustain its reactions with syndecan-2 independent of phosphorylation. The finding of syndecan-2 reactive with caveolin-2/Ras suggests the molecular complex most likely to obstruct RACK1 for functional attachment at syndecan-2, as revealed in cells transfected with oncogenic ras. We provided evidences to reinforce the view that molecular rearrangements upon transformation are specific and interesting.

Section snippets

Materials and methods

Materials. All reagents used were of the highest grade available commercially. [γ-³²P]Adenosine5′-triphosphate ([γ-³²P]ATP) was from Amersham (Buckinghamshire, UK). ATP and granylocyte-macrophage colony-stimulating factor (GM-CSF) were from Roche Molecular Biochemicals (Penzberg, Germany). Protein A and Protein G magnetic microbeads and μMAS (magnetic Sorting) columns for small scale (approximately 100 μl) molecular biology applications were purchased from Miltenyi Biotec (Bergisch Gladbach,

Syndecan-2 associates with Ras and caveolin-2 in cells transformed with oncogenic ras

Membrane proteins were effectively released in soluble form by treatment with warm Triton X-100 from membranes of BALB/3T3 cells transfected with plasmid pcDNA3.1 or pcDNA3.1-[S-ras(Q₆₁K)] [29], [30]. The membrane lysates of cells transformed with oncogenic ras or not were immunoprecipitated with antibodies against syndecan-2 and counterstained on a Western blot with antibodies against caveolin-1, and caveolin-2, and Ras (Fig. 1). The syndecan-2 of transformed cells was competent at recruiting

Discussions

By transformation with oncogenic ras and analysis with immunoprecipitation, syndecan-2 was found to detach from RACK1 but associate with caveolin-2 and Ras in transformed cells. In parallel, syndecan-2 was identified in a tyrosine phosphorylated freed form in transformed cells, the tyrosine kinase is presumably inactive, in contrast with tyrosine phosphorylated form in cells without transformation. This seems unlikely on the basis of current knowledge of kinases in signal-transduction systems