Tyrosine Phosphorylation of Sam68 by Breast Tumor Kinase Regulates Intranuclear Localization and Cell Cycle Progression

Kiven Erique Lukong,Daniel Larocque,Angela L Tyner,Stéphane Richard

doi:10.1074/jbc.m505802200

Abstract

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.

Highlights

Identified by aligning the first three members of this family [6]
We focused our results on the infiltrating ductal carcinomas (IDCs) because the array contained 175 different samples of this particular tumor type. 20/175 (11.4%) IDCs stained positively with anti-440pY antibodies, whereas 29/175 (16.6%) IDCs stained positive for breast tumor kinase (BRK) expression and 8 of the BRK-positive IDCs contained phosphorylated Tyr440
BRK Is Required for the Tyrosine Phosphorylation of Sam68 in Response to epidermal growth factor (EGF) Treatment—Because BRK has been shown to potentiate the mitogenic signals of EGF [33], we investigated first whether endogenous Sam68 was phosphorylated in response to EGF treatment

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Peptides comprising amino acids 432– 443 containing 3 tyrosine residues in the C-terminal of Sam were synthesized at W. After digestion with BamHI, the fragment was cloned into the BglII site of pADTR5-K7GFPq [37] These plasmids express myc-BRK and myc-Sam under the regulation of a tetracycline-inducible promoter and a second cassette that constitutively expresses GFP. The cells were pelleted by centrifugation, resuspended in PBS/bovine serum albumin/ Tween 20, and incubated for 1 h with a monoclonal antibody to BrdUrd (Chemicon). This was followed by incubation with a Cy5-labeled secondary antibody to mouse (1:500, Molecular Probes), resuspended in PBS containing 10 ␮g/ml propidium iodide, and analysis by flow cytometry. For BrdUrd labeling on astrocytes cultured on coverslips, infected astrocytes were pulsed for 8 h with 10 mM BrdUrd (Sigma) and fixed with 4% paraformaldehyde in phosphate-buffered saline and visualized by immunofluorescence using an Alexa 546-conjugated antibody (Molecular Probes, Inc.)

RESULTS

TABLE ONE

Metastatic breast adenocarcinoma

DISCUSSION