Abstract
BackgroundSmurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Methods: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation.ResultsCNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines.ConclusionsAltogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.
Highlights
Smad ubiquitin regulatory factor 2 (Smurf2) E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates
Smurf2 was readily detected in CNKSR2 immunoprecipitates (Fig. 1a) and reciprocally, CNKSR2 was found in Smurf2 immunoprecipitates (Fig. 1b)
Smurf2 interacts with CNKSR2 in a ligase independent manner To confirm that endogenous CNKSR2 associates with Smurf2 and to analyse if forced overexpression of Smurf2 increased CNKSR2 levels, Human embryonic kidney 293 (HEK293) cells were transfected with Flag-tagged Smurf2 WT, Flag-tagged Smurf2 C716A, and empty vector followed by immunoprecipitation and immunoblotting
Summary
Smurf E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. Smurf is a C2-WW-HECT E3 ubiquitin ligase that has the ability to ubiquitinate and degrade some proteins, e.g., the TGF-β receptor I and associated Smad proteins involved in TGF- β/BMP signaling, and have a unique ability to protect other substrates, e.g., spindle assembly checkpoint protein, MAD2 and NEDD9/HEF1 [5]. It contains WW domains, which directly bind to a PPXY motif ( known as PY motif ) in its targets which is further stabilized by the PY tail, a six-amino acid stretch immediately carboxylterminal to the PPXY motif, additional interactions exist [6]. Expanding the family of Smurf targets may provide further insights into regulatory role of Smurf E3 ubiquitin ligases in the cellular environment [5]
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