Characterization of RSK3 interactome in human cells

Cellular senescence is characterized by a proliferation arrest accompanied by a senescence-associated secretory phenotype (SASP) in normal epithelial cells. During carcinogenesis cells lose the ability to undergo senescence, at least the associated proliferation arrest, in response to stresses such as oncogenes, or TGFβ, a SASP molecule able to mediate autocrine and paracrine senescence. A genetic screen in human mammary epithelial cells (HMECs) identified that the serine threonine kinase RSK3 reverts TGFβ-induced senescence. Interestingly, RSK3 expression decreases in response to TGFβ in a SMAD3-dependent manner, and its constitutive expression rescues SMAD3-induced premature senescence, indicating that decrease of RSK3 itself contributes to TGFβ-induced senescence. Mechanistically, RSK3 regulates senescence by inhibiting NF-κΒ pathway through the decrease in proteasome-mediated IκBα degradation. To better understand how RSK3 might interfere with IκBα degradation, we conducted immunoprecipitation experiments to purify the RSK3-Flag protein and its partners. Mass spectrometry-based proteomics was then performed to identify potential partners.