Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation

Abstract

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas. A-Raf is a member of the Raf family of serine-threonine protein kinases, which comprises A-Raf, B-Raf, and Raf-1. Raf kinases are at the apex of the three-tiered Raf/MEK/ extracellular signal-regulated kinase (ERK) (mitogen-activated protein kinase (MAPK)) pathway regulating fundamental cellular functions, including differentiation, transformation, apoptosis, proliferation, and metabolism. 1,2 Activation of Ras GTPases at the cell membrane initiates Raf kinase activation and sequential phosphorylation and activation of the serinethreonine kinases MEK1/2 and ERK1/2. 3,4 In comparison to Raf-1 and B-Raf, A-Raf is only weakly activated by oncogenic H-Ras and Src 5 and is a poor MEK kinase, 5-8 which is due to unique non-conserved amino acid substitutions in the N-region. 9 Only recently, the first somatic oncogenic mutations of A-Raf were identified in lung adenocarcinomas 10 and Langerhans cell histiocytosis. 11,12 We recently showed that A-Raf and Raf-1, independent of their kinase activity, bind the proapoptotic mammalian sterile 20-like kinase (MST2) thereby suppressing MST2 activation and MST2-induced apoptosis. 13-17 MST2 employs several mechanisms to induce apoptosis including the transcriptional induction of PUMA, 14 a BH3 domain protein that causes mitochondrial depolarization and subsequent cell death. 18 Although Raf-1 counteracts MS...

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

“…Among these 10 proteins, the serine/threonine-protein kinase A-Raf and Apoptosis-inducing factor 1 (Aif), whose interaction with K-Ras4a-G12V might be increased by lysine defatty-acylation, attracted our attention. A-Raf is a member of the Raf family of serine/threonine-specific protein kinases, acts as a Ras effector and plays an important role in apoptosis ( Rauch et al, 2010 , 2016 ) and tumorigenesis ( Imielinski et al, 2014 ; Lee et al, 2010 ; Nelson et al, 2014 ). In response to apoptotic stimuli, Aif is released from the mitochondrial intermembrane space into the cytosol and nucleus, where it functions as a proapoptotic factor ( Hangen et al, 2010 ).…”

SIRT2 and lysine fatty acylation regulate the oncogenic activity of K-Ras4a

“…Among these 10 proteins, the serine/threonine-protein kinase A-Raf and Apoptosis-inducing factor 1 (Aif), whose interaction with K-Ras4a-G12V might be increased by lysine defatty-acylation, attracted our attention. A-Raf is a member of the Raf family of serine/threonine-specific protein kinases, acts as a Ras effector and plays an important role in apoptosis 62,63 and tumorigenesis 64–66 . In response to apoptotic stimuli, Aif is released from the mitochondrial intermembrane space into the cytosol and nucleus, where it functions as a proapoptotic factor 67 .…”

Spatial regulation of ARAF controls the MST2-Hippo pathway

“…1). 26 In highly proliferating cells of the basal cell layer of non-malignant stratified, non-keratinized squamous epithelia, ARAF controls MST2 in the cytoplasm and at the mitochondria. In contrast, differentiated cells of these squamous epithelia have ARAF localized at the plasma membrane.…”