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

Cell Death Differ. 2016 Aug;23(8):1283-95. doi: 10.1038/cdd.2016.2. Epub 2016 Feb 19.

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.

MeSH terms

  • Apoptosis*
  • Caspase 8 / metabolism
  • Cell Differentiation* / drug effects
  • Cell Membrane / metabolism
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • MCF-7 Cells
  • Microscopy, Fluorescence
  • Mitochondria / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Neuregulin-1 / pharmacology
  • Protein Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins A-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins A-raf / genetics
  • Proto-Oncogene Proteins A-raf / metabolism*
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Serine-Threonine Kinase 3

Substances

  • Neuregulin-1
  • RNA, Small Interfering
  • KSR2 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins A-raf
  • STK3 protein, human
  • Serine-Threonine Kinase 3
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Caspase 8