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A ceramide-binding C1 domain mediates kinase suppressor of ras membrane translocation

Xianglei Yin1, Mohammad Zafrullah, Hyunmi Lee

  • 1Laboratory of Signal Transduction, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology
|August 28, 2009
PubMed

Insights

Kinase Suppressor of Ras 1 (KSR1) binds ceramide, a lipid second messenger. This binding is crucial for KSR1

Area of Science:

  • Cellular signaling
  • Molecular biology
  • Biochemistry

Background:

  • Kinase Suppressor of Ras 1 (KSR1) is a key regulator of the Ras-Raf-MAPK pathway.
  • KSR1's CA3 domain is homologous to atypical PKC C1 lipid-binding domains and mediates membrane translocation for c-Raf-1 activation.

Purpose of the Study:

  • To biochemically characterize the KSR1 CA3 domain's lipid-binding properties.
  • To investigate the role of ceramide in KSR1 translocation and activation.

Main Methods:

  • Enzyme-linked immunosorbent assay (ELISA) to assess lipid binding of purified GST-KSR1-CA3 protein.
  • Stimulation of COS-7 cells with epidermal growth factor (EGF) to observe KSR1 translocation.
  • Pharmacologic inhibition of ceramide generation and disruption of key cysteines in KSR1.

Main Results:

  • Purified GST-KSR1-CA3 protein specifically binds ceramide, but not other tested lipids.
  • KSR1 translocates to glycosphingolipid-enriched plasma membrane platforms and is activated upon EGF stimulation.
  • Inhibiting ceramide generation or disrupting critical cysteines in KSR1 reduces its translocation and kinase activity.

Conclusions:

  • Ceramide acts as a specific lipid-binding moiety for the KSR1 CA3 domain.
  • Ceramide-mediated targeting of KSR1 to membrane platforms is essential for its activation in the Ras-Raf-MAPK pathway.
  • This study elucidates a mechanism for ceramide in regulating transmembrane signal transduction by controlling protein localization.

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