Functional proteomics identifies targets of phosphorylation by B-Raf signaling in melanoma

Mol Cell. 2009 Apr 10;34(1):115-31. doi: 10.1016/j.molcel.2009.03.007.

Abstract

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line, Tumor
  • Humans
  • MAP Kinase Signaling System
  • Mass Spectrometry
  • Melanoma / metabolism*
  • Mutation
  • Phosphoproteins / analysis
  • Phosphoproteins / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphoproteins / physiology
  • Phosphorylation
  • Proteomics*
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins B-raf / metabolism*
  • Proto-Oncogene Proteins B-raf / physiology
  • Substrate Specificity

Substances

  • Fam129B protein, human
  • Phosphoproteins
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf