Functional Proteomics Identifies Targets of Phosphorylation by B-Raf Signaling in Melanoma

• DOI: 10.1016/j.molcel.2009.03.007
• Publication Date: 2009-04-10T08:23:16Z
• Authors: William M. Old; John B. Shabb; Stephane Houel; Hong Wang; Kasey L. Couts; Chia-yu Yen; Elizabeth S. Litman; Carrie H. Croy; Karen Meyer-Arendt; Jose G. Miranda; Robert A. Brown; Eric S. Witze; Rebecca E. Schweppe; Katheryn A. Resing; Natalie G. Ahn
• Keywords: Proteomics; Proto-Oncogene Proteins B-raf; MAP Kinase Signaling System; Cell Biology; Phosphoproteins; Mass Spectrometry; Substrate Specificity; 3. Good health; Cell Line, Tumor; Mutation; Humans; Phosphorylation; Molecular Biology; Melanoma

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.