Functional proteomics identifies targets of phosphorylation by B-Raf signaling in 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.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
William M
WM
Old
John B
JB
Shabb
Stephane
S
Houel
Hong
H
Wang
Kasey L
KL
Couts
Chia-Yu
CY
Yen
Elizabeth S
ES
Litman
Carrie H
CH
Croy
Karen
K
Meyer-Arendt
Jose G
JG
Miranda
Robert A
RA
Brown
Eric S
ES
Witze
Rebecca E
RE
Schweppe
Katheryn A
KA
Resing
Natalie G
NG
Ahn
D045744:Cell Line, Tumor; D006801:Humans; D020935:MAP Kinase Signaling System; D013058:Mass Spectrometry; D008545:Melanoma / Q000378:metabolism*; D009154:Mutation; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D040901:Proteomics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013379:Substrate Specificity
C541423:NIBAN2 protein, human; D010750:Phosphoproteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf