Profiling the dynamics of a human phosphorylome reveals new components in HGF/c-Met signaling.

Crystal L. Woodard, C. Rory Goodwin, Jun Wan, Shuli Xia, Robert Newman, Jianfei Hu, Jin Zhang, S. Diane Hayward, Jiang Qian, John Laterra, Heng Zhu

Research output: Contribution to journalArticle

Abstract

Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.

Original languageEnglish (US)
Article numbere72671
JournalPloS one
Volume8
Issue number9
DOIs
StatePublished - 2013

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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