Profiling the Dynamics of a Human Phosphorylome Reveals New Components in HGF/c-Met Signaling

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.

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18

“…Because a common means for a pathogen to hijack the cellular pathways is by targeting multiple components of the same host signaling pathways (20), we first performed the STRING FIG. 2.…”

Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks

“…This has led to the development of several complementary approaches wherein more complex solutions, such as pre-formed protein complexes (J. Hu et al, 2015) or even whole cell lysates (Goodwin et al, 2016; Woodard et al, 2013), are used in place of purified proteins in the mobile phase. This can be combined with computational approaches to identify novel KSRs that are dependent on auxiliary interactions.…”

Discovering cancer biomarkers from clinical samples by protein microarrays

“…The later tendency is to monitor a dynamic global change of proteomics under certain conditions. As a case in point, Woodard et al compared phosphorylation profiles obtained from cell and xenograft tumor lysates under activation or inhibition of a receptor tyrosine by incubating these lysates with functional protein microarray [99]. Many proteins were discovered to be differentially phosphorylated when the related pathway was activated by tyrosine and many new components in the downstream of this signaling pathway were also identified.…”