Systems-level analyses of cell signaling in cancer by receptor tyrosine kinases

Signaling through members of the Receptor Tyrosine Kinase (RTK) family is critical for cellular communication in multicellular organisms. Signaling abnormalities in the Insulin Receptor family cause diseases such as diabetes while mutations in the Fibroblast Growth Factor Receptor family cause both developmental abnormalities (like dwarfism and achondroplasia) as well as cancer. Most RTKs function at the cellular membrane by binding to an extracellular growth factor that elicits either dimerization or a change in conformation of the receptors. This event then leads to the activation of intracellular tyrosine kinase activity and subsequent auto- and trans-phosphorylation of many sites on the receptor as well as on other intracellular proteins. Each of these phosphorylation sites generally serves as either an allosteric switch (of an activity contained by the protein) or a docking site for downstream signaling proteins containing Src Homology 2 (SH2) or Phosphotyrosine binding (PTB) domains.

While a personal search of the protein coding sequences of the intracellular regions of the 59 human RTK genes reveals 944 tyrosines, only 214 sites have been reported in the literature to be phosphorylated. In fact, many RTKs and RTK membrane accessory proteins such as Erbb4, FGFR4, ROS1, AXL, and insulin receptor substrates 2 and 3 have no reported tyrosine phosphorylation sites even though many of these signaling proteins are known to perform important biological functions. Because of the transient nature (~5 second off rate) of the downstream protein interactions, most are also unable to be detected using conventional mass spectrometry.

We have developed a protein microarray platform containing nearly every SH2 and PTB domain in the human genome arrayed into wells of microtiter plates. This platform allows for the quantitative determination of binding constants between our immobilized proteins (corresponding to SH2-containing signaling proteins) and soluble fluorescently labeled proteins or peptides (corresponding to the sites of phosphorylation on active RTKs). We used this platform to obtain a quantitative protein interaction network for the ErbB receptors.

Additional Developing Project Ideas:

Systems-level examination of phosphorylation patterns in cancer using array and mass spectrometric technology.

Systems-level analysis of transcriptional networks at the protein level using array technology.

Defining the phosphorylation sites on other RTKs using mass spectrometry.

Using the protein microarray platform to provide a biochemical definition of protein recruitment to other phosphorylation sites in vitro.

Systems-level analysis of signal transduction by serine-threonine phosphorylation.

Multiplexed, Real-time Analysis of Protein-Protein Interaction.

Sound interesting? Inquire within!

Keywords:

Systems Biology
Protein Micro Arrays
Genomics
Proteomics
Chemical Biology
Mass spectrometry
SH2 Domains
Phosphorylation
Cell Signaling
Cancer