Aberrations in proliferation and survival signaling pathways are common in
cancer cells, and are likely required for full oncogenic
transformation. The mutations resulting in dysregulated signaling commonly
involve the hyperactivation of tyrosine kinases. For many cancer types,
known kinase mutations can be found in a subset, but not all, of the cases.
Taken together, these observations strongly suggest that additional
cancer-driving tyrosine kinase mutations remain to be discovered. The
advent of highly specific kinase inhibitors, such as Gleevec, points to a
future of cancer therapy where treatments are customized based on the set
of kinase mutations detected in each patient – thus, further motivating the
identification of novel kinase mutations.
We have developed assays to enrich for tyrosine phosphorylated proteins by
affinity purification and find their identity and sites of phosphorylation
by mass spectrometry. We are applying this ‘phosphorylation profiling’ to
acute leukemia cells to help identify the kinase activity driving the
cancer phenotype. I will review our methods and results, with emphasis on
the issues that arise in mass spectrometry when analyzing post
translational modifications. I will also discuss approaches to integrate
known phosphorylation events, and empirically defined sequence motifs
surrounding phosphorylation sites, into the identification of
phosphorylated substrates in a particular sample.
This work was supported by grants from the NHGRI (NIH) and the DOE to
T.G.G. and D.E.
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