Src Homology 2 Domain Binding as a Phosphoproteomic Approach to Signaling in Acute Lymphoblastic Leukemia.

A general theme of orchestrated signal transduction is played by activated receptor phosphotyrosine kinases (PTK) and receptor PTK targets which propagate signals via recognition of sequence-specific phoshorylated tyrosines by so-called Src homology 2 (SH2) domains. SH2 domain interactions are used as a means of recruiting target proteins to activated PTKs and to translocate them to the plasma membrane where many effector proteins activated by receptor PTKs such as phospholipase C-γ or PI-3 kinase have their substrates. SH2 domains make up the most prevalent type of phosphotyrosine binding domains involved in signaling downstream of activated PTKs. SH2 domains are not only present in proteins with intrinsic enzymatic activity but also in adaptor proteins which shuttle effector enzymes to target signaling complexes. Increasing numbers of diseases are known to involve phosphotyrosine specific kinases and/or phosphatases going awry exemplified by the notorious ErbB2 receptor PTK in breast cancer or the Bcr-Abl PTK in CML. Currently, the tyrosine phosphorylation state in most acute lymphoblastic leukemias is undefined which is predicted to differ among the various subgroups and to be distinct from the signaling state of normal hematopoietic cells. To identify aberrant tyrosine kinase or phosphatase activity in the various types of acute lymphoblastic leukemia is of great interest since enzymes in general make good targets for drugs. A novel SH2 domain binding approach is presented which can detect distinctive profiles of tyrosine-phosphorylated proteins in complex mixtures of cellular proteins. A battery of SH2 domains is employed as probes in a competitive far-Western blot based assay to identify specific tyrosine-phosphorylated sites which reflect active signaling pathways in a cell. A further refinement of this technology is under way with DNA-tagged probes being developed which allow for multiplexing and high throughput quantitative assessment of SH2-domain binding by quantitative PCR or microarray technologies.