Middle T is the major transforming protein of polyomavirus. It is associated with membranes through a hydrophobic sequence at the C-terminus. Middle T has no known enzymatic function. Rather it serves to associate with a collection of cellular signal transducers. Many of these are associated as a result of phosphorylation MT associates with tyrosine kinase of the src family. MT then associates with PLC gamma, the adaptor SHC and phosphatidylinositol 3-kinase (PI3K) as a result of tyrosine phosphorylation at particular sites.

The binding sites on middle T are known for many of the signaling proteins.

Association of MT with PI3K is critical for transformation in vitro and tumor induction in vivo. PI3 kinase is a downstream target of growth factors and acute cell activators; it is important for cell growth and cell survival. It is a soluble enzyme that trafficks to membranes in response to tyrosine phosphorylation:

Analysis of the interaction of MT with PI3K has led us into structural studies. The association of MT with PI3K is mediated by the SH2 domains of the p85 subunit of PI3K. SH2 domains, modules of about 100 amino acids, function in tyrosine kinase signal transduction. SH2s bind tyrosine phosphorylated sequences based on the ptyr (white at the upper right in the figure) and residues +1 and +3 C-terminal to the ptyr (also white).

NMR Analysis can be used to map interactions of ligands with the SH2. The chemical shifts of amides perturbed by binding:

This makes it possible to compare a series of ligands. For example phosphotyrosine (pY) fills one pocket, pYM, and pYMPM show increasing interaction with the SH2 as mapped by NMR. The method can also be used to compare wild type to a point mutant P395. Such data show a point mutation can affect multiple residues. Since residues affected by the methionine of pYM are also affected by the mutation, the data provide an explanation for why such a mutant alters the specificities of the ligands that can bind to p85.