We previously identified a novel PTPase in human skeletal muscle by PCR and reported the cloning of the full-length cDNA from a HeLa library. This PTPase is called PTP-PEST due to the presence of four regions rich in pro, glu/asp and ser/thr. To determine if the PEST sequences serve any regulatory role, the full length protein (PTP-PEST) and a truncated version that contains the entire catalytic domain but lacks all the PEST sequences (PTP-PESTdelta) have been expressed in Sf9 cells using a recombinant baculovirus. To determine if these PTPs may be involved in insulin signalling, they were coexpressed with a recombinant baculovirus that produces a soluble form of the insulin receptor (SIR), (kindly provided by Melanie Cobb, UTSWMC). Co-expression of PTP-PEST or PTP- PESTdelta with the SIR caused a reduction in the P-tyr content of SIR; this decrease correlated with a decrease (40-60%) in the tyrosine kinase activity of the SIR measured against an articifial substrate, PEY. We reported that the PTPase activities of both PTP-PEST and PTP-PESTdelta were also inhibited 40-60% when coexpressed with the SIR. However, co- expression of the parental baculovirus with PTP-PESTs has since shown variable results, ranging from no effect on enzyme activity, to an inhibition of the same magnitude as seen with the SIR. These results question the specifity of the effect seen with the SIR. We are in the process of expressing PTP-PEST in mammalian cells that can be treated with insulin to address to this problem in a more physiological manner. For this goal, the cDNA for both PTP-PEST and PTP-PESTdelta were modified at the 3 prime end by PCR to encode for a FLAG tag. This FLAG tag is a short amino acid sequence that is recognized by a monoclonal antibody capable of Western blotting and immunoprecipitating the expressed protein. (The peptide antibody that we obtained against PTP-PEST is only capable of immunoprecipitating denatured protein, thus greatly limiting its usefulness.) These modified cDNAs will be placed into a mammalian expression vector for transfection onto cells. Recently, the binding regions for SH3 domains have been determined for a number of proteins and several consensus sequences have emerged that are rich in proline residues. PTP-PEST contains several of these consensus sequences, suggesting that it may interact with SH3 domain containing proteins, for example, grb2. This hypothesis can be tested by immunoprecipitating PTP- PEST from mammalian cells and blotting with antibodies to SH3 domain containing proteins.
