We have previously identified a novel PTPase in human skeletal muscle by PCR and reported the cloning of the full-length cDNA. This PTPase is called PTP-PEST due to the presence of four regions rich in pro, glu/asp and ser/thr. To assess 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. Each of these expressed proteins have been purified and found to have specific activities and affinities for an insulin receptor peptide substrate that do not vary by more than 2-fold. 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 tyrosine kinase (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 (40-600) in the tyrosine kinase activity of the SIR measured against an artificial substrate, PEY. Interestingly, there was also a decrease (40-60%) in the PTPase activities of both PTP-PEST and PTP- PESTdelta when coexpressed with the SIR. To determine if this effect of the SIR was a direct one, extracts of Sf9 cells were blotted with P-tyr antibodies. No prominent bands corresponding to the sizes of the PTPs were detected using this method. In addition, preliminary results of phosphoamino acid analysis of PTP-PESTs from 32Pi labeled Sf9 cells indicate only ser/thr phosphorylation. In support of these in vivo data, the in vitro stoichiometry of phosphorylation of the PTPs by SIR was negligible; 0.08 mol/mol for PTP-PEST and 0.01 mol/mol for PTP-PESTdelta. We have very recently obtained an antiserum against a PTP-PEST peptide that recognizes both forms of the PTPs by Western blot, and early indications are that it will also be very useful for immunoprecipitation. This antibody should allow us to determine protein levels and possibly enzyme activities of PTP-PEST in skeletal muscle from insulin sensitive and resistant Pimas.
