Full-length protein tyrosine phosphatase 1C (PTP1C), the catalytic domain of PTP1C (delta PTP1C), and the N-terminal SH2 domain truncated PTP1C (delta NPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity. Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs. There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP. Kinetic studies were carried out using all seven different phosphotyrosyl IRPs. Saturation kinetics were observed for PTP1C using the triphosphotyrosyl IRP only. In contrast, for delta PTP1C, saturation was achieved for all seven phosphotyrosyl IRPs. The best substrate for delta PTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 microM, about 3-4-fold lower than either the mono- or diphosphotyrosyl IRPs. However, in contrast to delta PTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP. Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with delta PTP1C. The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes. These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity.

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This study is the first to examine the effects of polyunsaturated fatty acids and some of their metabolites on [3H]thymidine incorporation into vascular smooth muscle explants from the coronary artery of rainbow trout (Oncorhynchus mykiss). At a concentration of 120 pM, eicosapentaenoic acid (EPA; 20:5w3), arachidonic acid (AA; 20:4w6), and eicosatrienoic acid (ETA; 20:3w6) all approximately doubled [3H]thymidine incorporation relative to controls. At a conceqtration of 20 pM, EPA had no significant effect, while ETA inhibited and AA caused an almost 5-fold increase in ["]thymidine incorporation. The large mitogenic effect of 20 pM AA was completely inhibited by simultaneous addition of EPA to the culture medium. ETA only partially inhibited the mitogenic effect of 20 pM AA. Four AA-derived eicosanoids (or their stable analogues) were also tested. [3H]Thymidine incorporation was at least doubled with 1000 ng/mL carbacyclin (a prostacyclin analogue), 120 ng/mL prostaglandin F,,, and U-46619 (a thromboxane A, analogue), but did not reach the level of stimulation produced by 20 pM AA. Leukotriene C, had no significant effect. We conclude that dietary modulation of polyunsaturated fatty acids (PUFAs) in salmonids could have significant effects on coronary vascular smooth muscle mitosis through the incorporation of PUFAs into cell membranes and the production of eicosanoids.