Use of an antisense strategy to dissect the signaling role of protein-tyrosine phosphatase α

The protein-tyrosine phosphatase PTP alpha has been proposed to play an important role in controlling the dephosphorylation of a number of key signaling proteins and in regulating insulin signaling, To examine the potential cellular functions and physiological substrates of PTP alpha a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTP alpha from 3T3-L1 adipocytes. The antisense probe, alpha AS1, achieved PTP alpha depletion levels normally of greater than or equal to 85% and which varied up to levels where PTP alpha was not detected at all. Elimination of PTP alpha by 85% inhibited c-Src activity by 80%. Abolishing PTP alpha to levels undetected did not alter the tyrosine dephosphorylation of the insulin receptor or insulin receptor substrate proteins. Moreover, the ability of insulin to activate ERK2 or to stimulate DNA synthesis was not altered by alpha AS1, It is concluded that endogenous PTP alpha is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTPa! is not required for the dephosphorylation of the insulin receptor or the insulin receptor substrate proteins or for the regulation of several downstream insulin signaling events in 3T3-L1 adipocytes, Finally, the development of the antisense probe, alpha AS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTP alpha.