Use of an antisense strategy to dissect the signaling role of protein-tyrosine phosphatase alpha.
The protein-tyrosine phosphatase PTPalpha 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 PTPalpha, a potent phosphorothioate oligonucleotide-based antisense strategy was developed that specifically depleted endogenous PTPalpha from 3T3-L1 adipocytes. The antisense probe, alphaAS1, achieved PTPalpha depletion levels normally of >/=85% and which varied up to levels where PTPalpha was not detected at all. Elimination of PTPalpha by 85% inhibited c-Src activity by 80%. Abolishing PTPalpha 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 alphaAS1. It is concluded that endogenous PTPalpha is a key regulator of c-Src activity in 3T3-L1 adipocytes and that PTPalpha 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, alphaAS1, provides an important molecular tool of general applicability for further dissecting the roles and precise targets of endogenous PTPalpha.
Arnott
C H
CH
Division of Biochemistry and Molecular Biology, School of Biological Sciences, Southampton, SO16 7PX, United Kingdom.
Sale
E M
EM
Miller
J
J
Sale
G J
GJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007328:Insulin / Q000378:metabolism; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D015398:Signal Transduction
D017931:DNA Primers; D007328:Insulin; D016376:Oligonucleotides, Antisense; D016392:Proto-Oncogene Proteins pp60(c-src); D017027:Protein Tyrosine Phosphatases