ABSTRACT
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.
Subject(s)
Oligonucleotides, Antisense/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Signal Transduction , 3T3 Cells , Animals , Base Sequence , DNA Primers , Insulin/metabolism , Mice , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolismABSTRACT
The signalling pathways by which insulin triggers protein synthesis were studied using an antisense strategy to deplete ERK1/ERK2 and rapamycin to inhibit the p70S6K pathway. The results indicated that ERK1/ERK2 principally regulated the amount of the protein synthesis machinery available in the cell while the p70S6K pathway contributed to modulating its activation in response to insulin. ERK1/ERK2 also mediated in a small proportion of insulin-stimulated protein synthesis which included the induction of c-fos protein. When c-fos induction was blocked the majority of insulin-stimulated protein synthesis still occurred and thus did not require transcriptional regulation of c-fos or its targets.
Subject(s)
Adipocytes/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Mitogen-Activated Protein Kinases , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/drug effects , Animals , Cell Line , Hypoglycemic Agents/metabolism , Insulin/metabolism , Mice , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Protein BiosynthesisSubject(s)
Adipocytes/cytology , Adipocytes/physiology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carrier Proteins , Glucose/metabolism , Insulin/pharmacology , Mitogen-Activated Protein Kinases , Oligonucleotides, Antisense/pharmacology , Signal Transduction/physiology , 3T3 Cells , Adaptor Proteins, Signal Transducing , Adipocytes/drug effects , Animals , Cell Cycle Proteins , Eukaryotic Initiation Factors , Mice , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Phosphoproteins/metabolism , Phosphorylation , Repressor Proteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , ThionucleotidesSubject(s)
Gene Expression Regulation, Viral , Neurons, Afferent/virology , Simplexvirus/physiology , Animals , Base Sequence , Cell Line , Cells, Cultured , Chloramphenicol O-Acetyltransferase/genetics , Humans , Immediate-Early Proteins/genetics , Neurons, Afferent/cytology , Promoter Regions, Genetic , Simplexvirus/genetics , TransfectionABSTRACT
The phosphorylation state of PHAS-I is thought to be important in the regulation of protein synthesis initiation. PHAS-I phosphorylation significantly increases in response to growth factors and insulin. ERK1/ERK2 have previously been implicated as PHAS-I kinases. Present work utilised a specific phosphorothioate oligonucleotide antisense strategy against ERK1/ERK2 to determine whether ERK1/ERK2 mediate FBS-stimulated PHAS-I phosphorylation in vivo. Depleting > 90% of cellular ERK1/ERK2 had no effect on FBS-stimulated PHAS-I phosphorylation. However, treatment of cells with a specific p70S6k pathway inhibitor, rapamycin, markedly attenuated FBS-stimulated PHAS-I phosphorylation. These results indicate that PHAS-I phosphorylation in response to FBS occurs through an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes.