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1.
Biochem Biophys Res Commun ; 288(1): 205-11, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11594774

ABSTRACT

We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.


Subject(s)
Insulin-Like Growth Factor I/pharmacology , Insulin/pharmacology , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Muscle, Skeletal/enzymology , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases , Actins/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cytoskeleton/metabolism , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Mitogen-Activated Protein Kinase Kinases/physiology , Muscle, Skeletal/drug effects , Organophosphonates/pharmacology , Phosphatidylinositol 3-Kinases/physiology , Phosphorylation , Protein Kinase C/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/metabolism
2.
Arch Biochem Biophys ; 384(2): 311-8, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11368318

ABSTRACT

The cytochromes P450 are a superfamily of enzymes that can carry out a wide range of oxidative reactions. While the transcriptional control of the cytochrome P450 genes has been relatively well-studied, posttranscriptional regulatory mechanisms that contribute to the regulation of P450s are much less well understood. We followed the decay of CYP1A1, CYP1A2, and CYP1B1 mRNAs after induction by the AH receptor ligand 2,3,7,8,-tetrachlorodibenzo-p-dioxin. CYP1A2 and CYP1B1 mRNAs were long-lived in this cell line (to > 24 h). In contrast, the CYP1A1 mRNA decays remarkably quickly. To determine if this rapid decay was unique to CYP1A1, we assessed the decay of selected human P450 and liver-specific mRNAs in HepG2 cells as a comparison. We analyzed albumin, phosphofructokinase, and GAPDH mRNAs and found that they were long-lived, with half-lives >24 h. We show that CYP2E1 mRNA can be detected in HepG2 cells by RT-PCR and that this mRNA also has a basal half-life of >24 h. Thus the CYP1A1 mRNA with its half-life of 2.4 h was one of the shortest-lived mRNA studied and is the most unstable of the cytochrome P450 mRNAs we have tested. The rapid decay of CYP1A1 mRNA is associated with a rapid loss in poly(A) tail length, suggesting that deadenylation is the first step in the decay pathway. The short half-life appears to be conserved across species, which suggests that this characteristic of the CYP1A1 mRNA is important for its function.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A1/genetics , 3' Untranslated Regions , Carcinoma, Hepatocellular , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/genetics , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1B1 , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic , Half-Life , Humans , Liver/enzymology , Polychlorinated Dibenzodioxins/pharmacology , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Tumor Cells, Cultured
3.
Chem Biol Interact ; 109(1-3): 43-52, 1998 Feb 20.
Article in English | MEDLINE | ID: mdl-9566732

ABSTRACT

During studies designed to subclone human phenol sulfotransferase (STP and STM) sequences for use in heterologous E. coli-based expression systems, we designed two oligonucleotide primers that would allow for the simultaneous PCR amplification of expression cassettes containing the coding regions of the STP1, STP2 and STM cDNAs. Following total RNA isolation from human liver, reverse transcription of cDNA, PCR amplification under standard conditions, plasmid subcloning and restriction analysis to select for suitable ST recombinants, we recovered plasmids containing inserts corresponding to STP1, STP2 and STM. However, ten additional, closely related but apparently novel ST sequences were also isolated. Alignments of the three known ST sequences (and one published allelic variant) with these new clones revealed that each one appears to be a PCR-generated modular chimera possessing a combination of DNA segments derived from STP1, STP2 and STM. This observation should serve as an alert to the potential pitfalls of using PCR techniques for the cloning of highly related genes and their cDNA products, especially when PCR primer design allows for the amplification of multiple products in a single reaction.


Subject(s)
Polymerase Chain Reaction/methods , Sulfotransferases/genetics , Cloning, Molecular , DNA/genetics , DNA/metabolism , Humans , Liver/enzymology , Liver/metabolism , RNA/genetics , RNA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sulfotransferases/metabolism , Transcription, Genetic
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