ABSTRACT
The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Animals , Base Sequence , Cell Line , Cloning, Molecular , Culture Media, Conditioned , DNA Primers , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase Kinases , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein-Tyrosine Kinases/metabolism , Rats , Recombinant Proteins/metabolism , Substrate Specificity , Transfection , p21-Activated KinasesABSTRACT
To assess the influence of the 3' long terminal repeat (LTR) on the promoter/enhancer activity of the 5' LTR, a set of isogenic retroviral vectors differing only in the U3 region of the 3' LTR was constructed. These U3 elements were derived from viruses with different tissue tropism. The 5' LTR originated from Moloney murine leukemia virus and directed the transcription of a reporter gene (chloramphenicol acetyltransferase [CAT] gene), giving rise to plasmids of the general configuration LTR-CAT-LTR'. Following transfection of these chimeric constructs into various cell types, the CAT activity in a given cell line was inversely related to the activity of the downstream U3 region when used in a single-LTR construct in that cell type, indicating negative regulation of the 5' LTR by the chimeric 3' LTR'. Our data indicate that a highly active 3' LTR interferes with gene expression from the 5' LTR. Potential mechanisms for this down-regulation are discussed.
Subject(s)
Gene Expression Regulation, Viral , Moloney murine leukemia virus/genetics , Proviruses/genetics , Repetitive Sequences, Nucleic Acid/genetics , Animals , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Down-Regulation , Genetic Vectors/genetics , Humans , Mice , RNA, Antisense/analysis , RNA, Messenger/analysis , Recombinant Fusion Proteins/biosynthesis , Transcription, Genetic , Transduction, Genetic , Virus Integration/geneticsABSTRACT
The observed rate of dissociation of radioactive estradiol from nuclear estrogen-receptor complexes from hen oviduct has been shown to depend to a large extent on the method used to initiate dissociation. The present study indicates that nuclear progesterone receptors display the same pattern of behavior. Dissociation kinetics of nuclear progesterone receptor extracted from hen oviducts were affected by the method of initiation of dissociation; the rate of dissociation at 25 degrees C when dissociation was initiated with unlabeled steroid was three times that observed when dissociation was initiated by addition of a charcoal/dextran suspension. In contrast to nuclear receptors, both estrogen and progesterone receptors prepared from cytosol displayed only a single rate of dissociation, no matter what the method of initiation of dissociation. These results strengthen the idea that nuclear receptors contain a factor or subunit which may be removed by charcoal, which alters the rate of dissociation of steroid from the complex.
