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1.
J Biol Chem ; 276(37): 35042-8, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11470791

ABSTRACT

In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4.14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4.14-3-3 complex.


Subject(s)
Histone Deacetylases/metabolism , Repressor Proteins/metabolism , 14-3-3 Proteins , Animals , Biological Transport , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Differentiation , Cell Nucleus/enzymology , Cytoplasm/metabolism , Histone Deacetylases/chemistry , Humans , Phosphorylation , Repressor Proteins/chemistry , Tyrosine 3-Monooxygenase/metabolism
2.
J Biol Chem ; 274(19): 12990-5, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10224048

ABSTRACT

The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.


Subject(s)
Fungal Proteins/genetics , Nerve Tissue Proteins/genetics , Phosphoric Monoester Hydrolases/genetics , Saccharomyces cerevisiae/genetics , Fungal Proteins/metabolism , Humans , Inositol Polyphosphate 5-Phosphatases , Nerve Tissue Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology
3.
Biochemistry ; 37(40): 14204-12, 1998 Oct 06.
Article in English | MEDLINE | ID: mdl-9760258

ABSTRACT

TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Long Terminal Repeat/drug effects , HIV-1/genetics , RNA, Viral/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Aminoglycosides , Anti-Bacterial Agents/pharmacology , Anti-HIV Agents/metabolism , Binding, Competitive/drug effects , Binding, Competitive/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Design , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/metabolism , HIV-1/drug effects , HIV-1/physiology , Humans , Quinazolines/pharmacology , Quinoxalines/pharmacology , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Transcriptional Activation/drug effects , Virus Replication/drug effects , Virus Replication/genetics , tat Gene Products, Human Immunodeficiency Virus
4.
Bioorg Med Chem ; 5(6): 1185-95, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9222512

ABSTRACT

Self-splicing group I intron RNA was chosen as a potential therapeutic target for small-molecule intervention. High-throughput screening methodologies have been developed to identify small organic molecules that regulate the activities of these catalytic introns. Group introns derived from pathogenic Pneumocystis carinii and phage T4 were used as model systems. Inhibitors identified from a library of approximately equal to 150,000 compounds were shown to regulate biochemical reactions including the two-step intron splicing and an RNA ligation catalyzed by the group I introns. These inhibitors provide a unique opportunity to understand small-molecule recognition of the self-splicing RNA. The methodologies developed for group I introns should be applicable to studies of other RNA systems.


Subject(s)
Enzyme Inhibitors/pharmacology , Introns/drug effects , RNA Splicing/drug effects , RNA, Catalytic/antagonists & inhibitors , RNA, Catalytic/metabolism , RNA/antagonists & inhibitors , RNA/metabolism , Bacteriophage T4/enzymology , Bacteriophage T4/genetics , Base Sequence , Enzyme Inhibitors/metabolism , Molecular Sequence Data , Molecular Weight , Pneumocystis/enzymology , Pneumocystis/genetics , RNA/genetics , RNA Precursors/antagonists & inhibitors , RNA Precursors/metabolism , RNA, Fungal/antagonists & inhibitors , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Viral/antagonists & inhibitors , RNA, Viral/metabolism
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