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.Phosphoinositides are essential components of eukaryotic membranes and are key regulators of membrane trafficking and actin cytoskeleton (1-3). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 1 serves as a precursor to second messengers and as a signaling molecule itself by regulating protein activities and through interactions with protein modules (3-5). Additionally, roles for PI(3)P, PI(3,5)P 2 and PI(3,4,5)P 3 in membrane movement have been defined (1, 6 -10). Homeostasis of phosphoinositides occurs both spatially and temporally via a plethora of lipase, kinase, and phosphatase activities, thereby providing several unique points of regulation (for reviews see .A role for inositol lipid phosphatases in membrane trafficking has come from the characterization and cloning of synaptojanin, a mammalian neuronal inositol polyphosphate 5-phosphatase (5-ptase) involved in synaptic vesicle recycling (18). Additionally, studies of three yeast INP5 gene products (also known as SJLs) demonstrate that although they are collectively essential, the individual proteins also have nonredundant roles in regulating membrane trafficking, cell wall synthesis, osmo-sensitivity, and actin cytoskeleton structure (19 -22). Synaptojanin and the Inp5ps are members of a large 5-ptase gene family, conserved from ...

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 HDAC4⅐14-3-3 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 HDAC4⅐14-3-3 complex.Accumulating evidence indicates that active transcriptional repression is an important component of many physiological events regulated at the level of gene expression, including muscle differentiation (1). The repression of transcription is manifest at the level of chromatin structure where histone deacetylases (HDACs) 1 are recruited to deacetylate histones and create a repressive chromatin structure (reviewed in Ref.2). Of the ten human HDACs identified so far (3), 2 HDAC4 and its closely related family member HDAC5 have been specifically implicated in regulating muscle differentiation ((1) and see below).The functional link between HDAC4/5 and muscle differentiation was first uncovered by the cloning of MITR, a transcriptional repressor identified as an interactive partner for myocyte enhancer factor 2 (MEF-2) transcription factor family members, which are important for muscle differentiation (4). MITR shows extensive homology to the non-catalytic N terminus of HDAC4 and -5 (4). Indeed both HDAC4 and HDAC5 interact with MEF-2. It was reported that overexpression of HDAC4 or HDAC5 represses MEF-2 transcriptional activity (5) and suppresses C2C12 myoblast differentiation (1). It was also found that the HDAC4/5⅐MEF-2 interaction and the effect of this complex on muscle differentiation could be reversed by a constitutively active form of a calcium/calmodulin-dependent protein kinase (CaMK) (6). However, the mechanism by which CaMK regulates HDAC4 and HDAC5 is not entirely clear.When ectopically expressed, HDAC4 can be found in either the nucleus or cytoplasm whereas the closely relat...

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. Drug discovery has traditionally involved a search for inhibitors of protein complexation to small molecules (e.g., substrates or ligands) or macromolecules (e.g., other proteins, nucleic acids, or polysaccharides). While agonists or antagonists of macromolecular receptors that become useful drugs must display a wide range of other attributes, including the appropriate physicochemical properties, pharmacokinetic and pharmacodynamic properties, stability, etc., they must first be protein ligands. The vast majority of available drugs act by binding noncovalently to a protein, preventing or (less often) stimulating that protein's complexation to a complement (1).Complexes of nucleic acid and proteins are key intermediates in all transcriptional and translational processes. Some nucleic acids (e.g., ribozymes) even form functional complexes with small molecules (2). Nonetheless, nucleic acids are widely viewed as ineffective targets for the discovery of low-molecular weight inhibitors. One reason is that the linear motif in single-stranded DNA and the repetitive motif in double-stranded DNA provide attractive targets for large, linear binding molecules (3), but unattractive targets for the small molecules that lead to orally available medications. Another reason is that the lack of tertiary structure in DNA does not afford the diverse topology associated with folded proteins.However, single-stranded RNA often folds into welldefined tertiary structures (4). Furthermore, such structures serve as docking sites for transcriptional activators (5) and substrates for self-splicing reactions (6, 7). Can such structured nucleic acids display sufficient shape diversity to permit the complexation of a small molecule at one site in a virtual sea of nucleic acid material? This is the essential question whose presumed negative answer hinders drug discovery at the nucleic acid level.Certain cis-acting RNA elements are essential for the gene expression of human immunodeficiency virus type 1 (HIV-1) (8). The functions and sequences of these RNA molecules have been well characterized (9-11). A segment of HIV-1 mRNA (residues 1-59), identified as the transactivation responsive element (TAR), adopts a stem-loop secondary structure consisting of a highly cons...