Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca 2؉ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160 ROCK ) can induce Ca 2؉-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 ؎ 12% (n ‫؍‬ 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca 2؉ -independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

Genetic defects in glycosyltransferases are responsible for a number of developmental defects and diseases known as congenital disorders of glycosylation (CDGs). Peters'-plus syndrome, a rare autosomal recessive disorder, is now known to be a CDG. This syndrome is characterized by a specific malformation of the eye that includes corneal opaqueness and iridocorneal adhesions (Peters' anomaly). Affected individuals are short in stature and have short limbs, and may have cleft lip/palate, defects in the central nervous system, heart, and various other organs. The phenotype varies in severity, ranging from death in early childhood to a general delay in growth and development, and is often associated with mental retardation. The mutations responsible for Peters'-plus syndrome inactivate a beta1,3-glucosyltransferase whose function is to add a glucose moiety to O-linked fucose, forming a rare glucose-beta1,3-fucose disaccharide. This disaccharide modification is specific to thrombospondin type 1 repeats (TSRs), domains found in extracellular proteins that function in cell-cell and cell-matrix interactions and signalling. Some ninety human proteins contain TSRs, but thus far the disaccharide has been demonstrated on only thrombospondin 1, properdin, F-spondin, ADAMTS-13, and ADAMTSL-1. These proteins perform essential functions in embryonic development, tissue remodelling, angiogenesis, neurogenesis, and complement activation. Identification of the beta1,3-glucosyltransferase and its substrate proteins is a key step towards understanding their roles in human development, and to uncovering the molecular and cellular mechanisms underlying the clinical manifestations of Peters'-plus syndrome.

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Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B′ (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 269, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a K d of 0.13 × 10 -6 M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22-and 31-fold (K d ) 2.9 × 10 -6 and 4.0 × 10 -6 M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol -1 , respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a K d of 6.1 × 10 -6 M and destabilizes the complex by 2.17 kcal mol -1 . On the other hand, mutation of Trp 722 at site B′ to Ala causes a much smaller decrease in affinity (K d ) 0.6 × 10 -6 M) and results in a destabilization energy of 0.87 kcal mol -1 . To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A+B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (K d ) 1.7 × 10 -6 M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (K d ) 4.1 × 10 -6 M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmoncalmodulin interaction and that Trp 722 in site B′ plays a minor role.