The product of the retinoblastoma (Rb) susceptibility gene ( RB-1 ) regulates expression of a variety of growth control genes via discrete promoter elements termed retinoblastoma control elements (RCEs). We have previously shown that RCEs are bound and regulated by a common set of ubiquitously expressed nuclear proteins of 115, 95 and 80 kDa, termed retinoblastoma control proteins (RCPs). We have also previously determined that Sp3 and Sp1, two members of the Sp family of transcription factors, encode the 115 and 95 kDa RCPs respectively and that Rb stimulates Sp1/Sp3-mediated transcription in vivo. In this report we have extended these results by determining that the 80 kDa RCP arises from Sp3 mRNA via translational initiation at two internal sites located within the Sp3 trans -activation domain. Internally initiated Sp3 proteins readily bind to Sp1 binding sites in vitro yet have little or no capacity to stimulate transcription of Sp-regulated genes in vivo. Instead, these Sp3-derived proteins function as potent inhibitors of Sp1/Sp3- mediated transcription. Since cell cycle- or signal- induced expression of a variety of genes, including p21 waf1/cip1, p15 INK4B, CYP11A, mdr1 and acetyl-CoA carboxylase, have been mapped to GC-rich promoter elements that bind Sp family members, we speculate that alterations of the protein and/or DNA binding activities of internally initiated Sp3 isoforms may account in part for the regulation of such differentially expressed genes.

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK acti-

We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAF II s) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAF II 70 and TAF II 40, were identified. We conclude that Sp3-mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors.Sp1 is the founding member of a family of five transcription factors, Sp1-5, that govern the expression of a wide variety of mammalian genes (for review, see Ref. 1). Sp1 encodes a ubiquitously expressed nuclear phosphoprotein that has been divided into five sub-domains based upon their respective functions (2, 3). The Sp1 trans-activation domain is composed of three sub-domains termed A-C, each of which is capable of stimulating transcription if tethered to DNA via a DNA-binding domain. Sub-domains A and B are composed by serine-and threonine-rich regions as well as glutamine-rich regions. The glutamine-rich portions of A and B are believed to be required for trans-activation, whereas the function(s) of the serine/threonine-rich sub-regions is(are) less well understood. Domain C carries a number of charged amino acids and weakly stimulates transcription in the absence of domains A or B. Carboxylterminal to the domain C is a region featuring three Cys 2 -His 2 zinc "fingers" required for sequence-specific DNA binding to GC-rich promoter elements. A carboxyl-terminal domain, termed D, facilitates protein multimerization and is essential for synergistic trans-activation of promoters with multiple Spbinding sites. Sp1 associates with a large number of transcription-associated proteins, including components of the basal transcription complex (e.g. hTAF II 130/dTAF II 110 and hTAF II 55; Refs. 4 -7), sequence-specific DNA-binding proteins (e.g. E2F, YY1, p53, and AP-2; Refs. 8 -13), and transcriptional regulators (e.g. p107, HDAC-1, and VHL-1; Refs. 14 -16). As might be expected given the variety of proteins with which it interacts, protein-binding sites have been identified th...