The secreted morphogen, Sonic hedgehog (Shh) is a significant determinant of brain size and craniofacial morphology1-4. In humans, SHH haploinsufficiency results in holoprosencephaly (HPE)5, a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a combination of genetic and biochemical experiments to uncover a critical pair of cis and trans acting determinants of Shh forebrain expression. A rare nucleotide variant located 460kb upstream of SHH was discovered in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity capture assay we screened SBE2 sequence for DNA binding proteins and identified members of the Six3/Six6 homeodomain family as candidate regulators of Shh transcription. Six3 and Six6 showed reduced binding affinity for the mutant compared to wild type SBE2 sequence. Moreover, HPE causing mutations in Six3 failed to bind and activate SBE2, whereas, Shh forebrain expression was unaltered in Six6 −/− embryos. These data provide a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE. Previous efforts to address this issue focused on determining the genomic location of functional Shh regulatory elements13. These experiments identified six enhancers distributed over a 500 kb interval surrounding the Shh gene that directed reporter activity to most areas of Shh expression in the mouse CNS, including the ventral forebrain ( Fig. 1). In particular, the highly conserved Shh brain enhancer-2 (SBE2), located 460 kb upstream of the SHH coding sequence, was identified as unique in its ability to regulate Shh-like expression throughout the hypothalamus. KeywordsTo identify functionally relevant nucleotides in SBE2, we screened the 1.1 kb sequence mediating its activity for mutations in humans with HPE. We reasoned that HPE causing variants in SBE2 could aid in identifying critical cis and trans determinants of SHH expression in the forebrain. Similar resequencing approaches have been successful in identifying common and rare coding sequence variants in genes associated with common diseases, but have not been routinely applied to the study of remote noncoding regions in rare diseases such as HPE (1:16,000 livebirths)12,14.From 474 HPE patients, we identified one individual who was heterozygous for a C to T base change at nucleotide position 444 of the enhancer sequence. The C/T variant is situated within a block of 10 nucleotides that have been maintained in human, mouse, chicken and frog for over 350 million years ( Fig. 1). This C/T nucleotide variant was not observed in DNA samples from 450 unrelated control individuals. The affected female exhibited features of semilobar HPE including microcephaly, midfacial hypoplasia, cleft-lip and palate, diabetes insipidus, and moderate fus...

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.

Exposure to sources of UV radiation, such as sunlight, induces a number of cellular alterations that are highly dependent on its ability to affect gene expression. Among them, the rapid activation of genes coding for two subfamilies of proto-oncoproteins, Fos and Jun, which constitute the AP-1 transcription factor, plays a key role in the subsequent regulation of expression of genes involved in DNA repair, cell proliferation, cell cycle arrest, death by apoptosis, and tissue and extracellular matrix remodeling proteases. Besides being regulated at the transcriptional level, Jun and Fos transcriptional activities are also regulated by phosphorylation as a result of the activation of intracellular signaling cascades. In this regard, the phosphorylation of c-Jun by UV-induced JNK has been readily documented, whereas a role for Fos proteins in UV-mediated responses and the identification of Fosactivating kinases has remained elusive. Here we identify p38 MAPKs as proteins that can associate with c-Fos and phosphorylate its transactivation domain both in vitro and in vivo. This phosphorylation is transduced into changes in its transcriptional ability as p38-activated c-Fos enhances AP1-driven gene expression. Our findings indicate that as a consequence of the activation of stress pathways induced by UV light, endogenous c-Fos becomes a substrate of p38 MAPKs and, for the first time, provide evidence that support a critical role for p38 MAPKs in mediating stress-induced c-Fos phosphorylation and gene transcription activation. Using a specific pharmacological inhibitor for p38␣ and -␤, we found that most likely these two isoforms mediate UV-induced c-Fos phosphorylation in vivo. Thus, these newly described pathways act concomitantly with the activation of c-Jun by JNK/ MAPKs, thereby contributing to the complexity of AP1-driven gene transcription regulation.Repeated and prolonged exposure to sunlight and hence to UV radiation causes skin damage that may induce alterations in the DNA and ultimately evolve into skin cancer. Extensive investigation of the response of mammalian cells to UV light has shown that exposure to UV light results in the rapid activation of a group of enzymes known as stress-activated protein kinases (SAPKs) 1 (1, 2) and the induction of expression of a set of immediate early genes (ergs) (3-6), which in turn participate in the cellular responses to this type of environmental stress.SAPKs is the common denomination for a subgroup of highly homologous proteins, JNKs and p38s, that belong to a superfamily of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs) (7-10). These kinases play an essential role in the transduction of environmental stimuli to the nucleus, as they are capable of regulating the expression of genes involved in a variety of cellular processes, including cell proliferation, differentiation, programmed cell death, and neoplastic transformation (11-13). MAPKs have been classified into at least six subfamilies, among which the Erk/MAPKs (Erk1 and -2), JNKs (JN...

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore-and receptorguided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKC␣ and PKC␦ mediated the apoptotic effect of PMA, whereas only PKC␣ was involved in the effect of the DAG-lactone. The PKC␣ selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKC␣ and PKC␦. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKC␣ to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKC␦ predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.The phorbol esters and related diterpenes are natural compounds that have been for many years the preferred pharmacological tools for studying protein kinase C (PKC), 1 a key family of kinases implicated in growth factor-and G-proteincoupled receptor signaling. These compounds mimic the action of the lipid second messenger diacylglycerol (DAG), a relatively simple and highly flexible molecule generated by cellular phospholipases. The higher potency of phorbol esters and their greater stability compared with the second messenger DAG makes these agents the preferred activators of PKC (1, 2). Phorbol esters regulate a variety of cellular functions, including cell cycle progression, differentiation, cytoskeleton remodeling, and malignant transformation. Although phorbol esters promote mitogenesis in several cell types, accumulating data indicate that activation of PKC leads to inhibition of cell growth in many cells (3-6). Interestingly, phorbol esters induce apoptosis in several cell lines, including thymocytes, breast cancer cells, and prostate cancer cells (7-12).The heterogeneity of effects of the phorbol esters is related to the presence of multiple phorbol ester/DAG receptors, including PKC isozymes and "nonkinase" PKC receptors. The PKC family comprises at least 10 related kinases with differential expression, ...