Notch transmembrane receptors direct essential cellular processes such as proliferation and differentiation through direct cell-to-cell interactions. the complex is assembled. Chlorogenic acid In this study we demonstrate that NICD multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly we demonstrate that the assembly is mediated by NICD multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA. INTRODUCTION Numerous intricate cellular processes are implemented through direct cell-to-cell interactions. Depending on the cell type the Notch signal transduction pathway initiates a variety of cellular processes including proliferation differentiation and apoptosis through these cell-to-cell interactions (2 9 23 24 28 32 For proper development and cellular homeostasis tight regulation of Notch signaling is essential. Inappropriate Notch signaling is observed in neoplasms of many tissue types indicating that deregulation of Notch signaling is Chlorogenic acid involved in the initiation and/or maintenance of the neoplastic phenotype (3-6 11 14 31 Therefore understanding the mechanisms governing the tight regulation of Notch signaling is essential. In mammals there are four known Notch genes (Notch1 to -4 genes) that encode single transmembrane-spanning cell surface receptors (2). Current models of the Notch signal transduction pathway suggest that the extracellular domain of Notch interacts with the extracellular domains of ligands found on adjacent cells. Ligands from the DSL (Delta Serrate and Lag-2) family of proteins interact with Notch receptor and these interactions dictate a series of proteolytic events that release the intracellular domain of Notch (NICD) from the plasma membrane. NICD translocates into the nucleus where it interacts with the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) and transcriptional coactivators of the Mastermind-like family to regulate transcription (20 30 37 38 Other proteins have been postulated to be associated with this complex one of which is the Ski-interacting protein (Skip). Skip was initially identified as a bifunctional nuclear receptor of vitamin D and as a repressor of Notch signaling in association with the protein SMRT (8). Subsequently Skip was shown to interact with NICD and also Chlorogenic acid function as a coactivator for Notch transcriptional activation although no mechanistic detail is known (13 39 In naturally occurring tumors and in model systems cells transformed by Notch contain two distinct high-molecular-weight NICD complexes (16). One of these complexes is CCNG2 localized predominantly in the nucleus were NICD associates with Mastermind-like 1 (Maml1) and CSL to form the activation complex (termed the A complex). The second of these NICD complexes Chlorogenic acid is localized predominantly in the cytoplasm and does not contain either Maml1 or CSL (termed the P complex). The relationship between these complexes is unclear; however one intriguing possibility is that the formation of the activation complex is derived from and perhaps depends on the smaller P complex. Some of the molecular details Chlorogenic acid of this trimeric complex were revealed in the crystal structure of CSL and truncated polypeptides of NICD and Maml1 (26 36 Although these structures reveal important interactions between NICD Maml1 and CSL they do little to reveal the molecular events that lead to the assembly of this transcriptional activation complex. Here we report that NICD forms multimers and that this multimerization is the initial step in transcriptional activation complex assembly. Subsequently the NICD multimer forms a complex with Skip which then provides a docking site to recruit Maml1 and form a preactivation complex. The interaction between the preactivation complex and CSL results in the loading of NICD and Maml1 onto CSL to form the transcriptional activation complex on DNA. These data reveal the molecular events of a stepwise assembly that lead to the formation of the Notch transcriptional activation complex. MATERIALS AND METHODS Cell culture. H1299 and 293T cells were propagated in Dulbecco’s modified Eagle medium (Life Technologies) supplemented with 10% fetal bovine serum 2 mM l-glutamine 100 U of penicillin per ml and 100 μg of streptomycin per ml (Life Technologies) under standard conditions. IPLB-Sf21 (Sf21) cells were maintained in Sf-900 II SFM medium (GibcoBRL) supplemented with 100 U penicillin per ml and 100 μg streptomycin per ml (Life Technologies). Generation of.