Notch ankyrin repeat domain variation influences leukemogenesis and Myc transactivation

Background

The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival.

Principal Findings

We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL.

Conclusions/Significance

We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.     

Evolutionary analysis of vertebrate Notch genes

We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.     

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.     

Role of STRAP in regulating GSK3β function and Notch3 stabilization

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.     

Role of STRAP in regulating GSK3β function and Notch3 stabilization

Glycogen synthase kinase 3β (GSK3β) can regulate a broad range of cellular processes in a variety of cell types and tissues through its ability to phosphorylate its substrates in a cell- and time-specific manner. Although it is known that Axin and presenilin help to recruit β-catenin/Smad3 and tau protein to GSK3β, respectively, it is not clear how many of the other GSK3β substrates are recruited to it. Here, we have established the binding of GSK3β with a novel scaffold protein, STRAP, through its WD40 domains. In a new finding, we have observed that STRAP, GSK3β and Axin form a ternary complex together. We show for the first time that intracellular fragment of Notch3 (ICN3) binds with GSK3β through the ankyrin repeat domain. This binding between STRAP and GSK3β is reduced by small-molecule inhibitors of GSK3β. Further studies revealed that STRAP also binds ICN3 through the ankyrin repeat region, and this binding is enhanced in a proteasomal inhibition-dependent manner. In vivo ubiquitination studies indicate that STRAP reduces ubiquitination of ICN3, suggesting a role of STRAP in stabilizing ICN3. This is supported by the fact that STRAP and Notch3 are co-upregulated and co-localized in 59% of non-small cell lung cancers, as observed in an immunohistochemical staining of tissue microarrays. These results provide a potential mechanism by which STRAP regulates GSK3β function and Notch3 stabilization and further support the oncogenic functions of STRAP.Key words: STRAP, GSK3β, Notch3, axin, lung cancer, ubiquitination     

Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding.

To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.     

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.