Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

Proteolytic processing of the amyloid precursor protein by ␤-secretase yields A4CT (C99), which is cleaved further by the as yet unknown ␥-secretase, yielding the ␤-amyloid (A␤) peptide with 40 (A␤ 40 ) or 42 residues (A␤ 42 ). Because the position of ␥-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the A␤ domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of ␥-secretase (A␤ 42 The main proteinaceous component of the amyloid plaques found in the brains of patients with Alzheimer's disease (AD) is ␤-amyloid (A␤; refs. 1 and 2), an Ϸ4-kDa peptide that is derived from the larger amyloid precursor protein (APP; ref.3). APP processing by the as yet unidentified protease activities, termed ␣-, ␤-, and ␥-secretases, leads to a variety of different soluble and membrane-bound proteins (for reviews, see refs. 4 and 5). The ␣-secretase activity cleaves APP within the A␤ domain and thus precludes the generation of A␤. This cleavage yields secretory ␣-APPs, comprising most of the N-terminal ectodomain of APP, and the remaining membrane-bound C-terminal fragment p3CT. Alternatively, APP can be cleaved by the ␤-secretase activity at the N terminus of A␤, generating a truncated, soluble ␤-APPs and a C-terminal fragment of 99 residues (A4CT, C99). The ␤-secretase product A4CT contains the entire A␤ domain, the transmembrane domain, and the cytoplasmic tail of APP and represents the direct precursor for A␤ (6, 7).Both membrane-bound C-terminal fragments of APP, A4CT and p3CT, are cleaved by the ␥-secretase activity within their transmembrane domains at the C terminus of A␤ or p3, thus releasing the 40-and 42-residue A␤ peptides (A␤ 40 and A␤ 42 ) and the 24-26 residue p3 peptides (p3 40 and p3 42 ) (8-11). Most cells secrete both peptides A␤ and p3 into the conditioned medium. In neuronal cells, as in primary hippocampal neurons and in kidney 293 cells, A␤, but not p3, also can be found intracellularly and does not seem to be secreted (12-16).The major A␤ species secreted by cultured cells expressing wild-type (wt) APP is A␤ 40 ; the minor species is A␤ 42 (17). Mutations in the APP close to the ␥-cleavage site have been shown to alter the cleavage specificity of the ␥-secretase activity (A␤ 42 ͞A␤ 40 ratio; refs. 14 and 18-20). However, the factors that determine this cleavage specificity are unknown. Experiments with inhibitors of ␥-secretase activity suggest that distinct proteases generate the A␤ 40 and A␤ 42 peptides (11,(21)(22)(23)), but it is not known whether these enzymes are related or not.Furthermore, although ␥-cleavage occurs in the transmembrane domain of A4CT, it is not clear whether the cleavage occurs while A4CT is inserted into the membrane or after release of A4CT from the membrane. Understanding the substrate specificity of the ␥-secretase activity is of great importance, because the cleavage at residue 42 ...

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPα), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPα degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPα. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPα, which account for their differential ability to induce leukemia.

We present the crystal structure of the catalytic core of human DNA polymerase kappa (hPolkappa), the first structure of a human Y-family polymerase. hPolkappa is implicated in the proficient extension of mispaired primer termini on undamaged DNAs, and in the extension step of lesion bypass. The structure reveals a stubby "fingers" subdomain, which despite its small size appears to be tightly restrained with respect to a putative templating base. The structure also reveals a novel "thumb" subdomain that provides a basis for the importance of the N-terminal extension unique to hPolkappa. And, most surprisingly, the structure reveals the polymerase-associated domain (PAD) juxtaposed on the dorsal side of the "palm" subdomain, as opposed to the fingers subdomain. Together, these properties suggest that the hPolkappa active site is constrained at the site of the templating base and incoming nucleotide, but the polymerase is less constrained following translocation of the lesion.

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for IntroductionTribbles (Trib) pseudokinases have recently emerged as important contributors to dysregulated signaling in malignant hematopoiesis. All 3 mammalian Trib homologs, Trib1, Trib2, and Trib3 (collectively termed "Trib1-3"), are associated with human malignancies. [1][2][3] Trib1 and Trib2 function as oncogenes in acute myelogenous leukemia (AML) and cooperate with homeobox gene 9 (HoxA9) to accelerate the onset of AML in murine AML models. 2,4 In particular, Trib2 is highly expressed in a specific subset of human AMLs that are associated with impaired CCAAT/ enhancer-binding protein-alpha (C/EBP-␣) function. 2 These AMLs express both myeloid and T-cell markers, and some contain activating Notch1 mutations. 5 As Trib2 is a direct transcriptional target of oncogenic Notch1, we hypothesize that the oncogenic Notch1 mutations are activating Trib2 expression in some of these tumors. 5 These biphenotypic AMLs respond poorly to current therapies, suggesting that new therapeutic strategies are needed. 6 Trib proteins interact with various signaling molecules and transcription factors, including activating transcription factor 4 (ATF4), 7 p65, 8 C-terminal interacting protein (CtIP), 9 mitogenactivated protein kinase kinase (MAPKK), 10 protein kinase B (AKT), 11 and constitutive morphogenesis 1 (COP1). 12 We recently demonstrated that Trib2 binds to and facilitates the degradation of C/EBP-␣, and proposed that this process is a key feature of Trib2-induced AML. 2 Trib2 (also termed c5Fw, TRB-2, GS3955, TRB2, and AW319517) has 3 clearly distinguishable regions, including an N-terminal portion, a central serine/threonine kinase-like domain (KD), and a C-terminal region that contains a COP1-binding site. The Trib2 N-terminus is the most divergent region of mammalian Trib proteins and is characterized by a high serine and proline content. The C-terminus contains a DQxVPx motif that is shared by Trib1-3 and binds the E3 ligase, COP1. 12 Trib2 belongs to the pseudokinase family because it contains a central region that shares considerable homology with serine/ threonine kinases, but has deviations in the catalytic loop that are likely to eliminate catalytic activity. 13 Canonical kinases have an N-terminal lobe that binds adenosine triphosphate (ATP), a central hinge region, and a C-terminal lobe that binds substrates. 14 The N-terminal lobe contains an invariant lysine residue critical for ATP binding that is conserved in Trib2, but the highly conserved sequence, HRDLKPEN, in the C-terminal subdomain, which is critical for phosphate transfer in active kinases, is altered to LRD...

␥-Secretase activity is the final cleavage event that releases the amyloid ␤ peptide (A␤) from the ␤-secretase cleaved carboxyl-terminal fragment of the amyloid ␤ protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of ␥-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing A␤ production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. A␤ production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in ␥-secretase cleavage in the presence or absence of pepstatin, an inhibitor of ␥-secretase activity. These studies demonstrate that ␥-secretase's cleavage specificity is primarily determined by location of the ␥-secretase cleavage site of APP with respect to the membrane, and that ␥-secretase activity is due to the action of multiple proteases exhibiting both a pepstatinsensitive activity and a pepstatin-insensitive activity. Given that ␥-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of A␤ production that will direct efforts to isolate the ␥-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant ␥-secretase activities.The 4-kDa amyloid ␤ protein (A␤) 1 that is invariably deposited as amyloid in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid ␤ protein precursor (APP) (1-3). Generation of A␤ from APP requires two proteolytic events, one at the amino terminus referred to as ␤-secretase and one at the carboxyl terminus known as ␥-secretase (Fig. 1). To date, neither of the proteases responsible for these activities has been definitively identified. Comparison of the soluble A␤ secreted by cells, soluble A␤ in cerebrospinal fluid, and insoluble A␤ isolated from the AD brain has revealed that there are numerous A␤ species with extensive amino-and carboxyl-terminal heterogeneity. The major A␤ species in both conditioned cell culture media and human cerebrospinal fluid is A␤1-40 (ϳ50 -70%) although some A␤1-42 (5-20%) is also present along with minor amounts of other peptides (e.g. A␤1-28, A␤1-33, A␤1-34, A␤3-34, A␤1-37, A␤1-38, and A␤1-39) (4 -6). The importance of the longer forms of A␤, in particular A␤42, has been heightened by the fact that all of the familial Alzheimer's disease (FAD) linked mutations that have been analyzed result in an increase in the concentration of A␤42 in a wide variety of model systems (reviewed in Refs. 7 and 8). Biophysical studies have shown that the longer forms of A␤ aggregate at a much faster rate and at lower concentrations than forms ending at A␤40 suggesting that alterations in A␤42 concentration, as occurs in FAD linked forms of the disease, may account for the observation that the longer forms of A␤ are often the initial spe...

Summary COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. We report here structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β-propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β-propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.

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