Infection of human cancer cells with myxoma virus requires Akt activation via interaction with a viral ankyrin-repeat host range factor.
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We demonstrate that the susceptibility of human cancer cells to be infected and killed by an oncolytic poxvirus, myxoma virus (MV), is related to the basal level of endogenous phosphorylated Akt. We further demonstrate that nonpermissive tumor cells will switch from resistant to susceptible for MV infection after expression of ectopically active Akt (Myr-Akt) and that permissive cancer cells can be rendered nonpermissive by blocking Akt activation with a dominant-negative inhibitor of Akt. Finally, the activation of Akt by MV involves the formation of a complex between the viral host range ankyrin-repeat protein, M-T5, and Akt. We conclude that the Akt pathway is a key restriction determinant for permissiveness of human cancer cells by MV. (+info)
Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries.
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We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells. (+info)
Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites.
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The homozygous inv (inversion of embryonic turning) mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs (Xinv-1 and Xinv-2) of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites (IQ motifs) are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs of Xenopus inv and mouse inv proteins may regulate their function in different ways. (+info)
The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner.
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The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation. (+info)
Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2.
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Vaccinia virus (VV) K1L is a host-range gene and encodes a protein comprised of six ankyrin repeats (ANKs). We showed here that a large portion of the K1L protein, except ankyrin repeat 1 (ANK1) and C-terminal halves of ANK2 and ANK3, can be deleted or substituted with an unrelated ANK with no adverse effect on VV replication in human HeLa cells. In contrast, only ANK4 and ANK6 can be mutated without impairing VV replication in rabbit RK13 cells. The growth rate of VV in HeLa cells was reduced differentially by substituting phenylalanine 82 or serine 83 of ANK2 and abolished completely by substituting both residues. These substitutions, however, did not affect K1L's ability to bind ACAP2, a GTPase-activating protein for ARF6. Our data support the hypothesis that surface residues of a few consecutive K1L ANKs mediate the host-range function by interacting with protein factors that are distinct from ACAP2. (+info)
Crystal structure of the human TRPV2 channel ankyrin repeat domain.
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TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs. (+info)
The notch ankyrin domain folds via a discrete, centralized pathway.
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The Notch ankyrin repeat domain contains seven ankyrin sequence repeats, six of which adopt very similar structures. To determine if folding proceeds along parallel pathways and the order in which repeats become structured during folding, we examined the effect of analogous destabilizing Ala-->Gly substitutions in each repeat on folding kinetics. We find that folding proceeds to an on-pathway kinetic intermediate through a transition state ensemble containing structure in repeats three through five. Repeats two, six, and seven remain largely unstructured in this intermediate, becoming structured in a second kinetic step that leads to the native state. These data suggest that the Notch ankyrin domain folds according to a discrete kinetic pathway despite structural redundancy in the native state and highlight the importance of sequence-specific interactions in controlling pathway selection. This centralized pathway roughly corresponds to a low energy channel through the experimentally determined energy landscape. (+info)
ANKHD1, ankyrin repeat and KH domain containing 1, is overexpressed in acute leukemias and is associated with SHP2 in K562 cells.
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In the present study, increased levels of ANKHD1 mRNA and protein expression in leukemia cell lines are reported, as compared with normal hematopoietic cells. Furthermore, a higher expression of ANKHD1 mRNA was detected in primary acute leukemia samples than in normal hematopoietic cells (P=0.002). ANKHD1 was detected in the cytosolic and membrane fraction of cells and was co-immunoprecipitated with SHP2 in protein extracts of K562 and LNCaP cell lines. These findings suggest a role for ANKHD1 as a scaffolding protein that may be associated with the abnormal phenotype of leukemia cells. (+info)