The <i>Drosophila</i> ankyrin repeat protein cactus has a predominantly α‐helical secondary structure

Ntwasa, Monde

doi:10.1016/0014-5793(93)80720-f

Cited by 15 publications

(24 citation statements)

References 23 publications

(24 reference statements)

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“…Since ankyrin repeat proteins are diverse in their function, with their target proteins being quite different, the conservation in sequence most likely reflects important structural elements (Gay & Ntwasa, 1993). It has been suggested that the less conserved positions in the ankyrin repeat sequence are responsible for specificity in protein-protein interactions (Gay & Ntwasa, 1993).…”

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that the less conserved positions in the ankyrin repeat sequence are responsible for specificity in protein-protein interactions (Gay & Ntwasa, 1993).…”

Section: Discussionmentioning

confidence: 99%

“…The first of these, the 85,000 Da membraneinteracting domain of ankyrin, part of a multigene family, contains 24 ankyrin repeats and is approximately 16% helical by CD (Michaely & Bennett, 1993). The second protein, a 53,000 Da Drosophila protein called cactus (Gay & Ntwasa, 1993), contains six ankyrin-like repeats and was reported to be 77% helical by CD. Closer examination of the CD spectrum for cactus suggests that the H u m a n c o n 0 e n 0 u 0 :…”

Section: Discussionmentioning

confidence: 99%

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Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Boice¹,

Fairman²

1996

Protein Science

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The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 pM. The CD spectrum indicates that the protein is composed of 33% a-helix, 22% P-sheet, 19% p-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a AG of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogendeuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.Keywords: analytical ultracentrifugation; ankyrin-like repeat; cyclin; circular dichroism; cyclin dependent kinase inhibitor Control of the eukaryotic cell cycle is a highly regulated process that involves the interaction of a large number of macromolecules. Critical checkpoints in this process are found during the entry of cells into both the GUS and G2/M phases of the cell cycle (for reviews, see Draetta, 1994;Sherr, 1995). The cyclin-dependent kinases (CDKs) are enzymes whose activities govern these checkpoints. CDK holoenzymes, which consist of a CDK catalytic subunit and a cyclin regulatory subunit, are regulated, in part, by their temporal expression, degradation, phosphorylation, and dephosphorylation. This regulation throughout the cell cycle serves to drive the cell through the successive phases.Recent studies have identified CDK inhibitors, a new class of proteins, adding yet another level of control to CDK regulation (for review, see Elledge & Harper, 1994). These CDK inhibitors are thought to function by blocking and/or disrupting CDK-cyclin association. p16, one of the first of these inhibitors to be discovered, was originally cloned from a yeast-two hybrid interaction screen using CDK4 as the bait protein (Serrano et al., 1993).

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Section: Discussionmentioning

confidence: 99%

“…It has been suggested that the less conserved positions in the ankyrin repeat sequence are responsible for specificity in protein-protein interactions (Gay & Ntwasa, 1993).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Boice¹,

Fairman²

1996

Protein Science

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“…Phosphorylation of cactus by CKII is unaffected by the presence in the reaction mixture of dorsal. The dorsal and cactus proteins bind together under these conditions [12] and thus both free and bound forms of cactus appear to be substrates for CKII.…”

Section: The Cactus Protein Is a Substrate For Ckii In Vitromentioning

confidence: 99%

“…Cactus was purified as previously described [12] except that the gel filtration step was omitted and ion exchange chromatography was carried out in 6 M urea. The preparation did not have significant amounts of purine nucleotide binding or hydrolysis activities.…”

Section: Expression and Purification Of Maternallzygotic Cactus Proteinmentioning

confidence: 99%

Casein kinase II phosphorylates Ser⁴⁶⁸ in the PEST domain of the Drosophila IκB homologue cactus

1997

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Cactus protein is a Drosophila homologue of the mammalian ΙκΒ family of cytoplasmic anchor proteins. In unstimulated cells they function to retain rel/NFx:B transcription factors in the cytoplasm but are rapidly degraded in response to signalling. The destruction of cactus or ΙκΒα allows the rel/ NFKB transcription factor to relocalise to the nucleus. Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. In this paper we show that, like mammalian ΙκΒα, the PEST domain of cactus is phosphorylated by casein kinase Π. We have localised the site of modification to a single residue, Ser 468 , and find no evidence for additional phosphorylation sites. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase Π may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.

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Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin

et al. 1997

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Abstract:Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 118 amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two-and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly "N-labeled or uniformly '5N/'3C-labeled protein to obtain the 'H, "N, and I3C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOES, NH exchange data, 3JHNa coupling constants, and chemical shifts of 'Ha, "C,, and I3Cp. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.

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The Drosophila ankyrin repeat protein cactus has a predominantly α‐helical secondary structure

Cited by 15 publications

References 23 publications

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Casein kinase II phosphorylates Ser⁴⁶⁸ in the PEST domain of the Drosophila IκB homologue cactus

Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin

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The Drosophila ankyrin repeat protein cactus has a predominantly α‐helical secondary structure

Cited by 15 publications

References 23 publications

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IκB homologue cactus

Nuclear magnetic resonance assignment and secondary structure of an ankyrin‐like repeat‐bearing protein: Myotrophin

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Casein kinase II phosphorylates Ser⁴⁶⁸ in the PEST domain of the Drosophila IκB homologue cactus