A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function

Aurora kinase-A and -B are key regulators of the cell cycle and tumorigenesis. It has remained a mystery why these 2 Aurora kinases, although highly similar in protein sequence and structure, are distinct in subcellular localization and function. Here, we report the striking finding that a single amino acid residue is responsible for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa cells, Aurora-AG198N was recruited to the inner centromere in metaphase and the midzone in anaphase, reminiscent of the Aurora-B localization. Moreover, Aurora-AG198N compensated for the loss of Aurora-B in chromosome misalignment and cell premature exit from mitosis. Furthermore, Aurora-AG198N formed a complex with the Aurora-B partners, INCENP and Survivin, and its localization depended on this interaction. Aurora-AG198N phosphorylated the Aurora-B substrates INCENP and Survivin in vitro. Therefore, we propose that the presence of Gly or Asn at a single site assigns Aurora-A and -B to their respective partners and thus to their distinctive subcellular localizations and functions.

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Faculty Opinions recommendation of The Aurora A and Aurora B protein kinases: a single amino acid difference controls intrinsic activity and activation by TPX2.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1028650.342243 ◽

2005 ◽

Author(s):

William Earnshaw

Keyword(s):

Amino Acid ◽

Protein Kinases ◽

Single Amino Acid ◽

Intrinsic Activity ◽

Aurora B ◽

Amino Acid Difference ◽

Aurora A

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Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

Journal of General Virology ◽

10.1099/vir.0.009449-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1741-1747 ◽

Cited By ~ 11

Author(s):

Tahir H. Malik ◽

Candie Wolbert ◽

Laura Nerret ◽

Christian Sauder ◽

Steven Rubin

Keyword(s):

Amino Acid ◽

Clinical Isolate ◽

Amino Acid Change ◽

Mumps Virus ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Supporting Evidence ◽

L Protein ◽

Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

Journal of Virology ◽

10.1128/jvi.00510-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 3

Author(s):

Sujit K. Mohanty ◽

Bryan Donnelly ◽

Phylicia Dupree ◽

Inna Lobeck ◽

Sarah Mowery ◽

...

Keyword(s):

Amino Acid ◽

Biliary Atresia ◽

Amino Acid Change ◽

Reverse Genetics ◽

Single Amino Acid ◽

Wild Type ◽

Neonatal Mice ◽

Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

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A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1751 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1751-1759 ◽

Cited By ~ 16

Author(s):

O R Choi ◽

C Trainor ◽

T Graf ◽

H Beug ◽

J D Engel

Keyword(s):

Amino Acid ◽

Precursor Cells ◽

Single Amino Acid ◽

Predicted Amino Acid Sequence ◽

Temperature Sensitive ◽

Wild Type ◽

Erythroid Precursor ◽

Single Amino Acid Change ◽

Avian Erythroblastosis Virus

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

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A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1751-1759.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1751-1759

Author(s):

O R Choi ◽

C Trainor ◽

T Graf ◽

H Beug ◽

J D Engel

Keyword(s):

Amino Acid ◽

Precursor Cells ◽

Single Amino Acid ◽

Predicted Amino Acid Sequence ◽

Temperature Sensitive ◽

Wild Type ◽

Erythroid Precursor ◽

Single Amino Acid Change ◽

Avian Erythroblastosis Virus

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A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

Journal of Virology ◽

10.1128/jvi.79.12.7327-7337.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7327-7337 ◽

Cited By ~ 60

Author(s):

Valery Z. Grdzelishvili ◽

Sherin Smallwood ◽

Dallas Tower ◽

Richard L. Hall ◽

D. Margaret Hunt ◽

...

Keyword(s):

Amino Acid ◽

Vesicular Stomatitis Virus ◽

In Vitro Transcription ◽

Single Amino Acid ◽

Wild Type ◽

L Protein ◽

Critical Residue ◽

Single Amino Acid Change ◽

Vesicular Stomatitis

ABSTRACT The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5′-cap structures methylated at the guanine-N7 and 2′-O-adenosine positions (7mGpppAm). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5′-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein.

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Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

Journal of Bacteriology ◽

10.1128/jb.184.2.390-399.2002 ◽

2002 ◽

Vol 184 (2) ◽

pp. 390-399 ◽

Cited By ~ 43

Author(s):

James C. Comolli ◽

Audrey J. Carl ◽

Christine Hall ◽

Timothy Donohue

Keyword(s):

Amino Acid ◽

Rhodobacter Sphaeroides ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Response Regulator ◽

Single Amino Acid ◽

Single Amino Acid Change ◽

Wide Range

ABSTRACT Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

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