A conserved helix‐unfolding motif in the naturally unfolded proteins

Zetina, Carlos R.

doi:10.1002/prot.1113

Cited by 24 publications

(26 citation statements)

References 34 publications

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“…On the other hand, CD studies indicate that phosphorylation of p27 by CK2 has direct structural and functional effects. These results may be interpreted by postulating that p27 in solution behaves like a naturally unfolded protein type II, according to the classification proposed by Zetina [Zetina, 2001]. This author has also suggested the existence of a helix motif that promotes protein unfolding.…”

Section: Kip2mentioning

confidence: 72%

Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2

Tapia

Bolaños-García

Sayed

et al. 2004

J of Cellular Biochemistry

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The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity.

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Section: Kip2mentioning

confidence: 72%

Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2

Tapia

Bolaños-García

Sayed

et al. 2004

J of Cellular Biochemistry

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“…Calcium-dependent VLS formation is affected by changes in DxDxD motifs and the C-terminal ␣-helix. Two potential calcium-coordinating domains upstream of the C-terminal ␣-helix could be responsible for the observed calcium switch in NSP5-directed VLS formation (12,24,35,45,(49)(50)(51). Here, we examined whether mutations in the potential calcium binding motifs and ␣-helical domains alter VLS formation and the calcium switch.…”

Section: Resultsmentioning

confidence: 99%

The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain

A¹,

Sen²,

Mackow

2007

J Virol

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The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic ␣-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 ␣-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted ␣-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.Rotavirus infection is the single most important cause of severe dehydrating diarrhea in infants and young children, causing approximately 600,000 deaths every year (20). Rotaviruses are icosahedral viruses with a segmented doublestranded RNA genome that infect villous-tip epithelial cells of the proximal small intestine (31). Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms (32,33). Viroplasms are punctate perinuclear structures that are sites of rotavirus protein assembly, RNA packaging, and replication (13,14,25,26,30,46). Viroplasm formation is a characteristic of several members of the Reoviridae family, including...

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“…Although there are already more than 300 substrates known for CK2, regulation of the kinase has been indicated, but on the other hand, CK2 has been reported to be constitutively active and not regulated (29,33). An investigation by Zetina, who has analyzed sequence-dependent helix unfolding in proteins, suggested that phosphorylation of the conserved motif (LS/SL)(D/ E)(D/E)(D/E)X(D/E) could stabilize helix unfolding (47). The sequence context of phosphorylated serine residue 249 ( 248 LS DDDDQ 254 ) in NS2 conforms to this conserved sequence except for the last residue.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

Modrof

Lymperopoulos

Roy

2005

J Virol

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In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.Protein phosphorylation is a ubiquitous protein modification that controls a number of intracellular processes. In eukaryotic systems, phosphorylation occurs almost exclusively on serine, threonine, or tyrosine residues (26). Also for RNA viruses, including vesicular stomatitis virus, ebola virus, human immunodeficiency virus type 1 (HIV-1), and rubella virus, protein phosphorylation has been shown to regulate vital processes such as virus transcription and replication, RNA binding activity, and virus assembly (9,22,28,34). The nonstructural protein 2 (NS2) is the only phosphorylated protein of the 10 viral proteins synthesized during a bluetongue virus (BTV) infection (25).BTV is the prototype of the Orbivirus genus in the Reoviridae family and hence characteristically possesses a doublestranded RNA genome enclosed by three consecutive capsid layers of multiple proteins. The BTV genome consists of 10 segments, each encoding one protein. There are seven structural proteins (viral polymerase 1 [VP1] through VP7), of which two are outer capsid proteins (VP2 and VP5) and the remaining five are associated with the BTV core. The viral core consists of a double-layered shell composed of VP3 and VP7, and within the core, there are three virus-encoded proteins (VP1, VP4, and VP6), all of which are enzymatically involved in viral transcription and replication. VP4 is the mRNA-capping enzyme, exhibiting guanyltransferase and methyltransferase activities, VP6 is a double-stranded RNA helicase, and the largest protein, VP1, is the viral RNA-dependent RNA polymerase (5,32,36,41). Core particles are transcriptionally active, producing and releasing mRNA. The remaining three BTV proteins are nonstructural...

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A conserved helix‐unfolding motif in the naturally unfolded proteins

Cited by 24 publications

References 34 publications

Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2

Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2

The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain

Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

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A conserved helix‐unfolding motif in the naturally unfolded proteins

Cited by 24 publications

References 34 publications

Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2

Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2

The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain

Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

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Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2

Cell cycle regulatory protein p27^KIP1 is a substrate and interacts with the protein kinase CK2