Linked Life Data

12033928

Source:http://linkedlifedata.com/resource/pubmed/id/12033928

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
22
pubmed:dateCreated
2002-5-29
pubmed:abstractText
Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
4
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6955-66
pubmed:dateRevised
2005-11-17
pubmed:meshHeading
pubmed-meshheading:12033928-Active Transport, Cell Nucleus, pubmed-meshheading:12033928-Amino Acid Sequence, pubmed-meshheading:12033928-Binding Sites, pubmed-meshheading:12033928-Cell Nucleus, pubmed-meshheading:12033928-Crystallization, pubmed-meshheading:12033928-Fluorescent Dyes, pubmed-meshheading:12033928-HeLa Cells, pubmed-meshheading:12033928-Humans, pubmed-meshheading:12033928-Macromolecular Substances, pubmed-meshheading:12033928-Magnetic Resonance Spectroscopy, pubmed-meshheading:12033928-Molecular Sequence Data, pubmed-meshheading:12033928-Mutation, pubmed-meshheading:12033928-Peptide Hydrolases, pubmed-meshheading:12033928-Protein Structure, Secondary, pubmed-meshheading:12033928-RNA-Binding Proteins, pubmed-meshheading:12033928-beta Karyopherins, pubmed-meshheading:12033928-ran GTP-Binding Protein
pubmed:year
2002
pubmed:articleTitle
Uncoupling Kapbeta2 substrate dissociation and ran binding.
pubmed:affiliation
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
pubmed:publicationType
Journal Article