Source:http://linkedlifedata.com/resource/pubmed/id/15591319
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
8
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pubmed:dateCreated |
2005-2-21
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pubmed:abstractText |
The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1-548 identifies two stable truncations: 129-548 and 129-369. Analytical ultracentrifugation suggests that 1-548 and 129-548 are dimers, whereas 129-369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1-548 is partially relieved by RhoA, inhibition by 129-548 or 129-369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1-548 by 20,000-fold. Synthetic DAD peptide binds both 1-548 and 129-548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129-369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Feb
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pubmed:issn |
0021-9258
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
25
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pubmed:volume |
280
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
6986-92
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:15591319-Actins,
pubmed-meshheading:15591319-Amino Acid Sequence,
pubmed-meshheading:15591319-Animals,
pubmed-meshheading:15591319-Binding Sites,
pubmed-meshheading:15591319-Carrier Proteins,
pubmed-meshheading:15591319-Mice,
pubmed-meshheading:15591319-Protein Structure, Tertiary,
pubmed-meshheading:15591319-Sequence Deletion,
pubmed-meshheading:15591319-rhoA GTP-Binding Protein
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pubmed:year |
2005
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pubmed:articleTitle |
Dissecting requirements for auto-inhibition of actin nucleation by the formin, mDia1.
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pubmed:affiliation |
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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