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PDBsum entry 3dvh
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Motor protein
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PDB id
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3dvh
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References listed in PDB file
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Key reference
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Title
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Biochemical and structural characterization of the pak1-Lc8 interaction.
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Authors
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C.M.Lightcap,
S.Sun,
J.D.Lear,
U.Rodeck,
T.Polenova,
J.C.Williams.
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Ref.
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J Biol Chem, 2008,
283,
27314-27324.
[DOI no: ]
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PubMed id
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Abstract
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Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed
in breast cancer, and their direct interaction has been proposed to regulate
tumor cell survival. These effects have been attributed in part to Pak1-mediated
phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders
Ser(88) inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding
sequence compared with other characterized LC8 binding sequences. Together,
these observations raise the question whether the Pak1/LC8 interaction is
distinct (i.e. enabled by a unique interface independent of LC8 dimerization).
Herein, we present results from biochemical, NMR, and crystallographic studies
that show that Pak1 (residues 212-222) binds to LC8 along the same groove as
canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants
K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding
sequences and identify a key hydrogen bond network that compensates for the loss
of the conserved glutamine in the consensus sequence. We also show that the
target binding interface formed through LC8 dimerization is required to bind to
Pak1 and precludes phosphorylation of LC8 at Ser(88). Consistent with this
observation, in vitro phosphorylation assays using activated Pak1 fail to
phosphorylate LC8. Although these results define structural details of the
Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they
do not support the current model whereby Pak1 binds to and subsequently
phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest
that LC8 binding modulates Pak1 activity and/or nuclear localization.
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Figure 3.
Mutational analysis of Pak1 binding sequence. Analytical SEC
of WT-LC8 (A) and individual point mutants (B-K) in
SMT3-Pak1-(204-226) was used to qualitatively assess the role of
each residue in the LC8-binding region of Pak1. SDS-PAGE of
fractions eluting between 10.1 and 10.5 ml is shown for each
experiment. The expected position of LC8 through SDS-PAGE is
indicated with an asterisk. Each experiment was performed three
times.
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Figure 7.
Role of Thr^67 on LC8-Pak1 interaction. A, stereoview of the
LC8-Pak1 hydrogen bond network. A conserved hydrogen bond
network in LC8 is formed through the side chains of Lys^43,
Asp^47, and Thr^67′ and the backbone of Trp^54 in LC8 and
interacts with Pak1 through the side chain of Asp^216 through a
short hydrogen bond (dotted red lines). B and C, analysis of
T67A mutant by analytical SEC. Analytical SEC assays demonstrate
that mutation of LC8-Thr^67 to alanine abrogates the Pak1-LC8
interaction. The dynein intermediate chain, on the other hand,
encodes threonine at the same position (Asp^216 in Pak1) and
does not form a significant hydrogen bond (distance is 5.54
Å based on Protein Data Bank entry 2PG1). Analytical SEC
assays show that the dynein IC binds to the mutant, T67A-LC8.
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The above figures are
reprinted
from an Open Access publication published by the ASBMB:
J Biol Chem
(2008,
283,
27314-27324)
copyright 2008.
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