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Title: Biochemical and Structural Characterization of the Pak1-LC8 Interaction

Abstract

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 Ser88 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 Ser88. Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although thesemore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959581
Report Number(s):
BNL-82567-2009-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US201016%%725
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 283; Journal Issue: 40; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; DIMERIZATION; GLUTAMINE; HYDROGEN; IN VITRO; MAMMARY GLANDS; MUTANTS; NEOPLASMS; PHOSPHORYLATION; RESIDUES; SERINE; TARGETS; TUMOR CELLS; national synchrotron light source

Citation Formats

Lightcap, C, Sun, S, Lear, J, Rodeck, U, Polenova, T, and Williams, J. Biochemical and Structural Characterization of the Pak1-LC8 Interaction. United States: N. p., 2008. Web. doi:10.1074/jbc.M800758200.
Lightcap, C, Sun, S, Lear, J, Rodeck, U, Polenova, T, & Williams, J. Biochemical and Structural Characterization of the Pak1-LC8 Interaction. United States. https://doi.org/10.1074/jbc.M800758200
Lightcap, C, Sun, S, Lear, J, Rodeck, U, Polenova, T, and Williams, J. 2008. "Biochemical and Structural Characterization of the Pak1-LC8 Interaction". United States. https://doi.org/10.1074/jbc.M800758200.
@article{osti_959581,
title = {Biochemical and Structural Characterization of the Pak1-LC8 Interaction},
author = {Lightcap, C and Sun, S and Lear, J and Rodeck, U and Polenova, T and Williams, J},
abstractNote = {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 Ser88 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 Ser88. 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.},
doi = {10.1074/jbc.M800758200},
url = {https://www.osti.gov/biblio/959581}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 40,
volume = 283,
place = {United States},
year = {Tue Jan 01 00:00:00 EST 2008},
month = {Tue Jan 01 00:00:00 EST 2008}
}