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Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Lightcap CM, Kari G, Arias-Romero LE, Chernoff J, Rodeck U, Williams JC - PLoS ONE (2009)

Bottom Line: In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells.Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site.Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

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Related in: MedlinePlus

Model of Pak1 translocation.Pak1 is a stable homodimer in its inactive state. Multiple signals from different pathways (GPCRs, RTKs, and lipids) act on Pak1 and activate it. One potential pathway suggests that the N-terminus of Pak1 binds to Nck or Grb2, which is associated with the activated EGFR. This permits Cdc42/Rac1 to bind to Pak1, allowing it to trans-autophosphorylate. We propose this permits LC8 binding, which localizes the weak NLS to act as a bipartite ligand for importin binding and ultimately nuclear translocation.
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pone-0006025-g006: Model of Pak1 translocation.Pak1 is a stable homodimer in its inactive state. Multiple signals from different pathways (GPCRs, RTKs, and lipids) act on Pak1 and activate it. One potential pathway suggests that the N-terminus of Pak1 binds to Nck or Grb2, which is associated with the activated EGFR. This permits Cdc42/Rac1 to bind to Pak1, allowing it to trans-autophosphorylate. We propose this permits LC8 binding, which localizes the weak NLS to act as a bipartite ligand for importin binding and ultimately nuclear translocation.

Mentions: Taken together, these observations suggest that EGF-induced nuclear localization of Pak1 requires a significant conformational change accompanying the transition from the inactive-to-active state, dimerization through LC8-binding sequences in close proximity to the NLS, and alignment of weak NLS signals in Pak1 monomers through LC8-dependent orientation followed by importin recognition and nuclear import (Fig. 6).


Interaction with LC8 is required for Pak1 nuclear import and is indispensable for zebrafish development.

Lightcap CM, Kari G, Arias-Romero LE, Chernoff J, Rodeck U, Williams JC - PLoS ONE (2009)

Model of Pak1 translocation.Pak1 is a stable homodimer in its inactive state. Multiple signals from different pathways (GPCRs, RTKs, and lipids) act on Pak1 and activate it. One potential pathway suggests that the N-terminus of Pak1 binds to Nck or Grb2, which is associated with the activated EGFR. This permits Cdc42/Rac1 to bind to Pak1, allowing it to trans-autophosphorylate. We propose this permits LC8 binding, which localizes the weak NLS to act as a bipartite ligand for importin binding and ultimately nuclear translocation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698211&req=5

pone-0006025-g006: Model of Pak1 translocation.Pak1 is a stable homodimer in its inactive state. Multiple signals from different pathways (GPCRs, RTKs, and lipids) act on Pak1 and activate it. One potential pathway suggests that the N-terminus of Pak1 binds to Nck or Grb2, which is associated with the activated EGFR. This permits Cdc42/Rac1 to bind to Pak1, allowing it to trans-autophosphorylate. We propose this permits LC8 binding, which localizes the weak NLS to act as a bipartite ligand for importin binding and ultimately nuclear translocation.
Mentions: Taken together, these observations suggest that EGF-induced nuclear localization of Pak1 requires a significant conformational change accompanying the transition from the inactive-to-active state, dimerization through LC8-binding sequences in close proximity to the NLS, and alignment of weak NLS signals in Pak1 monomers through LC8-dependent orientation followed by importin recognition and nuclear import (Fig. 6).

Bottom Line: In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells.Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site.Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.

ABSTRACT
Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Show MeSH
Related in: MedlinePlus