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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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LC8 facilitates Pak1 nuclear import.(A) MCF-7 cells transiently transfected with either wild-type (WT), kinase active (T423E) GFP-Pak1, Pak1-LC8mut or Pak1-NLSmut mRNA. Scalebar shown is 10 microns. Mutations of either the NLS or the LC8 binding sequence in WT-Pak1 or T423E-Pak1 markedly reduced EGF-dependent nuclear import and stained with DAPI to visualize nuclei. (B) Quantification of nuclear accumulation of MCF-7 cells harboring either Pak1 or Pak1 mutants. Each bar represents percentage of cells with nuclear localized GFP (50 cells per experiment, done in triplicate). (C) The fraction of GFP located in cytoplasmic (C) and nuclear (N) fractions of MCF-7 cells after stimulation with EGF. Potential cross contamination of nuclear and cytoplasmic fractions was assessed by immunoblot analysis of Laminin A&C and Vinculin, respectively. (D) Nuclear import of T423E-Pak1 mutants after EGF stimulation. Nuclear percentages were calculated as in B. (E) Western Blot analysis of cytoplasmic and nuclear fractions of MCF-7 cells expressing T423E-Pak1 mutants after stimulation with EGF using an anti-GFP antibody.
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pone-0006025-g002: LC8 facilitates Pak1 nuclear import.(A) MCF-7 cells transiently transfected with either wild-type (WT), kinase active (T423E) GFP-Pak1, Pak1-LC8mut or Pak1-NLSmut mRNA. Scalebar shown is 10 microns. Mutations of either the NLS or the LC8 binding sequence in WT-Pak1 or T423E-Pak1 markedly reduced EGF-dependent nuclear import and stained with DAPI to visualize nuclei. (B) Quantification of nuclear accumulation of MCF-7 cells harboring either Pak1 or Pak1 mutants. Each bar represents percentage of cells with nuclear localized GFP (50 cells per experiment, done in triplicate). (C) The fraction of GFP located in cytoplasmic (C) and nuclear (N) fractions of MCF-7 cells after stimulation with EGF. Potential cross contamination of nuclear and cytoplasmic fractions was assessed by immunoblot analysis of Laminin A&C and Vinculin, respectively. (D) Nuclear import of T423E-Pak1 mutants after EGF stimulation. Nuclear percentages were calculated as in B. (E) Western Blot analysis of cytoplasmic and nuclear fractions of MCF-7 cells expressing T423E-Pak1 mutants after stimulation with EGF using an anti-GFP antibody.

Mentions: Collectively, these results led us to test whether nuclear import of Pak1 depends on LC8 binding. We generated a double mutant Pak1 (A218Q and T219E [LC8mut-Pak1]; see Fig. 1B) to abrogate LC8 binding and tested whether these mutations would affect Pak1 nuclear import. Wild-type (WT) and LC8mut-Pak1 constructs were tagged N-terminally with GFP or Myc sequences. MCF-7 cells were transiently transfected with either GFP-WT-Pak1 or GFP-LC8mut-Pak1 and stimulated with EGF for 25 minutes. We observed markedly lower levels of nuclear GFP-LC8mut-Pak1 as compared to GFP-WT-Pak1 (Fig. 2). Co-immunoprecipitation experiments using MCF-7 cells that coexpressed the Myc-Pak1 mutant and HA-LC8 verified that LC8mut-Pak1 did not interact with LC8 whereas Myc-WT-Pak1 did (Fig. S1A). To confirm the role of the NLS in EGF-dependent nuclear import of Pak1, we mutated all three lysine residues in the Pak1 NLS sequence to alanines and evaluated nuclear Pak1 accumulation upon EGF stimulation (Figs. 1B and 2). Similar to the LC8mut-Pak1 construct, the NLS mutant, NLSmut-Pak1, also showed much less EGF-stimulated nuclear accumulation (Fig. 2). Quantitative analysis of Pak1 nuclear accumulation by confocal microscopy revealed that 24% of the EGF-stimulated cells transfected with GFP-WT-Pak1 contained GFP in the nucleus, in contrast to only 8% or 10% of EGF-stimulated MCF-7 cells transfected with the GFP-LC8mut or GFP-NLSmut Pak1 constructs, respectively. Unstimulated MCF-7 cells transiently transfected with any of the three constructs contained GFP in less than 1% of nuclei.


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)

LC8 facilitates Pak1 nuclear import.(A) MCF-7 cells transiently transfected with either wild-type (WT), kinase active (T423E) GFP-Pak1, Pak1-LC8mut or Pak1-NLSmut mRNA. Scalebar shown is 10 microns. Mutations of either the NLS or the LC8 binding sequence in WT-Pak1 or T423E-Pak1 markedly reduced EGF-dependent nuclear import and stained with DAPI to visualize nuclei. (B) Quantification of nuclear accumulation of MCF-7 cells harboring either Pak1 or Pak1 mutants. Each bar represents percentage of cells with nuclear localized GFP (50 cells per experiment, done in triplicate). (C) The fraction of GFP located in cytoplasmic (C) and nuclear (N) fractions of MCF-7 cells after stimulation with EGF. Potential cross contamination of nuclear and cytoplasmic fractions was assessed by immunoblot analysis of Laminin A&C and Vinculin, respectively. (D) Nuclear import of T423E-Pak1 mutants after EGF stimulation. Nuclear percentages were calculated as in B. (E) Western Blot analysis of cytoplasmic and nuclear fractions of MCF-7 cells expressing T423E-Pak1 mutants after stimulation with EGF using an anti-GFP antibody.
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pone-0006025-g002: LC8 facilitates Pak1 nuclear import.(A) MCF-7 cells transiently transfected with either wild-type (WT), kinase active (T423E) GFP-Pak1, Pak1-LC8mut or Pak1-NLSmut mRNA. Scalebar shown is 10 microns. Mutations of either the NLS or the LC8 binding sequence in WT-Pak1 or T423E-Pak1 markedly reduced EGF-dependent nuclear import and stained with DAPI to visualize nuclei. (B) Quantification of nuclear accumulation of MCF-7 cells harboring either Pak1 or Pak1 mutants. Each bar represents percentage of cells with nuclear localized GFP (50 cells per experiment, done in triplicate). (C) The fraction of GFP located in cytoplasmic (C) and nuclear (N) fractions of MCF-7 cells after stimulation with EGF. Potential cross contamination of nuclear and cytoplasmic fractions was assessed by immunoblot analysis of Laminin A&C and Vinculin, respectively. (D) Nuclear import of T423E-Pak1 mutants after EGF stimulation. Nuclear percentages were calculated as in B. (E) Western Blot analysis of cytoplasmic and nuclear fractions of MCF-7 cells expressing T423E-Pak1 mutants after stimulation with EGF using an anti-GFP antibody.
Mentions: Collectively, these results led us to test whether nuclear import of Pak1 depends on LC8 binding. We generated a double mutant Pak1 (A218Q and T219E [LC8mut-Pak1]; see Fig. 1B) to abrogate LC8 binding and tested whether these mutations would affect Pak1 nuclear import. Wild-type (WT) and LC8mut-Pak1 constructs were tagged N-terminally with GFP or Myc sequences. MCF-7 cells were transiently transfected with either GFP-WT-Pak1 or GFP-LC8mut-Pak1 and stimulated with EGF for 25 minutes. We observed markedly lower levels of nuclear GFP-LC8mut-Pak1 as compared to GFP-WT-Pak1 (Fig. 2). Co-immunoprecipitation experiments using MCF-7 cells that coexpressed the Myc-Pak1 mutant and HA-LC8 verified that LC8mut-Pak1 did not interact with LC8 whereas Myc-WT-Pak1 did (Fig. S1A). To confirm the role of the NLS in EGF-dependent nuclear import of Pak1, we mutated all three lysine residues in the Pak1 NLS sequence to alanines and evaluated nuclear Pak1 accumulation upon EGF stimulation (Figs. 1B and 2). Similar to the LC8mut-Pak1 construct, the NLS mutant, NLSmut-Pak1, also showed much less EGF-stimulated nuclear accumulation (Fig. 2). Quantitative analysis of Pak1 nuclear accumulation by confocal microscopy revealed that 24% of the EGF-stimulated cells transfected with GFP-WT-Pak1 contained GFP in the nucleus, in contrast to only 8% or 10% of EGF-stimulated MCF-7 cells transfected with the GFP-LC8mut or GFP-NLSmut Pak1 constructs, respectively. Unstimulated MCF-7 cells transiently transfected with any of the three constructs contained GFP in less than 1% of nuclei.

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