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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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Overexpression of human Pak1 produces embryonic abnormalities in zebrafish that are contingent upon nuclear import of Pak1.(A) Images of zebrafish embryos at 4 dpf. Embryos were injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants (80 ng/µL). (B) Quantitative analysis of differential zebrafish body axis malformation at 4 dpf in embryos injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants. (C) Immunoblot analysis of lysates from zebrafish injected with either human Pak1-WT, NLSmut, or LC8mut mRNA.
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pone-0006025-g005: Overexpression of human Pak1 produces embryonic abnormalities in zebrafish that are contingent upon nuclear import of Pak1.(A) Images of zebrafish embryos at 4 dpf. Embryos were injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants (80 ng/µL). (B) Quantitative analysis of differential zebrafish body axis malformation at 4 dpf in embryos injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants. (C) Immunoblot analysis of lysates from zebrafish injected with either human Pak1-WT, NLSmut, or LC8mut mRNA.

Mentions: Next, we examined the effects of overexpressing human Pak1 in zebrafish embryos. Injection of hPak1 mRNA alone led to death and/or severe morphological aberrations in the injected embryos. Interestingly, these were similar to the aberrations observed in fish treated with Pak1-targeted MOs, and mainly consisted of aberrations in body axis development and pericardial edema. However, embryos singly injected either with the human NLSmut- or LC8mut-Pak1 mRNA showed very few morphological alterations, consistent with the view that deleterious effects of Pak1 overexpression on zebrafish morphology and survival depend on nuclear import (Figs. 5A and B). Western blot analysis using a Pak1 specific antibody verified that all three groups of human Pak1 mRNA injected fish expressed similar levels of Pak1 protein (Fig. 5C). In aggregate, these results support a crucial role of nuclear import of Pak1, facilitated by LC8, in development and survival of zebrafish embryos.


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)

Overexpression of human Pak1 produces embryonic abnormalities in zebrafish that are contingent upon nuclear import of Pak1.(A) Images of zebrafish embryos at 4 dpf. Embryos were injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants (80 ng/µL). (B) Quantitative analysis of differential zebrafish body axis malformation at 4 dpf in embryos injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants. (C) Immunoblot analysis of lysates from zebrafish injected with either human Pak1-WT, NLSmut, or LC8mut mRNA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006025-g005: Overexpression of human Pak1 produces embryonic abnormalities in zebrafish that are contingent upon nuclear import of Pak1.(A) Images of zebrafish embryos at 4 dpf. Embryos were injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants (80 ng/µL). (B) Quantitative analysis of differential zebrafish body axis malformation at 4 dpf in embryos injected with either Pak1 WT-mRNA or one of the mRNA of the Pak1 nuclear import mutants. (C) Immunoblot analysis of lysates from zebrafish injected with either human Pak1-WT, NLSmut, or LC8mut mRNA.
Mentions: Next, we examined the effects of overexpressing human Pak1 in zebrafish embryos. Injection of hPak1 mRNA alone led to death and/or severe morphological aberrations in the injected embryos. Interestingly, these were similar to the aberrations observed in fish treated with Pak1-targeted MOs, and mainly consisted of aberrations in body axis development and pericardial edema. However, embryos singly injected either with the human NLSmut- or LC8mut-Pak1 mRNA showed very few morphological alterations, consistent with the view that deleterious effects of Pak1 overexpression on zebrafish morphology and survival depend on nuclear import (Figs. 5A and B). Western blot analysis using a Pak1 specific antibody verified that all three groups of human Pak1 mRNA injected fish expressed similar levels of Pak1 protein (Fig. 5C). In aggregate, these results support a crucial role of nuclear import of Pak1, facilitated by LC8, in development and survival of zebrafish embryos.

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