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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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Functional domains of Pak1 constructs relevant to LC8 interaction and nuclear import.(A) Schematic representation of the domain structure of Pak1. The LC8 binding site and the nuclear localization sequence (NLS) shown to be critical for Pak1 nuclear import are highlighted. (B) Mutants used for immunofluorescent experiments with MCF-7 cells and reconstitution experiments in zebrafish. Mutations of A218Q and T219E in the LC8 binding site were generated to inhibit the LC8-Pak1 interaction and were based on the crystal structure 3DVP. The three lysine residues in the NLS were mutated to alanines.
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pone-0006025-g001: Functional domains of Pak1 constructs relevant to LC8 interaction and nuclear import.(A) Schematic representation of the domain structure of Pak1. The LC8 binding site and the nuclear localization sequence (NLS) shown to be critical for Pak1 nuclear import are highlighted. (B) Mutants used for immunofluorescent experiments with MCF-7 cells and reconstitution experiments in zebrafish. Mutations of A218Q and T219E in the LC8 binding site were generated to inhibit the LC8-Pak1 interaction and were based on the crystal structure 3DVP. The three lysine residues in the NLS were mutated to alanines.

Mentions: Previous studies have established that Pak1 translocates into the nucleus upon EGF stimulation of MCF-7 breast cancer cells and that EGF-induced nuclear import requires a weak nuclear localization sequence consisting of residues 243 to 245 [11]. Further, LC8 has been shown to enhance nuclear import of both the Rabies P protein and the p53 binding protein 53BP1 [12]. Finally, our recent structural and biochemical studies showed that residues 212 to 222 of Pak1 encode an LC8 binding site adjacent to the nuclear localization site (Fig. 1A) [9].


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)

Functional domains of Pak1 constructs relevant to LC8 interaction and nuclear import.(A) Schematic representation of the domain structure of Pak1. The LC8 binding site and the nuclear localization sequence (NLS) shown to be critical for Pak1 nuclear import are highlighted. (B) Mutants used for immunofluorescent experiments with MCF-7 cells and reconstitution experiments in zebrafish. Mutations of A218Q and T219E in the LC8 binding site were generated to inhibit the LC8-Pak1 interaction and were based on the crystal structure 3DVP. The three lysine residues in the NLS were mutated to alanines.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006025-g001: Functional domains of Pak1 constructs relevant to LC8 interaction and nuclear import.(A) Schematic representation of the domain structure of Pak1. The LC8 binding site and the nuclear localization sequence (NLS) shown to be critical for Pak1 nuclear import are highlighted. (B) Mutants used for immunofluorescent experiments with MCF-7 cells and reconstitution experiments in zebrafish. Mutations of A218Q and T219E in the LC8 binding site were generated to inhibit the LC8-Pak1 interaction and were based on the crystal structure 3DVP. The three lysine residues in the NLS were mutated to alanines.
Mentions: Previous studies have established that Pak1 translocates into the nucleus upon EGF stimulation of MCF-7 breast cancer cells and that EGF-induced nuclear import requires a weak nuclear localization sequence consisting of residues 243 to 245 [11]. Further, LC8 has been shown to enhance nuclear import of both the Rabies P protein and the p53 binding protein 53BP1 [12]. Finally, our recent structural and biochemical studies showed that residues 212 to 222 of Pak1 encode an LC8 binding site adjacent to the nuclear localization site (Fig. 1A) [9].

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