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Kank attenuates actin remodeling by preventing interaction between IRSp53 and Rac1.

Roy BC, Kakinuma N, Kiyama R - J. Cell Biol. (2009)

Bottom Line: Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect.Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth.Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.

ABSTRACT
In this study, insulin receptor substrate (IRS) p53 is identified as a binding partner for Kank, a kidney ankyrin repeat-containing protein that functions to suppress cell proliferation and regulate the actin cytoskeleton. Kank specifically inhibits the binding of IRSp53 with active Rac1 (Rac1(G12V)) but not Cdc42 (cdc42(G12V)) and thus inhibits the IRSp53-dependent development of lamellipodia without affecting the formation of filopodia. Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect. Moreover, insulin-induced membrane ruffling is inhibited by overexpression of Kank. Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth. Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

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Kank inhibits insulin-induced membrane ruffling. The effectof Kank on insulin-induced membrane ruffling in NIH3T3 cells wasexamined. The cells were transfected and treated as described inMaterials and methods. The images were obtained by confocal lasermicroscopy. The number of cells with membrane ruffling is shown on theright as a percentage of the total number of transfected cells. Theresults are shown as the mean ± SD for triplicate experimentsin which 100 cells per experiment were counted. *, P <0.001 compared with lane 2. Bar, 10 µm.
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fig7: Kank inhibits insulin-induced membrane ruffling. The effectof Kank on insulin-induced membrane ruffling in NIH3T3 cells wasexamined. The cells were transfected and treated as described inMaterials and methods. The images were obtained by confocal lasermicroscopy. The number of cells with membrane ruffling is shown on theright as a percentage of the total number of transfected cells. Theresults are shown as the mean ± SD for triplicate experimentsin which 100 cells per experiment were counted. *, P <0.001 compared with lane 2. Bar, 10 µm.

Mentions: Lamellipodia and filopodia are formed at the leading edge in migrating cells inresponse to growth factors, chemokines, and extracellular matrix molecules(Ridley et al., 2003). Insulin actsas a growth factor, and insulin-stimulated KB cells, a strain derived from humanepidermoid carcinoma, show membrane ruffling and Rac1 activation (Nishiyama et al., 1994). Overexpressionof Kank inhibited active Rac1-dependent lamellipodial development (Fig. 5), and KD of Kank enhanced theformation of lamellipodia (Fig. 6). Wethen investigated whether Kank can inhibit insulin-induced membrane ruffling(Fig. 7). Whereas serum-starvedNIH3T3 cells without treatment showed little membrane ruffling (Fig. 7, lane 1), insulin-stimulated cellsexhibited extensive membrane ruffling (Fig.7, lane 2). When Kank was overexpressed, the number of cells withmembrane ruffling was significantly decreased (Fig. 7, lane 3). However, the effect of overexpression of Kank wasnot observed when IRSp53 was coexpressed (Fig.7, lane 5). This result suggests that IRSp53 abrogates the inhibitoryeffect of Kank on insulin-induced membrane ruffling and, thus, that Kankinhibits insulin-induced membrane ruffling through IRSp53.


Kank attenuates actin remodeling by preventing interaction between IRSp53 and Rac1.

Roy BC, Kakinuma N, Kiyama R - J. Cell Biol. (2009)

Kank inhibits insulin-induced membrane ruffling. The effectof Kank on insulin-induced membrane ruffling in NIH3T3 cells wasexamined. The cells were transfected and treated as described inMaterials and methods. The images were obtained by confocal lasermicroscopy. The number of cells with membrane ruffling is shown on theright as a percentage of the total number of transfected cells. Theresults are shown as the mean ± SD for triplicate experimentsin which 100 cells per experiment were counted. *, P <0.001 compared with lane 2. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654296&req=5

fig7: Kank inhibits insulin-induced membrane ruffling. The effectof Kank on insulin-induced membrane ruffling in NIH3T3 cells wasexamined. The cells were transfected and treated as described inMaterials and methods. The images were obtained by confocal lasermicroscopy. The number of cells with membrane ruffling is shown on theright as a percentage of the total number of transfected cells. Theresults are shown as the mean ± SD for triplicate experimentsin which 100 cells per experiment were counted. *, P <0.001 compared with lane 2. Bar, 10 µm.
Mentions: Lamellipodia and filopodia are formed at the leading edge in migrating cells inresponse to growth factors, chemokines, and extracellular matrix molecules(Ridley et al., 2003). Insulin actsas a growth factor, and insulin-stimulated KB cells, a strain derived from humanepidermoid carcinoma, show membrane ruffling and Rac1 activation (Nishiyama et al., 1994). Overexpressionof Kank inhibited active Rac1-dependent lamellipodial development (Fig. 5), and KD of Kank enhanced theformation of lamellipodia (Fig. 6). Wethen investigated whether Kank can inhibit insulin-induced membrane ruffling(Fig. 7). Whereas serum-starvedNIH3T3 cells without treatment showed little membrane ruffling (Fig. 7, lane 1), insulin-stimulated cellsexhibited extensive membrane ruffling (Fig.7, lane 2). When Kank was overexpressed, the number of cells withmembrane ruffling was significantly decreased (Fig. 7, lane 3). However, the effect of overexpression of Kank wasnot observed when IRSp53 was coexpressed (Fig.7, lane 5). This result suggests that IRSp53 abrogates the inhibitoryeffect of Kank on insulin-induced membrane ruffling and, thus, that Kankinhibits insulin-induced membrane ruffling through IRSp53.

Bottom Line: Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect.Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth.Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.

ABSTRACT
In this study, insulin receptor substrate (IRS) p53 is identified as a binding partner for Kank, a kidney ankyrin repeat-containing protein that functions to suppress cell proliferation and regulate the actin cytoskeleton. Kank specifically inhibits the binding of IRSp53 with active Rac1 (Rac1(G12V)) but not Cdc42 (cdc42(G12V)) and thus inhibits the IRSp53-dependent development of lamellipodia without affecting the formation of filopodia. Knockdown (KD) of Kank by RNA interference results in increased lamellipodial development, whereas KD of both Kank and IRSp53 has little effect. Moreover, insulin-induced membrane ruffling is inhibited by overexpression of Kank. Kank also suppresses integrin-dependent cell spreading and IRSp53-induced neurite outgrowth. Our results demonstrate that Kank negatively regulates the formation of lamellipodia by inhibiting the interaction between Rac1 and IRSp53.

Show MeSH
Related in: MedlinePlus