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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 has no effect on active cdc42-dependent filopodialdevelopment. The effect of Kank expression on activecdc42-dependent formation of filopodia was examined. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and Kank or its mutants (red). The number ofcells spreading with filopodia is shown on the right as a percentage ofthe total number of transfected cells. The results are shown as the mean± SD for triplicate experiments in which ∼100 cellsper experiment were counted. LM, phase-contrast light microscopic image.Bar, 10 µm.
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fig5: Kank has no effect on active cdc42-dependent filopodialdevelopment. The effect of Kank expression on activecdc42-dependent formation of filopodia was examined. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and Kank or its mutants (red). The number ofcells spreading with filopodia is shown on the right as a percentage ofthe total number of transfected cells. The results are shown as the mean± SD for triplicate experiments in which ∼100 cellsper experiment were counted. LM, phase-contrast light microscopic image.Bar, 10 µm.

Mentions: Kank affected the interaction between IRSp53 and Rac1 and partially affected thatbetween IRSp53 and cdc42 (Fig. 3). Thisled us to investigate the effect of Kank expression on the formation oflamellipodia or filopodia mediated by Rac1 or cdc42 (Figs. 4 and 5).Initially, IRSp53 was reported to be a downstream target of Rac1 that links Rac1activity to WAVE/Scar and the Arp2/3 complex (Miki et al., 2000; Etienne-Manneville and Hall, 2002). However, others reported thatIRSp53 preferentially binds to active cdc42 (Govind et al., 2001; Krugmann etal., 2001). Overexpression of Kank severely impaired the developmentof lamellipodia and cell spreading induced by a constitutively active form ofRac1, Rac1G12V (Fig. 4 A, lane4). Overexpression of KankS167A, which lacks the ability to bind to14-3-3 and has inhibitory effects on active RhoA and cell migration (Kakinuma et al., 2008), also impaired theformation of lamellipodia induced by Rac1G12V (Fig. 4 A, lane 5). However,KankΔcoil, which cannot interact with IRSp53, had no effecton the Rac1G12V-induced development of lamellipodia (Fig. 4 A, lane 6). To confirm that thiseffect of Kank depends on the interaction with IRSp53, we performed coexpressionof Kank with IRSp53 (Fig. 4 B).Coexpression of GFP-Rac1G12V and Kank with IRSp53 resulted in thedisappearance of Kank's inhibitory effect on theRac1G12V-dependent formation of lamellipodia (Fig. 4 B, lane 4). However, coexpression ofGFP-Rac1G12V and Kank with IRSp53R11E/Q23E orIRSp53K143E, which could not bind to Rac1 and could bind to Kank(Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805147/DC1; Suetsugu et al., 2006b), resulted in theretention of the inhibitory effect on the Rac1G12V-dependentdevelopment of lamellipodia (Fig. 4 B,lanes 5 and 6). This result implies that Kank inhibits Rac1–IRSp53signals for the formation of lamellipodia. Moreover, to confirm that thisRac1G12V-dependent formation depends on IRSp53, we performed KDof IRSp53 along with GFP-Rac1G12V expression (Fig. 4 C). We first examined the KD of mouse IRSp53 (mIRS)in NIH3T3 cells using various candidate plasmids (Fig. S2). These plasmidscontained an H1 promoter–based expression system for siRNAs inmammalian cells (Steffen et al., 2004).Based on the results, we used #513 siRNA for the KD of mIRS.Coexpression of GFP-Rac1G12V and mIRS-KD reduced theRac1G12V-dependent formation of lamellipodia (Fig. 4 C, lane 3), whereas on the coexpression of humanIRSp53 with GFP-Rac1G12V and mIRS-KD, GFP-Rac1G12Vretained its function (Fig. 4 C, lane 4).However, the coexpression of human IRSp53R11E/Q23E orIRSp53K143E with GFP-Rac1G12V and mIRS-KD had littleeffect on mIRS-KD's function in the Rac1G12V-dependentformation of lamellipodia (Fig. 4 C,lanes 5 and 6). Therefore, the interaction between IRSp53 and active Rac1 isimportant for lamellipodia to form. These results suggest that IRSp53 is one ofthe targets of Rac1G12V in the Rac1G12V-dependentdevelopment of lamellipodia. However, overexpression of Kank andKankS167A had little effect on the formation of filopodia induced bya constitutively active form of cdc42, cdc42G12V (Fig. 5, lanes 3 and 4), which may implythat Kank was not involved in the cdc42G12V-mediated formation ofmicrospikes.


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

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

Kank has no effect on active cdc42-dependent filopodialdevelopment. The effect of Kank expression on activecdc42-dependent formation of filopodia was examined. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and Kank or its mutants (red). The number ofcells spreading with filopodia is shown on the right as a percentage ofthe total number of transfected cells. The results are shown as the mean± SD for triplicate experiments in which ∼100 cellsper experiment were counted. LM, phase-contrast light microscopic image.Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Kank has no effect on active cdc42-dependent filopodialdevelopment. The effect of Kank expression on activecdc42-dependent formation of filopodia was examined. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and Kank or its mutants (red). The number ofcells spreading with filopodia is shown on the right as a percentage ofthe total number of transfected cells. The results are shown as the mean± SD for triplicate experiments in which ∼100 cellsper experiment were counted. LM, phase-contrast light microscopic image.Bar, 10 µm.
Mentions: Kank affected the interaction between IRSp53 and Rac1 and partially affected thatbetween IRSp53 and cdc42 (Fig. 3). Thisled us to investigate the effect of Kank expression on the formation oflamellipodia or filopodia mediated by Rac1 or cdc42 (Figs. 4 and 5).Initially, IRSp53 was reported to be a downstream target of Rac1 that links Rac1activity to WAVE/Scar and the Arp2/3 complex (Miki et al., 2000; Etienne-Manneville and Hall, 2002). However, others reported thatIRSp53 preferentially binds to active cdc42 (Govind et al., 2001; Krugmann etal., 2001). Overexpression of Kank severely impaired the developmentof lamellipodia and cell spreading induced by a constitutively active form ofRac1, Rac1G12V (Fig. 4 A, lane4). Overexpression of KankS167A, which lacks the ability to bind to14-3-3 and has inhibitory effects on active RhoA and cell migration (Kakinuma et al., 2008), also impaired theformation of lamellipodia induced by Rac1G12V (Fig. 4 A, lane 5). However,KankΔcoil, which cannot interact with IRSp53, had no effecton the Rac1G12V-induced development of lamellipodia (Fig. 4 A, lane 6). To confirm that thiseffect of Kank depends on the interaction with IRSp53, we performed coexpressionof Kank with IRSp53 (Fig. 4 B).Coexpression of GFP-Rac1G12V and Kank with IRSp53 resulted in thedisappearance of Kank's inhibitory effect on theRac1G12V-dependent formation of lamellipodia (Fig. 4 B, lane 4). However, coexpression ofGFP-Rac1G12V and Kank with IRSp53R11E/Q23E orIRSp53K143E, which could not bind to Rac1 and could bind to Kank(Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805147/DC1; Suetsugu et al., 2006b), resulted in theretention of the inhibitory effect on the Rac1G12V-dependentdevelopment of lamellipodia (Fig. 4 B,lanes 5 and 6). This result implies that Kank inhibits Rac1–IRSp53signals for the formation of lamellipodia. Moreover, to confirm that thisRac1G12V-dependent formation depends on IRSp53, we performed KDof IRSp53 along with GFP-Rac1G12V expression (Fig. 4 C). We first examined the KD of mouse IRSp53 (mIRS)in NIH3T3 cells using various candidate plasmids (Fig. S2). These plasmidscontained an H1 promoter–based expression system for siRNAs inmammalian cells (Steffen et al., 2004).Based on the results, we used #513 siRNA for the KD of mIRS.Coexpression of GFP-Rac1G12V and mIRS-KD reduced theRac1G12V-dependent formation of lamellipodia (Fig. 4 C, lane 3), whereas on the coexpression of humanIRSp53 with GFP-Rac1G12V and mIRS-KD, GFP-Rac1G12Vretained its function (Fig. 4 C, lane 4).However, the coexpression of human IRSp53R11E/Q23E orIRSp53K143E with GFP-Rac1G12V and mIRS-KD had littleeffect on mIRS-KD's function in the Rac1G12V-dependentformation of lamellipodia (Fig. 4 C,lanes 5 and 6). Therefore, the interaction between IRSp53 and active Rac1 isimportant for lamellipodia to form. These results suggest that IRSp53 is one ofthe targets of Rac1G12V in the Rac1G12V-dependentdevelopment of lamellipodia. However, overexpression of Kank andKankS167A had little effect on the formation of filopodia induced bya constitutively active form of cdc42, cdc42G12V (Fig. 5, lanes 3 and 4), which may implythat Kank was not involved in the cdc42G12V-mediated formation ofmicrospikes.

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