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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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Expression of Kank inhibits lamellipodial development mediated byactive Rac1 through IRSp53. (A) Effect of the expression ofKank on active Rac1-induced formation of lamellipodia. 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 lamellipodia is shown on the right as a percentageof the total number of transfected cells. *, P < 0.001compared with lane 3. (B) Overexpression of IRSp53 abolished the effectof Kank on Rac1G12V-dependent lamellipodial formation. NIH3T3cells were transfected as indicated, treated as described in Materialsand methods, and stained for Kank (red) and human IRSp53 or its mutants(purple). GFP is shown in green. The number of cells spreading withlamellipodia is shown as in A. *, P < 0.001 compared withlane 1; **, P < 0.01 compared with lane 1 or 4.IRSR,Q/E, human IRSp53 with arginine replaced by glutamicacid at position 11 and glutamine replaced by glutamic acid at position23; IRSK/E, human IRSp53 with lysine replaced by glutamicacid at position 143. (C) KD of mIRS (mIRS-KD) decreasedRac1G12V-dependent lamellipodial formation. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and mIRS (endogenous) with human IRSp53(expressing transiently) or its mutants (red). The number of cellsspreading with lamellipodia is shown as in A. *, P <0.001 compared with lane 2; **, P < 0.01 comparedwith lane 2 or 4. LM, phase-contrast light microscopic image. Theresults are shown as the mean ± SD for triplicate experimentsin which ∼100 cells per experiment were counted. Bars, 10µm.
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fig4: Expression of Kank inhibits lamellipodial development mediated byactive Rac1 through IRSp53. (A) Effect of the expression ofKank on active Rac1-induced formation of lamellipodia. 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 lamellipodia is shown on the right as a percentageof the total number of transfected cells. *, P < 0.001compared with lane 3. (B) Overexpression of IRSp53 abolished the effectof Kank on Rac1G12V-dependent lamellipodial formation. NIH3T3cells were transfected as indicated, treated as described in Materialsand methods, and stained for Kank (red) and human IRSp53 or its mutants(purple). GFP is shown in green. The number of cells spreading withlamellipodia is shown as in A. *, P < 0.001 compared withlane 1; **, P < 0.01 compared with lane 1 or 4.IRSR,Q/E, human IRSp53 with arginine replaced by glutamicacid at position 11 and glutamine replaced by glutamic acid at position23; IRSK/E, human IRSp53 with lysine replaced by glutamicacid at position 143. (C) KD of mIRS (mIRS-KD) decreasedRac1G12V-dependent lamellipodial formation. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and mIRS (endogenous) with human IRSp53(expressing transiently) or its mutants (red). The number of cellsspreading with lamellipodia is shown as in A. *, P <0.001 compared with lane 2; **, P < 0.01 comparedwith lane 2 or 4. LM, phase-contrast light microscopic image. Theresults are shown as the mean ± SD for triplicate experimentsin which ∼100 cells per experiment were counted. Bars, 10µm.

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)

Expression of Kank inhibits lamellipodial development mediated byactive Rac1 through IRSp53. (A) Effect of the expression ofKank on active Rac1-induced formation of lamellipodia. 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 lamellipodia is shown on the right as a percentageof the total number of transfected cells. *, P < 0.001compared with lane 3. (B) Overexpression of IRSp53 abolished the effectof Kank on Rac1G12V-dependent lamellipodial formation. NIH3T3cells were transfected as indicated, treated as described in Materialsand methods, and stained for Kank (red) and human IRSp53 or its mutants(purple). GFP is shown in green. The number of cells spreading withlamellipodia is shown as in A. *, P < 0.001 compared withlane 1; **, P < 0.01 compared with lane 1 or 4.IRSR,Q/E, human IRSp53 with arginine replaced by glutamicacid at position 11 and glutamine replaced by glutamic acid at position23; IRSK/E, human IRSp53 with lysine replaced by glutamicacid at position 143. (C) KD of mIRS (mIRS-KD) decreasedRac1G12V-dependent lamellipodial formation. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and mIRS (endogenous) with human IRSp53(expressing transiently) or its mutants (red). The number of cellsspreading with lamellipodia is shown as in A. *, P <0.001 compared with lane 2; **, P < 0.01 comparedwith lane 2 or 4. LM, phase-contrast light microscopic image. Theresults are shown as the mean ± SD for triplicate experimentsin which ∼100 cells per experiment were counted. Bars, 10µm.
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Related In: Results  -  Collection

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fig4: Expression of Kank inhibits lamellipodial development mediated byactive Rac1 through IRSp53. (A) Effect of the expression ofKank on active Rac1-induced formation of lamellipodia. 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 lamellipodia is shown on the right as a percentageof the total number of transfected cells. *, P < 0.001compared with lane 3. (B) Overexpression of IRSp53 abolished the effectof Kank on Rac1G12V-dependent lamellipodial formation. NIH3T3cells were transfected as indicated, treated as described in Materialsand methods, and stained for Kank (red) and human IRSp53 or its mutants(purple). GFP is shown in green. The number of cells spreading withlamellipodia is shown as in A. *, P < 0.001 compared withlane 1; **, P < 0.01 compared with lane 1 or 4.IRSR,Q/E, human IRSp53 with arginine replaced by glutamicacid at position 11 and glutamine replaced by glutamic acid at position23; IRSK/E, human IRSp53 with lysine replaced by glutamicacid at position 143. (C) KD of mIRS (mIRS-KD) decreasedRac1G12V-dependent lamellipodial formation. NIH3T3 cells weretransfected as indicated, treated as described in Materials and methods,and stained for GFP (green) and mIRS (endogenous) with human IRSp53(expressing transiently) or its mutants (red). The number of cellsspreading with lamellipodia is shown as in A. *, P <0.001 compared with lane 2; **, P < 0.01 comparedwith lane 2 or 4. LM, phase-contrast light microscopic image. Theresults are shown as the mean ± SD for triplicate experimentsin which ∼100 cells per experiment were counted. Bars, 10µm.
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