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• W2156867189 abstract "Article1 July 2004free access The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK Suresh K Alahari Suresh K Alahari Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Peter J Reddig Peter J Reddig Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Rudy L Juliano Corresponding Author Rudy L Juliano Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Suresh K Alahari Suresh K Alahari Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Peter J Reddig Peter J Reddig Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Rudy L Juliano Corresponding Author Rudy L Juliano Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Author Information Suresh K Alahari1,‡, Peter J Reddig1,‡ and Rudy L Juliano 1 1Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA ‡These authors contributed equally to this work *Corresponding author. Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA. Tel.: +1 919 966 4583; Fax: +1 919 966 5640; E-mail: [email protected] The EMBO Journal (2004)23:2777-2788https://doi.org/10.1038/sj.emboj.7600291 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nischarin, a novel intracellular protein, was originally identified as a binding partner for the α5β1 integrin. Here we show that Nischarin also interacts with members of the PAK family of kinases. The amino terminus of Nischarin preferentially binds to the carboxy-terminal domain of PAK1 when the kinase is in its activated conformation. Nischarin binding to PAK1 is enhanced by active Rac, with the three proteins forming a complex, while expression of the α5β1 integrin also increases the Nischarin/PAK1 association. Interaction with Nischarin strongly inhibits the ability of PAK1 to phosphorylate substrates. This effect on PAK kinase activity closely parallels Nischarin's ability to inhibit cell migration. Conversely, reduction of endogenous levels of Nischarin by RNA interference promotes cell migration. In addition, PAK1 and Nischarin colocalize in membrane ruffles, structures known to be involved in cell motility. Thus, Nischarin may regulate cell migration by forming inhibitory complexes with PAK family kinases. Introduction Cell migration is a complex process that requires the spatial and temporal coordination of many proteins. During migration, cells extend membrane protrusions, establish adhesive contacts, exert force to move the cell body, and ultimately retract the rear portion of the cell. These events involve actin filament extension regulated by the Arp2/3 complex, as well as actinomyosin-mediated contractility (Pollard and Borisy, 2003). Members of the integrin family of heterodimeric cell surface receptors also play a key role in cell migration. Integrins and certain associated cytosolic proteins provide a structural linkage between the proteins of the extracellular matrix and the actin cytoskeleton (Liu et al, 2000; Sastry and Burridge, 2000). Further, integrins help to regulate an intricate network of signaling pathways needed for the control of migration (Hood and Cheresh, 2002; Juliano, 2002). Members of the Rho family of GTPases are critically important in regulating the actin cytoskeleton (Bishop and Hall, 2000; Ridley, 2001). However, a number of other signaling components have been implicated in cell migration, including focal adhesion kinase, Src, Crk, PI-3-kinase and MAP kinases, as has been reviewed elsewhere (Alahari et al, 2002). The Rho family GTPases Rac and CDC42 regulate the formation of membrane protrusions involved in motility (Ridley, 2001). Among the key downstream effectors of CDC42 and Rac are members of the PAK family of serine/threonine kinases (Kumar and Vadlamudi, 2002; Bokoch, 2003). Based on their structures, PAKs have been divided into two groups: group 1 consists of PAK1–3, while group 2 consists of PAK4–6. Group 1 PAKs have a Rac/CDC42-binding domain and an overlapping autoinhibitory domain in the amino terminus, and a kinase domain in the carboxy terminus (Bagrodia and Cerione, 1999). Inactive group 1 PAKs exist as autoinhibited dimers. Upon GTPase binding, PAKs undergo a conformational change that separates the autoinhibitory domain from the kinase domain (Parrini et al, 2002). This induces kinase activity and autophosphorylation at several sites, including Thr 423 in the activation loop (Buchwald et al, 2001; Chong et al, 2001). Outside of the kinase- and GTPase-binding domains, group 2 PAKs are quite different from group 1 and their regulation may be distinct (Dan et al, 2002). PAKs are primarily localized in the cytoplasm in resting cells; however, activated PAKs translocate to focal adhesions and membrane ruffles (Sells et al, 2000). The exact mechanism of PAK regulation of the actin cytoskeleton and cell migration is not fully understood. PAKs phosphorylate and inhibit myosin light-chain kinase, leading to reduced actinomyosin contractility (Sanders et al, 1999). In addition, PAKs phosphorylate and activate LIM kinase leading to increased phosphorylation of cofilin, which plays a role in actin severing (Edwards et al, 1999) and regulation of actin filament turnover (Carlier et al, 1999). However, kinase-independent contributions of PAKs to cell motility have also been described (Sells et al, 1999). In addition to Rac and CDC42, group 1 PAKs also associate with a variety of other proteins including Nck, filamin, paxillin, Merlin, p41-Arc, G-protein βγ subunits, Cool/Pix exchange factors, and certain cytoplasmic tyrosine kinases (Manser et al, 1998; Xia et al, 2001; Brown et al, 2002; Feng et al, 2002; Kumar and Vadlamudi, 2002; Vadlamudi et al, 2002; Bokoch, 2003; Kissil et al, 2003; Vadlamudi et al, 2004). Among the negative regulators of PAK are several kinases, including protein kinase A (Howe and Juliano, 2000), while the phosphatases PP2A and POPX1/2 dephosphorylate PAK and thus inhibit its activity (Koh et al, 2002; Kumar and Vadlamudi, 2002). In summary, PAKs interact with the cytoskeleton in several ways that influence cell motility; conversely, a number of proteins associate with PAKs and regulate their functions. One important way in which integrins influence cellular behavior is by the interaction of their cytoplasmic tails with intracellular proteins (Liu et al, 2000). Many investigators have sought proteins that bind to integrin tail regions and might thus be downstream effectors. Pursuing this approach, we identified a novel protein termed Nischarin that interacts preferentially with the α5 cytoplasmic domain. Overexpression of Nischarin in fibroblasts led to changes in cytoskeletal organization and to a profound inhibition of cell migration (Alahari et al, 2000). Nischarin also strongly inhibited Rac-driven cell migration and invasion of carcinoma cells, with the observations suggesting that Nischarin might act primarily by affecting PAK (Alahari, 2003). In the current report we demonstrate that Nischarin binds selectively to PAKs via its N-terminus, with preferential binding to the ‘open’ conformation of the kinase. Nischarin also inhibits the ability of PAK1 to phosphorylate substrates. The ability of Nischarin to inhibit PAK kinase activity closely parallels its inhibition of cell migration. Further, Nischarin and PAK1 colocalize in areas of membrane ruffling associated with cell motility. Finally, the reduction of endogenous Nischarin levels results in enhanced cell migration via a process that seems to involve PAK activation. These studies suggest that Nischarin is a key regulator of PAKs in the context of cell migration. Further, since Nischarin also binds the α5β1 integrin, this may provide a means for localized control of PAK function. Results The N-terminal domain of Nischarin binds to the C-terminal domain of PAK1 Previous work on the effect of Nischarin on Rac-driven cell migration suggested a functional linkage between Nischarin and PAKs (Alahari, 2003). To determine if the link was physical, the ability of Nischarin and PAK1 to form intracellular complexes was examined. Nischarin is a large protein with an integrin α5-binding region as its only functionally defined domain (Alahari et al, 2000) (see Figure 1A). To begin to isolate the region responsible for binding PAK, Nischarin was initially subdivided into two large domains, the N-terminus (aa 1–802) and the C-terminus (aa 970–1354), with the sites of truncation chosen based on a predicted lack of secondary structure (http://www.embl-heidelberg.de/predictprotein/predictprotein.html). Most of the proline- and alanine-rich region, residues 803–969, was not included in either construct because truncated proteins containing this region were not stable (data not shown). Cos-7 cells were transfected with Myc epitope-tagged Nischarin constructs and V5 epitope-tagged PAK1; Nischarin was immunoprecipitated with anti-Myc antibody and the immunoprecipitates were probed for associated PAK1. The presence of PAK1 was readily detectable upon probing the immunoblots with the anti-V5 antibody in immunoprecipitates of full-length Nischarin or the N-terminus, but not the C-terminus (Figure 1B). Reciprocal immunoprecipitation of V5-PAK1 with the anti-V5 antibody co-immunoprecipitated Myc-Nischarin or the N-terminus (Figure 1C). The unrelated protein β-galactosidase bound neither PAK1 nor Nischarin in these assays. Figure 1.Nischarin/PAK1 interactions. (A) Nischarin domains. Regions homologous to known protein motifs as defined by BLAST analysis are shown. These include the leucine-rich repeats, leucine zipper motifs, potential SH3 binding sites (PXXP) and the cytochrome P450 cysteine heme-iron ligand signature. The integrin-binding domain (IBD) as defined in initial studies (Alahari et al, 2000) is also noted. (B, C) Nischarin's PAK-binding region. Cos-7 cells were cotransfected with Myc-Nischarin, Myc-Nis 1–802, Myc-Nis 970–1354, or Myc-β-galactosidase and V5-PAK1. At 48 h after transfection, the cells were lysed and the tagged proteins were immunoprecipitated with a 1:100 dilution of monoclonal anti-Myc or anti-V5 antibody. The blots were probed for the Myc and V5 epitopes. (D) Interaction of endogenous PAK and Nischarin. PC12 cells were lysed in modified RIPA buffer and lysates were immunoprecipitated with an agarose-conjugated rabbit polyclonal anti-PAK1 (N20) or a control agarose-conjugated IgG overnight at 4°C. Immunoblots were probed with a monoclonal anti-Nischarin antibody or an irrelevant mAb. (E) Effect of α5β1 on PAK/Nischarin interactions. V5-PAK1 and Myc-Nischarin were expressed in Cos-7 cells with or without coexpression of the integrin α5 subunit. Immunoprecipitation and Western blotting were as described above. Download figure Download PowerPoint To isolate the region within the N-terminus that mediated complex formation, further deletion analysis was performed (Supplementary Figure 1). Thus the regions 1–415 and 416–624 exhibited strong binding, while 625–802 exhibited weaker binding. Division of the 1–415 region into two separate segments (1–217 and 218–415) disrupted PAK complex formation. Thus, several regions within the Nischarin amino terminus are able to contribute to the formation of PAK/Nischarin complexes. Supporting the importance of this interaction, the Nischarin–PAK association takes place between endogenous proteins as well as during protein overexpression. Thus immunoprecipitation of endogenous PAK from PC12 cells results in co-immunoprecipitation of endogenous Nischarin (Figure 1D). Since Nischarin interacts with the α5β1 integrin (Alahari et al, 2000), it is important to determine if the binding of Nischarin to PAK is affected by its interaction with integrin. As seen in Figure 1E, overexpression of the α5 subunit enhanced the binding of coexpressed Nischarin and PAK1. Further, all three components were found in the Nischarin immunoprecipitate, suggesting a simultaneous association between PAK, α5β1 and Nischarin. The regulatory domain of PAK1 spans residues 1–248 of its N-terminus, while amino acids from 248 to 545 comprise the kinase domain. To identify which region of PAK1 interacts with Nischarin, the two domains were separately expressed in Cos-7 cells along with full-length Myc-Nischarin. Nischarin interacted with the kinase domain of PAK1 (248–545), but not with the regulatory domain (Figure 2A); this was consistently seen in reciprocal immunoprecipitations of these proteins (Figure 2B). Since Nischarin specifically interacts with the kinase domain of PAK1, we hypothesized that the interaction of Nischarin with full-length PAK1 would be dependent on the activation state of the kinase. To test this, Myc-Nischarin was coexpressed with the PAK1 activation loop mutant V5-PAK1-T423E; the threonine to glutamic acid substitution mimics the phosphorylation of T423 and partially activates the kinase (Sells et al, 1997; King et al, 2000; Chong et al, 2001). Myc-Nischarin was also coexpressed with V5-PAK1-K299R, a kinase-dead version of the protein (Sells et al, 1997). Nischarin immunoprecipitated via its Myc tag showed enhanced co-precipitation of PAK1-T423E, as compared to wild-type (WT) PAK1, while co-precipitation of PAK1-K299R was dramatically reduced (Figure 2C). These data indicate that the activation of PAK1 facilitates its interaction with Nischarin. Figure 2.The Nischarin-binding domain of PAK1. (A, B) Nischarin interacts with the kinase domain of PAK1. Full-length V5-PAK1, V5-PAK-1-248 or V5-PAK1-248-545 was cotransfected with Myc-Nischarin. The extracts were immunoprecipitated with the anti-V5 (A) or anti-Myc (B) antibodies and the immunoprecipitates were immunoblotted for the indicated epitopes. (C) Nischarin interacts with active PAK1. Cos-7 cells were cotransfected with the Myc-Nischarin or Myc-β-galactosidase and V5-PAK1, constitutively active V5-PAK1-T423E, or kinase-dead V5-PAK1-K299R. After transfection, the Myc-tagged proteins were immunoprecipitated and the blots probed with anti-Myc and anti-V5 antibodies. Download figure Download PowerPoint PAK and Nischarin colocalize in membrane ruffles Since Nischarin both binds to PAK and affects cell migration, one might anticipate that PAK and Nischarin would be found together in subcellular compartments associated with cell movement. To test this, rat embryonic fibroblasts were cotransfected with GFP-Nischarin and with Myc-tagged PAK1. Transfected cells were plated on fibronectin substrata and the subcellular distributions of PAK1 and Nischarin were visualized by confocal fluorescence microscopy. Both PAK1 and Nischarin were widely distributed in the cytoplasm and seemed associated with vesicular structures in the perinuclear area. However, both proteins were also enriched in membrane ruffles, and image superposition clearly indicated regions of colocalization (Figure 3Aiii). When PAK1 mutants were examined, it was obvious that kinase-dead K299R PAK1 failed to show significant membrane colocalization with GFP-Nischarin (Figure 3B). By contrast, expression of the active T423E mutant resulted in enhanced colocalization with GFP-Nischarin (Figure 3C). Higher power images of cell protrusions suggested that PAK is present at the leading edge of the protrusion, while GFP-Nischarin overlaps with PAK just behind the edge (Figure 3Ciii′). These results show that PAK1 and Nischarin colocalize in ruffles, cellular structures known to be involved in cell migration, and that Nischarin preferentially localizes with active PAK1. Figure 3.Colocalization of PAK and Nischarin in membrane ruffles. Rat embryonic fibroblasts were transiently transfected with GFP-Nischarin and (A) Myc-PAK1 or (B) Myc-K299R-PAK1 or (C) Myc-T423E-PAK1. After serum starvation, the cells were replated on fibronectin-coated coverslips for 45 min, stained with anti-Myc antibody and observed using an Olympus confocal fluorescence microscope with a × 60 lens. (A) GFP fluorescence is shown in green (i); anti-Myc-PAK1 staining is shown in red (ii); an overlay image is shown in (iii) where yellow indicates colocalization of Nischarin and PAK1 (B, C) Similar images of GFP-Nischarin and (B) Myc-K299R-PAK1 or (C) Myc-T423E-PAK1. Images i′–iii′ show enlargements of part (yellow box) of the i–iii images for Myc-T423E-PAK1 (white arrowheads point to colocalization of PAK and Nischarin in ruffles; blue arrowheads show PAK staining at the far edges of the cell). Scale bar: 20 μm. Download figure Download PowerPoint Nischarin selectively inhibits PAK kinase activity Given that Nischarin preferentially associates with activated PAK1, we hypothesized that Nischarin may influence PAK kinase activity. To test this, we transiently cotransfected Cos-7 cells with V5-tagged PAK1 and with Myc-Nischarin or Myc-β-gal. Immunoprecipitated PAK proteins were tested for their kinase activity using myelin basic protein as a substrate. As shown in Figure 4A, serum stimulation strongly increased PAK kinase activity, while coexpression of full-length Nischarin blocked the increase in PAK activity; by contrast, coexpression of β-galactosidase had no effect. Interestingly, full-length Nischarin was required for effective inhibition of PAK kinase activity, whereas the amino-terminal (1–802) or carboxy-terminal (970–1354) fragments, or other fragments of Nischarin (data not shown) had no effect on PAK activation. Figure 4.Kinase activity assays. (A) Nischarin inhibits serum-stimulated PAK activity. Immunoprecipitates were made from Cos-7 cells transfected with the indicated constructs. The cells shown in lanes 2–5 were stimulated with serum. Upper panel: the immunoprecipitates were used in in vitro kinase assays using myelin basic protein (MBP) as a substrate; middle panel: the lysates were blotted with anti-Myc antibody; lower panel: the lysates were blotted with anti-V5 antibody. (B) Nischarin inhibits T423E PAK activity. Immunoprecipitates were made from Cos-7 cells transfected with the indicated constructs. Upper panel: the immunoprecipitates were used in in vitro kinase assays as above; middle panel: the lysates were blotted with anti-Myc antibody; lower panel: the lysates were blotted with anti-V5 antibody. (C) Nischarin does not affect JNK activity. Cell lysates made from Cos-7 cells transfected with the indicated combinations of pAX vector, HA-JNK1, pAX-RacQ61L and Myc-Nischarin were immunoprecipitated with anti-HA antibody and the immunoprecipitates were used to detect JNK activation as described (Alahari, 2003). Upper panel: phosphorylation of GST-JUN; lower panel: immunoblotting with anti-HA antibody. (D) Nischarin inhibits autophosphorylation of PAK1. This assay was similar to that of (A) except that the kinase assay was for 5 min and the gel was run to allow visualization of the PAK band. Upper panel: phosphorylation of V5-PAK1 and MBP; middle panel: PAK levels in the IP; lower panel: Nischarin or β-gal levels in the lysate. Download figure Download PowerPoint These experiments demonstrated that Nischarin blocks serum-mediated activation of PAK1. However, they did not distinguish between direct effects on PAK1 itself and blockade of the signaling pathway leading to PAK activation. To investigate whether Nischarin acts directly on PAK, a constitutive, partially activated form of PAK1 (PAK1-T423E) was used in kinase assays. As seen in Figure 4B, the kinase activity of immunoprecipitated PAK1-T423E was inhibited by Nischarin. Similar to the results with WT PAK1, the activity of T423E was inhibited only by full-length Nischarin, but not by truncated forms of Nischarin (data not shown). Thus Nischarin seems to inhibit directly the ability of PAK1 to phosphorylate substrates. To determine whether the effect of Nischarin on PAK kinase activity is selective, we examined the action of Nischarin on c-Jun kinase (JNK) (Bishop and Hall, 2000). Rac strongly increased JNK activity, but Nischarin had no inhibitory effect on this process (Figure 4C). This indicates that Nischarin selectively inhibits PAK activity without affecting other Rac-driven kinases. As an initial approach to understanding the mechanism by which Nischarin inhibits PAK1, we tested whether Nischarin blocks the autophosphorylation of PAK1 that is part of its activation process or, alternatively, whether Nischarin could serve as a competitive substrate for PAK1. As seen in Figure 4D, overexpression of Nischarin inhibited PAK autokinase activity in parallel with inhibition of substrate phosphorylation. Further, use of an antibody that recognizes the phosphorylated form of one of the autoactivation sites on PAK1 demonstrated that phosphorylation at this site was inhibited by Nischarin overexpression (Supplementary Figure 2). These observations suggest that Nischarin can inhibit the activation of PAK1. By contrast, we found little evidence that Nischarin could serve as a substrate for PAK1 and thus block phosphorylation of other substrates by competition. For example, partially purified, in vitro expressed Nischarin was not significantly phosphorylated by active GST-PAK1 under conditions where myelin basic protein was abundantly phosphorylated (Supplementary Figure 3). This suggests that Nischarin is a poor substrate for PAK1 and thus unlikely to compete with other substrates. Nischarin levels modulate PAK-induced cell migration As discussed above, PAK has been shown to play an important role in cell migration. Nischarin inhibits both cell migration (Alahari et al, 2000; Alahari, 2003) and PAK kinase activity; thus, it is important to understand the relationship between these two actions of Nischarin. To investigate this issue, we examined the effects of Nischarin and its fragments on migration stimulated by an active form of PAK1 (T423E). Thus, CHO B2 α27 cells (which express human alpha 5 integrin) were transfected with PAK1-T423E or with kinase-dead PAK (PAK1-K299R), and were cotransfected with Nischarin or the N-terminal (1–802) or C-terminal (970–1354) domains of Nischarin. Subsequent to transfection, cell migration assays were performed. As shown in Figure 5A, PAK1-T423E stimulated migration in the CHO cells, and this was strongly inhibited by overexpression of Nischarin. Interestingly, the kinase-dead version of PAK1 did not stimulate migration in this system. Consistent with our data on kinase inhibition, only full-length Nischarin was able to inhibit PAK1-T423E-driven migration (Figure 5A), while the N- or C-terminal domains had no effect. Thus there is a close parallel between Nischarin's ability to block PAK kinase activity and its ability to inhibit PAK-driven cell migration. Figure 5.Effects of Nischarin on PAK-induced migration. (A) Overexpression of full-length Nischarin inhibits PAK-driven migration. CHO B2-α27 cells were transiently transfected with vector alone, with V5-PAK1-T423E plus Myc-vector, full-length Myc-Nischarin, Myc-Nischarin (1–802) or Myc-Nischarin (970–1354). Other cells were transfected with V5-PAK1-K299R plus Myc-Nischarin or vector control. A β-gal plasmid was also used to mark all transfectants. Cells were plated in transwells, and the β-gal-expressing cells migrating through the transwells were counted. (B) Effects of siRNA on Nischarin levels. PC12 cells were transfected with pcDNA-CD4 and 150 nM anti-rat Nischarin siRNA or control siRNA (anti-human MDR1). At 48 h after transfection, the CD4-positive cells were selected with anti-CD4-coated Dynabeads®. The cells were lysed and equal amounts of protein were used for SDS–PAGE. An anti-Nischarin antibody was used for Western blotting. Two separate lanes are shown for cells treated with siRNA for Nischarin. (C) Effects of siRNA on cell migration. The haptotactic migration of PC12 cells was examined using a Transwell assay. Membrane inserts were coated with 10 μg/ml collagen. Cells were transfected with 150 nM anti-Nischarin siRNA or with control siRNA, as well as with a vector expressing β-gal. Some sets of cells were cotransfected with a construct that expresses the PAK1 autoinhibitory domain (AID). Cells were plated in transwells and the β-gal-expressing cells migrating through the transwells were counted. Results are the means and standard errors of six determinations. (*) The difference between the Nis siRNA and Nis siRNA+AID samples was significant at the 0.01 level. (D) Effects of siRNA on PAK activity. PC12 cells were transfected with 150 nM Nischarin siRNA or control siRNA. At 48 h after transfection, the cells were lysed and endogenous PAK was immunoprecipitated. The immunoprecipitate was used in an in vitro kinase assay with MBP as a substrate. The upper panel shows MBP phosphorylation, the middle panel the amount of PAK in the immunoprecipitate and the lower panel the amount of Nischarin in the lysate. Download figure Download PowerPoint Since overexpression of Nischarin inhibits PAK-driven cell migration, we wished to see if reducing endogenous levels of Nischarin would affect cell motility. Thus, PC12 cells (which have endogenous Nischarin) were transfected with an siRNA oligonucleotide targeted to Nischarin, or with a control oligonucleotide. The migratory ability of these cells was then tested. The anti-Nischarin siRNA, but not the control siRNA, caused a substantial reduction in the amount of endogenous Nischarin (Figure 5B). The anti-Nischarin siRNA, but not the control, also dramatically stimulated cell migration (Figure 5C). Interestingly, coexpression of a PAK fragment comprising the autoinhibitory domain significantly reversed the increase in migration caused by the anti-Nischarin siRNA. This domain can block PAK kinase activity, suggesting that the stimulation of migration caused by the siRNA was due to increased PAK activity. To pursue this further, we treated PC12 cells with anti-Nischarin siRNA or control siRNA, immunoprecipitated the endogenous PAK and tested its kinase activity. As seen in Figure 5D, transfection with anti-Nischarin siRNA caused an increase in the activity of endogenous PAK. It should be noted that only a fraction of the cells were transfected, so that only a portion of the total pool of PAK was affected by siRNA-mediated modulation of Nischarin. In summary, these results suggest that reducing levels of endogenous Nischarin can promote cell motility by permitting enhanced activation of PAK. The PAK/Nischarin interaction is modulated by active Rac and requires the open form of PAK Since the above observations suggest that the Nischarin–PAK interaction significantly affects cell migration behavior, we decided to further investigate other aspects of PAK/Nischarin binding. Binding of Rac1/CDC42 is an important step in the activation of PAK (Buchwald et al, 2001; Parrini et al, 2002). Since the data of Figure 2C suggest that Nischarin may bind preferentially to active PAK1, this implies that the binding of an active Rac1 to PAK1 should enhance the Nischarin/PAK1 interaction. Further, since introduction of a Y40C effector site mutation into Rac1 disrupts Rac1 binding to PAK1 (Westwick et al, 1997), this should reduce Rac's ability to enhance PAK1/Nischarin complex formation. As predicted, coexpression of active Rac1Q61L led to the enhancement of Nischarin co-immunoprecipitation with PAK1 (by about 3.5-fold) (Figure 6A and Supplementary Figure 4). By contrast, coexpression of mutant Rac1Q61L/40C resulted in less co-immunoprecipitation of PAK1 with Nischarin as compared to Rac1Q61L (Figure 6B). These data demonstrate that Rac activation of PAK1 enhances the formation of the Nischarin/PAK1 complex and this is dependent on the ability of Rac to bind to PAK1. Further, the presence of Nischarin does not inhibit the ability of PAK1 to bind Rac1, rather these proteins can form a tripartite complex. Figure 6.Effects of Rac and of PAK conformation on PAK/Nischarin binding. (A) Rac enhances Nischarin/PAK1 interaction. Myc-Nischarin and Myc-PAK1 were cotransfected into Cos-7 cells with HA-Rac1Q61L or pCGN vector control. After immunoprecipitation of PAK1 with polyclonal PAK1 antibody (N20), the levels of co-immunoprecipitating Myc-Nischarin were determined by Western blotting and were quantified on a Fluor-S MultiImager (Bio-Rad) and normalized to the vector control. The error bars show standard deviation (N=6). (B) Rac40C is less effective at promoting PAK–Nischarin interaction. Myc-Nischarin and V5-PAK1 were cotransfected with HA-Rac1Q61L or HA-Rac1Q61L/40C. Myc-Nischarin was immunoprecipitated from cytoplasmic lysates and immunoblots were performed to detect the indicated epitopes. (C) Rac increases the binding of both WT and kinase-dead PAK1 to Nischarin. Myc-Nischarin was coexpressed with HA-Rac1Q61L and with V5-PAK1 or V5-PAK1-K299R. The lysates were immunoprecipitated with anti-Myc and the immunoprecipitates blotted with antibodies to the indicated epitopes. (D) Nischarin binds to the open conformation of PAK1. Cos-7 cells were transfected with Myc-Nischarin or pcDNA and V5-PAK1, V5-PAK1-L107F, V5-PAK1-K299R or V5-PAK1-K299R-L107F. PAK1 was immunoprecipitated with the anti-V5 antibody. The input and immunoprecipitates were blotted for the respective epitope tags of t" @default.
• W2156867189 created "2016-06-24" @default.
• W2156867189 creator A5044182015 @default.
• W2156867189 creator A5049332865 @default.
• W2156867189 creator A5073729123 @default.
• W2156867189 date "2004-07-01" @default.
• W2156867189 modified "2023-10-18" @default.
• W2156867189 title "The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK" @default.
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