The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK - PubMed (original) (raw)

The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK

Suresh K Alahari et al. EMBO J. 2004.

Abstract

Nischarin, a novel intracellular protein, was originally identified as a binding partner for the alpha5beta1 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 alpha5beta1 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.

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Figures

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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 the transfected proteins.

Figure 7

Figure 7

In vitro binding of Nischarin and PAK1. (A) In vitro binding of Nischarin N-terminus to GST-PAKs. The 6xHis-tagged N-terminus and the transcription factor TGV-6xHis were expressed in the E. coli Expressway™ In Vitro Protein Synthesis System. Lysates containing the N-terminus-6xHis or TGV-6xHis were incubated with GST-PAK1-248-545-K299R or GST and bound proteins were isolated on glutathione–Sepharose 4B beads. The immunoblots were probed with anti-6xHis and anti-GST monoclonal antibodies. (B) Binding of rabbit reticulocyte expressed Nischarin N-terminus to GST-PAKs. The N-terminus Myc fragment was expressed in the TnT® rabbit reticulocyte transcription/translation system. Binding to GST fusion protein was conducted as in (A). The immunoblots were probed with anti-Myc and anti-GST monoclonal antibodies.

Figure 8

Figure 8

Interaction of Nischarin with PAK4 and PAK5. Nischarin-EGFP or EGFP and Myc-PAK1, Myc-PAK4 or Myc-PAK5 were coexpressed in Cos-7 cells. The PAK proteins were immunoprecipitated from lysates via the Myc epitope. The immunoprecipitates were immunoblotted for the presence of Myc and EGFP epitopes. The left column demonstrates binding of Nischarin-EGFP to PAK1, PAK4 and PAK5 (upper panel), whereas EGFP alone did not co-precipitate with the various PAKs (mid-panel). The right panels are loading controls.

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