Identification of proteins associated with overexpressed His-myc hSSH-1l, 1S, 2S, and 3S in HEK 293 cells. Lysates from 10 cm dishes of HEK293 cells infected 48 h earlier with adenoviruses expressing His-myc hSSH-1L, 1S, 2S, and 3S were passed over nickel resin, washed with lysis buffer and eluted with lysis buffer containing 250 mM imidazole. (A) Immunoblots of proteins eluted at pH<8. (B) Immunoblots of proteins eluted at pH >8.3. (C) Domain organization of SSH-1L and LIMK1, showing the nomenclature of the parental fragments used in this study. Most of these fragments were tagged with His, myc, Flag (F), GST, GFP or HA when used for expression experiments.

Purified LIMK1 and hSSH-1L interact directly in vitro through the kinase domain of LIMK1 and the N-terminal half of hSSH-1L. (A) Silver-stained gel (left) and immunoblots of purified LIMK1 (right) (lane 1) and hSSH-1L (lane 2). In lane 3 the 2.5 M urea wash prior to elution of hSSH-1L was omitted. Note that the SSH-1L and LIMK1 preparations used for in vitro binding (lanes 1 and 2) are devoid of both actin and 14-3-3 immunoreactivity. Both showed minor contaminating bands by SDS–PAGE that are probably degradation products. (B) Nickel resin and glutathione-Sepharose pulldown assays of purified His-myc hSSH-1L and GST-LIMK1. The His-myc hSSH-1L is pulled down with glutathione beads only in the presence of GST-LIMK1, and the GST-LIMK1 is pulled down on nickel beads only in the presence of His-myc hSSH-1L. (C) Immunoblots of pulldown experiments showing direct interaction between bacterially expressed LIMK1 kinase domain (His-kinase) and truncated SSH (GST-SSH-t) proteins.

SSH-t downregulates the activity of LIMK1 towards cofilin by dephosphorylating Thr 508 and other phosphorylated serine residues. (A) Lysates of GST-LIMK1s expressing 293T cells were subjected to Western blotting before and after incubation with mammalian expressed purified GST-SSH-t or GST-SSH-t (CS). The filter was probed with anti-pLIMK1 antibody, stripped and reprobed with rat anti-LIMK1 mAbs. Incubation with SSH-t, but not with SSH-t (CS), resulted in decreased level of p-LIMK1. The numbers at the bottom represent the ratio between p-GST-LIMK1 and GST-LIMK1 as determined by densitometry. (B) Constitutively active LIMK1-EE508 and GST-LIMK1 were immunoprecipitated or pulled down from overexpressing mammalian cells and subjected to in vitro kinase assay. In the presence of bacterially expressed GST-SSH-t, the levels of p-LIMK1 and p-LIMK1-EE508 proteins were reduced by five- and three-fold, respectively. (C) Lysate of F-LIMK1-expressing cells was incubated with glutathione-bound GST-SSH-t, GST-SSH-t (CS) or GST expressed in mammalian cells. After centrifugation, the supernatants were precipitated with anti-Flag beads followed by in vitro phosphatase assay with [³²P]cofilin as a substrate. In the presence of GST-SSH-t, the levels of p-cofilin and p-LIMK1 were greatly reduced.

The phosphorylation state of hSSH-1L affects its binding to 14-3-3ζ, but not to F-actin. (A) SDS–PAGE analysis of pellets (400 000 g × 20 min) from an F-actin sedimentation (2.5 μM actin and 1.5 μM GST-14-3-3) along with hSSH-1L (single example of triplicates). hSSH-1L sediments only with F-actin (lanes 1 and 3) but not alone (lane 2). Lanes 5–6: numbers above lanes are order of addition. In lane 5, hSSH-1L and 14-3-3ζ were incubated together for 10 min prior to addition of the actin. In lane 6, the actin and hSSH-1L were incubated 10 min before adding 14-3-3ζ. (B) Dephosphorylated hSSH-1L does not bind 14-3-3ζ. After dephosphorylation with λ-phosphatase, the repurified His-hSSH-1L was incubated with GST-14-3-3ζ and reciprocal pulldowns were performed with nickel and glutathione resins. Immunoblots of the pulled down proteins were compared with the pull down of hSSH-1L before λ-phosphatase treatment. (C) 14-3-3ζ added to hSSH-1L isolated from mammalian cells (phosphorylated form) does not activate its phosphatase activity, nor does it inhibit its activity in the presence of F-actin. All assay time points shown are 15 min incubations. The pADF signal in the right two lanes was not visible at 30 s, so a 2 min exposure is shown. The signal remaining in samples plus and minus 14-3-3ζ is less than 5%.

Schematic representation of the regulatory interactions between the new players that regulate the actin cytoskeleton. PAK4 activates LIMK1 and inactivates SSH-1L by phosphorylation. SSH also plays a dual role. It dephosphorylates and inactivates LIMK1 and dephosphorylates and activates ADF/cofilin, resulting in a net increase in ADF/cofilin activity and actin filament turnover. The other players in this pathway are F-actin and 14-3-3ζ. F-actin is necessary for SSH-1L activity and the interaction of pSSH-1L and F-actin is modulated by 14-3-3ζ. Stimulatory (arrows) and inhibitory interactions (blocked lines) are shown.

Localization of LIMK1, hSSH-1L and 14-3-3 in neuronal growth cones and Saos-2 cells. (A) Endogenous LIMK1 and endogenous SSH-1L localization in the growth cone of an E18 rat hippocampal neuron. (a) Phase, and epi-fluorescence images of same growth cone labelled with (b) rat anti-LIMK1 and (c) rabbit anti-hSSH-1L (Supplementary Figure 1). (d) Overlay of fluorescence images. Bar in Aa for rows A and B=5 μm. (B) Endogenous SSH-1L and 14-3-3 in the growth cone of an E18 hippocampal neuron. (a) Phase, and epi-fluorescence images of same growth cone labelled with (b) 14-3-3 pan antibody and (c) rabbit anti-hSSH-1L. (d) Overlay of fluorescence images. (C) Saos-2 cells immunostained for endogenous (a, g, j) or overexpressed (d) SSH-1L, LIMK1 (b, e, h, k) and an overlay (c) showing little co-localization. (d–f) Saos-2 cells overexpressing hSSH-1L which now co-localizes with LIMK1 at focal adhesion (arrow) (d) as seen in the overlay (f). (g–l) Saos-2 cell expressing the constitutively active V12rac. Overlay in (i) shows co-localization to a cortical band (arrow in h) that contains F-actin (not shown) and to membrane ruffles (arrows in k) seen at a higher focal plane of a different cell (j–l). Bars=10 μm in a–f and 5 μm in j–l.

Analysis of LIMK1 and SSH interaction. (A) Interaction between endogenous LIMK1 and endogenous SSH-1L. MEF lysates immunoprecipitated with mouse anti-LIMK1 mAb were subjected to Western blotting with anti-SSH antibodies (top panel). The filter was reprobed with rat anti-LIMK1 mAb to show that LIMK1 was present in the IP and in the lysate (bottom panel). As negative control, the lysate was immunoprecipitated with IgG2. (B) Flag-tagged full-length (F-LIMK1) and the kinase domain of LIMK1 (F-kinase) but not F-LIM1&2 and F-PDZ domains interact with the full-length myc-SSH-1L. F-kinase bound 10 × more myc-SSH-1L protein than full-length LIMK1. The filter was reprobed with rabbit anti-myc polyclonal antibodies (middle panel) to demonstrate that equal amounts of myc-SSH were immunoprecipitated, whereas the bottom panel shows expression of the different LIMK1 proteins in lysates prepared from transfected COS-7 cells. (C) F-LIMK1 interacts with GST-SSH-t, the N-terminal A+B domain, and the phosphatase domain, but not with GST alone. (D) GST-SSH-t interacts with GFP-tagged active (GFP-LIMK1) or inactive full-length and kinase domains of LIMK1 but not with GFP alone. GFP-LIMK1M1 represents kinase-dead LIMK1 (D470A), GFP-K is GFP-tagged kinase domain, and GFP-Ks is the GFP-tagged dominant-negative splice variant kinase domain of LIMK1.

Slingshot is a LIMK1 phosphatase. Active and inactive myc-SSH-1L proteins were immunopurified with anti-myc mAb and GST-LIMK1 was affinity-purified with glutathione Sepharose. The purified proteins were incubated in the presence of 5 μCi [γ³²P]ATP for 30 min, followed by Western blotting and autoradiography. (A) The level of GST-LIMK1 phosphorylation is greatly decreased only in the presence of active myc-SSH-1L (top panel). The levels of GST-LIMK1 (middle panel) and myc-SSH (bottom panel) were analysed by Western blotting. (B) Time course of in vitro phosphatase assay with [³²P]LIMK1 as substrate. The relative level of phosphorylated LIMK1 was calculated from the ratio of p-LIMK1/LIMK1 values determined by densitometry in the presence of active SSH. The level of [³²P] released during the assay was measured and plotted. The level of [³²P]LIMK1 after 60 min incubation with myc-SSH-1L (CS) is shown as an open square and the level of [³²P] released as an open diamond. (C) Comparison of the activities of full-length and C-terminal truncated mammalian and bacterial SSH expressed proteins. Each point was calculated taking into consideration the amounts of protein present in the assay. (D) The effect of bacterially and mammalian expressed GST-SSH-t on the phosphorylation of HA-LIMK1 and HA-LIMK2 by in vitro kinase assay. Bacterially expressed SSH-t(B) is more active than SSH-t(M) expressed in mammalian cells (top panel). Note that SSH-t expressed in mammalian cells is phosphorylated, probably by a kinase that is present in the protein complex. After phosphorImaging, the filter was probed with anti-HA mAb (middle panel) and with rabbit anti-GST antibodies (bottom panel) to demonstrate the levels of HA-LIMK1&2 and the SSH proteins, respectively.

hSSH-1L is a phosphorylated on serine residues and phosphorylation inhibits its activity toward pADF and is necessary for binding 14-3-3ζ. (A) Coomassie Blue stained nickel-resin purified hSSH isoforms isolated from overexpressing HEK 293 cells (left panels) are phosphorylated as shown by staining with the Pro-Q Diamond phosphoprotein stain (right two panels). Treatment of hSSH-1L with bacterial λ-phosphatase for 60 min markedly reduced the phosphoprotein staining (far right panel) although the total protein loaded remains constant (Coomassie Blue panel). The images of the phosphoprotein stain have been inverted for easier visualization of the bands. (B) Phosphoamino analysis of GST-SSH-t, purified from overexpressing mammalian cells that had been labelled with [³²P]orthophosphate (top), and of bacterially expressed GST-SSH-t labelled in vitro by PAK4 and [γ³²P]ATP (bottom). (C) Following λ-phosphatase treatment, hSSH-1L is repurified on nickel resin and is devoid of phosphatase activity (including λ-phosphatase) in the absence of actin (far right lane). In the presence of actin, the mammalian cell derived hSSH-1L is active both before and after dephosphorylation. (D) Time course of dephosphorylation of pADF during incubation with hSSH-1L before and after treatment with λ-phosphatase. (E) PAK4 is a SSH-1 kinase. PAK4Δ is active as it increases phosphorylation of its known substrate LIMK1 by 1.8-fold. Phosphorylation of SSH-1L by an equal amount of PAK4Δ is even greater. (F) SSH-t phosphorylated in vitro by PAK4 is unable to dephosphorylate cofilin. [³²P]cofilin was greatly reduced by mammalian and bacterially expressed GST-SSH-t, but not when treated with bacterially expressed GST-SSH-t phosphorylated by PAK4Δ (top panel). The level of GST-SSH-t proteins was determined by probing with anti-GST (middle panel) and that of cofilin by staining with amido black.

Overexpressed SSH-1L alters cellular F-actin structure in a phosphatase-independent manner and co-expression of 14-3-3ζ inhibits these alterations. Overexpression of myc-SSH-1L (CS) results in abnormal accumulations that co-stain for F-actin and myc (A–C) but also for SSH-1L (D). Co-expression of SSH-1L (CS) and 14-3-3ζ (E) reduces abnormal structures and results in more normal slingshot distribution. Bars=10 μm.