Arg kinase-binding protein 2 (ArgBP2) interaction with α-actinin and actin stress fibers inhibits cell migration

Abstract

Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2-0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192-228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2.

Keywords: 14-3-3 Protein; Actin Stress Fibers; Actinin; ArgBP2; Cell Migration; Cytoskeleton; Protein kinase A (PKA); Tumor Metastasis.

FIGURE 1.

ArgBP2 localizes to actin stress fibers. A, schematic exon map of ArgBP2α, ArgBP2β, nArgBP2, and ArgBP2γ. Exon numbers are as indicated. The sorbin homology and SH3 domains, and conserved regions denoted A, B, and C as described in this study are marked accordingly. B, Western analysis of ArgBP2 expression in various cell lines. FLAG-ArgBP2γ was immunoprecipitated and run on 9% SDS-PAGE, then stained with Coomassie Blue to determine its mobility (top of panel). ArgBP2γ (red arrow) and ArgBP2α/β (blue arrow) were detected in HeLa and COS-7, whereas MDCK cell express only ArgBP2α/β. Typical staining patterns of F-actin and ArgBP2 in COS-7 cells (C) and HeLa cells (D) with detectable (yellow plus) or undetectable (blue dot) ArgBP2 expression are shown. Cells that are ArgBP2 positive cells present with thicker actin stress fibers. ArgBP2 is localized in distinct puncta along the dorsal and ventral actin stress fibers in HeLa cells. The fluorescent signal was inverted (white to black) for greater clarity. E and F, control U2OS cell (E) and GFP-ArgBP2γ localization (F) in U2-OS imaged by SIM. Distribution of the GFP-tagged protein closely resembles that of endogenous ArgBP2 in HeLa cells. GFP-ArgBP2γ expressing cells are characterized by denser and more cross-linked actin stress fibers. The red box marks the region enlarged to highlight the increased bundling at the puncta. The fluorescent signal is inverted (white to black) for greater clarity.

FIGURE 2.

Clonal analysis of ArgBP2 expression in COS-7 and HeLa cells. A, COS-7 cells demonstrate a mosaic expression for ArgBP2 as assessed by indirect immunofluorescence. Red and yellow dots denote ArgBP2 high and medium/low expression, respectively, and blue dots denote cells with undetectable ArgBP2 levels. The fluorescent signal is inverted (white to black) for greater clarity. B, HeLa and COS-7 colonies derived from single cells in culture were fixed and analyzed at day 6. The cell outlines (using binary mask of anti-α-actinin staining) illustrates the typical cell morphology of these clonal populations, with either detectable (ArgBP2 positive) or undetectable (ArgBP2 negative) expression. ArgBP2-positive colonies are more compacted and less dispersed than ArgBP2-negative colonies.

FIGURE 3.

ArgBP2 colocalizes with α-actinin-rich puncta along actin stress fibers. As indicated, HeLa cells were stained with (A) anti-ArgBP2 and anti-vinculin to stain focal adhesions or (B) anti-ArgBP2 and anti-α-actinin to stain actin stress fibers. Line scan analysis of signal from each channel was performed along the white arrow in the indicated direction. The white box shows a smaller region enlarged with the signal inverted to highlight the localization more clearly. The blue arrowhead in B highlights a ruffle at the cell edge where α-actinin is enriched but ArgBP2 is not detected. C, GFP-ArgBP2 expressing U2-OS cells were stained for endogenous Myosin IIa and imaged by SIM for better resolution. A line scan was performed along the white arrow in the indicated direction. The individual channels were inverted for greater clarity.

FIGURE 4.

ArgBP2 expression increases the level of actinin on stress fibers and increases the cross-linking and organization of the actomyosin network. COS-7 (A), HeLa (B), and U2-OS (C) cells were stained for endogenous α-actinin and for endogenous ArgBP2 in A and B or GFP-ArgbP2 in C. The detection of endogenous ArgBP2 in COS-7 (A) and HeLa (B) correlates with more α-actinin present along the actin stress fibers. C, U2-OS cells stably expressing GFP-ArgBP2 also increased the α-actinin recruitment to the actin stress fibers. Yellow plus signs denote ArgBP2-positive cells and blue dots denote ArgBP2-negative cells. The fluorescent signal was inverted (white to black) for greater clarity. The increase in intensity of α-actinin at these puncta was detected by selecting an identically sized region from ArgBP2 positive and negative cells. The background was subtracted for these regions in ImageJ and the signal intensity was quantified and plotted (D). These regions were then thresholded and objects were separated by a watershed algorithm in ImageJ. The area of the objects were calculated and plotted in E. Both graphs display one S.D.

FIGURE 5.

ArgBP2γ binds to actin stress fibers through an N terminally located sequence. A, the table summarizes the ArgBP2 construct localization in HeLa cells indicated as “−” (absent), “+” (weak), “++” (moderate), and “+++” (strong). B, the micrographs show representative images of FLAG-tagged proteins in HeLa cells. The full-length ArgBP2γ(1–573), the N-terminal half (1–340), and ArgBP2γ lacking regions A and C localized prominently to puncta along stress fibers. However, ArgBP2γ lacking domain B was concentrated in focal adhesions (blue arrows) with only residual localization to the actin stress fibers with no observable increase in puncta size. Domain B (last panel) was clearly localized to α-actinin-rich puncta on actin stress fibers (red arrows). The fluorescent signal was converted to black on white for clarity.

FIGURE 6.

ArgBP2γ region B interacts with α-actinin. A, the schematic illustrates the constructs used and amino acid sequence alignment of regions that are conserved (B in red and C in orange) comparing human, rat, and zebrafish ArgBP2. B, proteins encoding FLAG-tagged ArgBP2γ, ponsin, Vinexin, ArgBP2γ-ΔB, ArgBP2γ-ΔC, or ArgBP2γ-(S259A) as indicated were transfected into COS-7 cells. The 2% SDS-soluble cell lysate and 2% Triton X-100-soluble cell lysate were probed for either FLAG or α-actinin. The solubility of α-actinin was reduced only in the presence of ArgBP2γ and ArgBP2γ-ΔC. C, FLAG-tagged ArgBP2γ, ArgBP2γ-ΔB, ArgBP2γ-C, ArgBP2γ-(S259A), or ponsin were mixed with an equal volume of cell lysate containing GFP-α-actinin and subjected to anti-FLAG immunoprecipitation. GFP-α-actinin bound FLAG-ArgBP2γ only in the presence of domain B. Ponsin displayed poor binding to GFP-α-Actinin despite its ∼4-fold higher expression compared with ArgBP2γ. D, the schematic shows the domain structure of α-actinin. Various domains of α-actinin were tested for their ability to interact with endogenous ArgBP2; only CH1 appeared necessary to bind ArgBP2. E, FLAG-ArgBP2γ binding to α-actinin is enhanced by F-actin. The Triton X-100-soluble lysates were treated latrunculin A, phalloidin, or Ca²⁺ (10 μm) for 30 min prior to anti-FLAG immunoprecipitation (IP). F, the lysates from COS-7 cells expressing FLAG-ArgBP2, U2-OS cells transfected with either control siRNA or actinin siRNA were probed for actin and actinin levels. The actinin siRNA reduced the level of actinin by 70%. FLAG-ArgBP2γ containing lysates were mixed with those from control or α-actinin siRNA knockdown cells. The level of actin co-immunoprecipitated with ArgBP2γ was reduced when lysates contained less α-actinin. G, ArgBP2 was tested for F-actin co-sedimentation (see “Experimental Procedures”) with or without recombinant α-actinin (CH1 + 2). The top panel shows the blot for the GST-HA-ArgBP2 protein (as indicated) that includes the B domain, and the bottom panel shows recombinant GST-HA-ArgBP2 lacking domain B. The recombinant protein was clarified before incubation with buffer alone, with F-actin alone, or with F-actin + α-actinin(CH1 + 2) as indicated by the horizontal bar. Equal volumes of supernatant (S) or re-suspended pellet (P) fractions (100,000 × g, 1 h) were analyzed by SDS-PAGE and Western blotting.

FIGURE 7.

ArgBP2 binding to 14-3-3 is PKA dependent and interferes with α-actinin interaction. A, immunoprecipitated FLAG-tagged ArgBP2γ, ArgBP2γ-ΔA, ArgBP2γ-ΔB, ArgBP2γ-ΔC, or ArgBP2γ-(S259A) were probed for bound 14-3-3. The region C and, in particular, Ser-259 were required for 14-3-3 binding. B, a solid-phase phosphopeptide array contained 13-mer peptides with a central phosphoserine derived from human ArgBP2 with Cdc25c Ser-216 as control was probed for 14-3-3 binding. The sequences surrounding Ser(P)-207 and Ser(P)-259 were more efficient than the control Cdc25c peptide at binding 14-3-3. C, the effects of forskolin (25 μm) or H89 (20 μm) on 14-3-3 binding to FLAG-ArgBP2γ. An increase in 14-3-3 binding induced by forskolin was blocked by prior H89 treatment, confirming the involvement of PKA. Forskolin treatment typically results in a 4-fold increase in 14-3-3 binding (n = 4, error bars are 1 S.D.). D, cells expressing either ArgBP2γ or ArgBP2γ-(S259A) were treated with DMSO or forskolin (25 μm) for 30 min; Triton X-100-soluble lysate recovered from these cells was mixed with those from cells expressing (untreated) GFP-α-actinin, prior to immunoprecipitation (IP). ArgBP2γ-(S259A) did not bind 14-3-3 and, importantly, α-actinin binding was unaffected by forskolin treatment. E, cells transiently expressing either FLAG-ArgBP2γ(1–340) or FLAG-ArgBP2γ(1–340)-(S259A) were treated with DMSO or forskolin (25 μm) for 30 min. The 14-3-3 binding to ArgBP2γ(1–340) was increased by forskolin and the α-actinin binding was correspondingly reduced. The α-actinin binding to ArgBP2γ(1–340)-(S259A) was unaffected by forskolin as this mutant was unable to bind 14-3-3 even with forskolin treatment. F, cells expressing FLAG-ArgBPγ either untreated or treated with DMSO, forskolin (25 μm), or calyculin A (50 μm) for 30 min. Both forskolin and calyculin treatment increased 14-3-3 binding to FLAG-ArgBPγ and reduced α-actinin binding.

FIGURE 8.

Forskolin treatment leads to loss of ArgBP2 from stress fibers. A and B, a micrograph showing control cells treated with DMSO (A) and the effect of forskolin (B; 25 μm) on HeLa cells. Anti-ArgBP2, anti-α-actinin and phalloidin staining are shown; a loss of ArgBP2 from stress fibers is observed but the protein remains at focal adhesions. The fluorescent signals were inverted for greater clarity. The box marks the enlarged panel where stress fibers are clearly visible.

FIGURE 9.

Effect of forskolin on ArgBP2 localization. U2-OS cell lines stably expressing GFP-ArgBP2γ (A) or GFP-ArgBP2γ-(S259A) (B) were imaged with TIRFM to illuminate only the ventral stress fibers. The cells were imaged for 15 min before (gray box) forskolin (25 μm/ml) was added (orange dotted line), and imaged for a further 30 min after treatment (orange box). Two representative cells are shown for each condition. The large structures correspond to focal adhesions, whereas smaller puncta were distributed along stress fibers. Signals corresponding to GFP-ArgBP2-wt puncta in these arrays were often reduced after forskolin treatment, but not with GFP-ArgBP2γ-(S259A) cells. C, the intensity of signal along stress fibers (an example region of interest is highlighted by the magenta dotted box) was expressed as the percentage change from its original intensity and plotted in the graph (calculated across multiple cells, n = 8, error bars are S.E.). To determine the statistical significance of these observations, the intensity was averaged over the first 2 time points (pre-treatment) and the last 2 time points (30 min after treatment) as displayed in the graph. After forskolin treatment, there was a significant difference in the change in ArgBP2-wt (p = 0.0003) but not with the ArgBP2-(S259A) mutant.

FIGURE 10.

ArgBP2γ expression affects stress fiber turnover and cell migration inhibition by ArgBP2γ requires α-actinin binding. A, in our wound healing assay, control U2-OS cells and cells stably expressing GFP-ArgBP2 were plated at 100% confluence into a Chamlide 4-well device. After the cells were attached, the removal of the 4-well rubber separator created an even wound. The wound edges were tracked as depicted in the micrograph and the change in area was plotted for multiple wound areas (n = 6, error bars are 1 S.D.). The control cells invaded this wound at a rate 50% faster than cells expressing ArgBP2 (p < 0.0001). B, random cell migration tracks of U2-OS cell lines stably expressing vector, GFP-ArgBP2γ, GFP-ArgBP2γ-(S259A), GFP-ArgBP2γ-ΔB (a lacking α-actinin binding domain) or control cells. Cells were plated on a fibronectin-coated matrix for 2 h, then migration was monitored over 5 h (n = 40, error bars are 1 S.D.). Both GFP-ArgBP2γ and GFP-ArgBP2γ-(S259A) expressing cells exhibited on average a ∼50% reduction in migration speed relative to control cells (p < 0.0001 for both). The GFP-ArgBP2γ-ΔB cells exhibited migration behavior similar to control cells (p = 0.1948). C, model of ArgBP2 function. ArgBP2 is targeted to stress fibers through an interaction with α-actinin where it is able to increase cross-linking of the actomyosin network. Following PKA phosphorylation, ArgBP2 binds to 14-3-3, which displaces α-actinin interaction and its localization to the actomyosin network. The focal adhesions interaction is unaffected by PKA phosphorylation, which is mediated by the C-terminal SH3 domains. In metastatic cells where ArgBP2 is frequently lost, this regulation of stress fibers is absent causing less cross-linking by α-actinin and allowing for faster cell migration.