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doi: 10.1242/10.1242/jcs.00174

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Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

Catherine S. Chew^*, Xunsheng Chen, John A. Parente, Jr, Shannan Tarrer, Curtis Okamoto and Hai-Yen Qin

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-3175, USA
Present address: Genzyme Corporation, One Mountain Road, Framingham, MA 01701, USA
Present address: Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA

View larger version (20K): [in a new window]   Fig. 1. Native and recombinant his-tagged lasp-1 co-sediment with purified non-muscle F-actin but GST-tagged lasp-1 does not co-sediment with G-actin in cell lysates. (A) Coomassie-blue-stained SDS-PAGE gel showing typical actin co-sedimentation assay. Lasp-1 (8 µM, arrow) or -actinin (2 µM, star, positive control) were incubated with actin (23 µM, arrowhead). After actin polymerization, supernatants (lanes 1-5) and pellets (lanes 6-10) were prepared and resolved by electrophoresis. Lanes 1,2,6,7: lasp-1 without F-actin. Lanes 3,4,8,9: lasp-1+F-actin. Lanes 5,10: -actinin+F-actin. Std: BioRad precision Mr standard (100, 75, 50, 37, 25 kDa). Note that his-tagged lasp-1 migrates with an apparent molecular mass of 38 kDa. Previous analyses with less precise Mr standards reported an apparent molecular mass of 41 kDa for his-tagged lasp-1 and 40 kDa for native lasp-1 (Chew and Brown, 1987; Chew et al., 1998). (B) Western blot of actin co-sedimentation of duplicate samples of endogenous lasp-1 in parietal cell extracts. Lasp-1 was co-sedimented with 7.5 µM F-actin as described in Materials and Methods. Similar results were obtained in three independent experiments. (C) Quantitation of lasp-1 association with F-actin at different concentrations of lasp-1. The F-actin concentration was 14 µM. Corrections for nonspecific precipitation of his-tagged lasp-1 were performed for each concentration. Values are means±s.e.m. for n=4 independent experiments. (D) Western blot of GST pull-down assay fractions using an actin antibody showing that similar amounts of actin in samples with GST-sepharose vs GST-tagged lasp-1. As expected, no signal was detected in the absence of parietal cell lysate. Similar results were obtained in four independent experiments. See Materials and Methods for details.

View larger version (20K): [in a new window]   Fig. 2. Time course of phosphorylation of lasp-1 by cAMP-dependent protein kinase and tentative identification of the major in vitro phosphorylation sites. (A) Top panel: diagram showing location of the two known cAMP-dependent protein kinase consensus sites in nebulin repeat region. Lower panel: time course of phosphorylation of his-tagged lasp-1 by a recombinant subunit of cAMP-dependent protein kinase (see Materials and Methods for details). (B) Chromatogram of tryptic digest of lasp-1. 20 µg of his-tagged lasp-1 was phosphorylated with recombinant catalytic subunit of cAMP-dependent protein kinase using [-32P]ATP as a substrate. Radiolabeled protein was isolated on an SDS-PAGE gel, subjected to in-gel tryptic digest and labeled peptides resolved on a SMART system with an acetonitrile gradient (flow rate, 100 µl/minute). Peaks shown in the figure were further resolved using a slower flow rate (50 µl/minute) then microsequenced. Parentheses in the sequences locate the predicted position of arginine and other residues present in the deduced lasp-1 sequence. Since trypsin normally cleaves at arginine residues, these amino acids were not expected, nor were they identified in sequencing analyses. However, because it is known that trypsin does not readily cleave R-X-Ser(P), it is likely that the tryptic fragment in the second peak contained the RRDS sequence in lower abundance compared with the MGPSGGEGAEPE fragment.

View larger version (30K): [in a new window]   Fig. 3. Serine to alanine mutations of the predicted cAMP-dependent protein kinase consensus phosphorylation sites in lasp-1 inhibits the phosphorylation of lasp-1 by cAMP-dependent protein kinase in vivo and in vitro. (A) For in vitro analyses, his-tagged wild-type and mutated lasp-1 were phosphorylated and resolved on an SDS-PAGE gel as described in Fig. 2. Inset shows autoradiographic data. Bands were excised from the gel and radiolabel incorporation quantitated by Cerenkov counting (graph). Mutations were as follows: RRDA, Ser99; RGFA, Ser146; R/R, both sites mutated. Values are expressed as a percentage of total counts present in wild-type lasp-1. (B) Western blot analysis of expressed wild-type (WT) and mutated (RGFA146, RRDA99) lasp-1 constructs following transfection of pcDNA3 vectors into MDCK cells. Transfected and mock-transfected (Mock, empty vector) cells were incubated with DMSO vehicle or forskolin (10 µM, 15 minutes) and lysed; extracts were analyzed using the lasp-1 mab as described in Materials and Methods. Mr band shifts are known to correlate with increased phosphorylation in vivo and in vitro (Chew et al., 1998; Chew et al., 2000). (C) Two dimensional western blot analyses of extracts prepared from transfected MDCK cells as described in panel B and Materials and Methods. Note the acidic shift in the RRDA but not the double (R/R) mutant following forskolin stimulation. These data support the conclusion that both Ser99 and Ser146 are in vivo phosphorylation sites.

View larger version (17K): [in a new window]   Fig. 4. Phosphorylation of lasp-1 with PKA inhibits F-actin co-sedimentation. (A) Representative Coomassie blue-stained SDS-PAGE gel containing supernatants and pellets from an actin co-sedimentation assay. Arrow shows location of actin. Lanes 1,2,7,8: his-tagged lasp-1 alone; lanes 3,4,9,10: lasp-1 plus F-actin; lanes 5,6,11,12: phosphorylated lasp-1 + F-actin. (B) Scatchard analyses of lasp-1 and phosphorylated lasp-1 binding to F-actin. Values for each point represent averages from 4-5 independent experiments.

View larger version (66K): [in a new window]   Fig. 5. Subcellular distribution patterns of lasp-1 in gastric fibroblasts. (a,a',d) Lasp-1 immunoreactivity detected with Cy5-labelled secondary antibody; (b,b',e) F-actin staining with Oregon green phalloidin; (c,c',f) merged images (lasp-1 red; F-actin green). Bars, 10 µM (a,c); 5 µM (f).

View larger version (77K): [in a new window]   Fig. 6. Exposure of gastric fibroblasts to the adenylyl cyclase activator, forskolin, disrupts stress fibers and focal contacts and induces the translocation of lasp-1 from focal contacts to the cell interior. Control, a-c; forskolin, d-i. (a,d.g) lasp-1; (b,e,h) F-actin; (c-f) merged images (lasp-1, red; F-actin, green). Data are representative of five independent experiments. Bars, 10 µM.

View larger version (58K): [in a new window]   Fig. 7. In cultured gastric parietal cells, forskolin stimulation induces the recruitment of lasp-1 to the F-actin rich canalicular membrane region as well as to newly formed membrane extensions. Parietal cells in primary culture can be identified based on several different criteria including cell size and general morphology, autofluorescence when excited at lower wavelengths, lack of stress fibers or focal contacts, and the expansion of the canaliculi following secretagogue stimulation (Chew et al., 1989) (C.S.C., unpublished). (a,c,e,g) lasp-1; (b,d,f,h) F-actin. The cell in the top panel was treated with DMSO vehicle. Cells in the lower panels were stimulated with forskolin (10 µM, 30 minutes). Panels e-h contain images of the same cell acquired from the mid-section (e,f) and near the base (g,h). Arrows indicate position of expanded intracellular canaliculi. Arrowheads, newly formed cell extensions. Data representative of six independent experiments. Bars, 10 µM.

View larger version (35K): [in a new window]   Fig. 8. In transfected parietal cells, expression HA-tagged lasp-1, forskolin stimulation also induces the recruitment of this protein to the canalicular region. However, mutation of the two major cAMP-dependent phosphorylation sites, S99 and S146, to alanines appears to block this cAMP-dependent process and may also inhibit the acid secretory response. Panels a,c,e,g show signals derived from HA-tagged lasp-1 construct (a,c) and for HA-tagged double S99/S146 (RRDA/RGFA) mutants (e,g). Corresponding F-actin signals in the same cells are shown in panels on the right. Arrows indicate location of expended intracellular canaliculi; arrowheads indicate intracellular canaliculi in non-transfected cells. Note non-transfected cell in panel h (actin signal only)is clearly stimulated by forskolin addition but the transfected cell is not. Data representative of six independent experiments. Bars, 10 µM.

View larger version (90K): [in a new window]   Fig. 9. In gastric mucosal fibroblasts, lasp-1 is recruited to regions of active membrane-cytoskeletal rearrangements following the activation of cAMP-independent signaling pathways. Gastric fibroblasts in mixed gastric mucosal primary cultures were growth-factor deprived for 24 hours, treated with 100 nM PMA (0 minutes, a-c; 2 minutes, d-f; 5 minutes, g-i; 10 minutes, j-l) and then fixed and stained for lasp-1 and F-actin as described in Materials and Methods and Fig. 5. Left panels, lasp-1; center panels, F-actin; right panels, merged images (lasp-1, red; F-actin, green). Arrows indicate locations of microspikes and nascent focal complexes. Data representative of three independent experiments. Bars, 5 µM.