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First published online April 16, 2004
doi: 10.1242/10.1242/jcs.00955

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Leukocyte-specific protein 1 targets the ERK/MAP kinase scaffold protein KSR and MEK1 and ERK2 to the actin cytoskeleton

¹ Cell Biology Programme, The Hospital for Sick Children Research Institute, Toronto, Ontario, M5G 1X8, Canada
² Toronto Western Research Institute, Cell and Molecular Biology Division, University Health Network, and Department of Immunology, University of Toronto, Toronto, Ontario, M5T 2S8, Canada

View larger version (69K): [in a new window]   Fig. 1. LSP1 interacts with ERK/MAP kinase pathway components. (A) W10 cells were lysed in 50 mM Tris pH 7.5, 5 mM EDTA, 0.5% CHAPS, and PKCßI, MEK1 and ERK2 were immunoprecipitated and analyzed for the presence of LSP1 by western blotting. No antibodies were added to control assays (cntrl). (B) Pull down experiments were performed using GST, GST-ERK2 or GST-MEK1 recombinant proteins and CHAPS-soluble W10 cell lysates and analyzed for the presence of LSP1. (C) To check recovery of GST and GST fusion proteins the same western blot shown in B was reprobed with an anti-GST antibody. All samples were analyzed on one gel but only the relevant portion of each lane containing the recovered GST or GST fusion protein is shown.

View larger version (65K): [in a new window]   Fig. 2. LSP1 associates with the ERK scaffold KSR. Immunoprecipitates of W10 cell lysates were prepared using anti-KSR (lane 3) or anti-MEK1 antibodies (lane 5). No antibodies were added to the control assay (lane 1). Precipitated proteins were first analyzed by western blotting for the presence of LSP1 (A) and subsequently for the presence of KSR (C). The bands visible in lanes 2 and 4 of A represent spill-over from the anti-KSR immunoprecipitate. The position of LSP1 in the anti-KSR and anti-MEK1 precipitates was identified using an anti-LSP1 immunoprecipitate (B). The position of KSR was identified using a whole cell extract of W10 cells (D). KSR, LSP1 and precipitating IgG are indicated at the right. Molecular mass markers (in kDa) are indicated at the left.

View larger version (42K): [in a new window]   Fig. 3. LSP1 targets KSR, MEK1 and ERK2 to peripheral actin filaments. The LSP1neg BW5147 cells (upper panels) and the LSP1+ transfectant T22 (lower panels) were stained for LSP1, KSR, MEK1, ERK2 and F-actin. Merged images show that LSP1, KSR, MEK1 or ERK2 (red) co-localize with F-actin (green) in the presence of LSP1 (lower panels) but not in the absence of LSP1 (upper panels). Some randomly chosen areas of co-localization indicated by a yellow color are identified by arrows. The insets show staining for LSP1 (blue). Scale bar: 10 µm.

View larger version (31K): [in a new window]   Fig. 4. LSP1-associated MEK1 is activated by anti-IgM. Anti-MEK1 (A) or anti-LSP1 (B) immunoprecipitates were analyzed for kinase activity using a peptide containing the MEK phosphorylation site of human ERK1 as substrate. Similarly, anti-ERK2 (C) or anti-LSP1 (D) immunoprecipitates were analyzed for kinase activity using an EGF-derived peptide. Precipitates were prepared from CHAPS-soluble lysates from unstimulated cells or from cells stimulated for 5, 10 or 20 minutes with 50 µg/ml anti-IgM (, W10; , TW10.1; , W10 cells pretreated for 20 minutes with 10 µM U0126). Results are expressed as the average fold increase over unstimulated cells±s.e.m. of three experiments. Anti-MEK1 (E) and anti-LSP1 (F) immunoprecipitates were assayed for MEK1 activity using recombinant His-ERK2 as substrate. Anti-ERK2 (G) and anti-LSP1 (H) were assayed for ERK2 activity using myelin basic protein (MBP) as a substrate.

View larger version (57K): [in a new window]   Fig. 5. ERK2 dissociates from the LSP1 complex after anti-IgM stimulation. (A) Anti-MEK1 and anti-ERK2 immunoprecipitates from W10 cells (unstimulated or stimulated for 20 minutes with 50 µg/ml anti-IgM) were analyzed for the presence of LSP1. No antibodies were added to control assays using unstimulated cells (cntrl). Equal recovery of MEK1 or ERK2 in unstimulated and stimulated samples was confirmed by western blot analysis using anti-MEK1 (B) or anti-ERK2 (C) antibodies. (D) Anti-LSP1 immunoprecipitates from unstimulated or anti-IgM-stimulated cells were analyzed for the presence of ERK2 without (lanes1-3) or with pretreatment of the cells with 10 µM U0126 (lanes 4-6). Recovery of LSP1 was determined by western blotting of parallel samples using anti-LSP1 (E).

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