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Phosphorylation of MAP4 affects microtubule properties and cell cycle progression

¹ Departments of Biological Sciences, Anatomy & Cell Biology, and Pathology, Colleges of Arts & Sciences and Physicians & Surgeons, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027-2450, USA
² Integrated Program in Cell, Molecular & Biophysical Studies, College of Physicians & Surgeons, Columbia University, 1212 Amsterdam Avenue, New York, NY 10027-2450, USA
³ Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachiohji, Tokyo 192-0397, Japan
^* Author for correspondence (e-mail:jcb4{at}columbia.edu )

View larger version (45K): [in a new window]   Fig. 1. Conservation of phosphorylation site sequences within the MAP4 MT-binding domain. Comparison of amino acid sequences from human (Chapin et al., 1995), mouse (West et al., 1991), bovine (Aizawa et al., 1990), and Xenopus (Shiina et al., 1999) shows conservation of serines among species (696 and 787 positions in human MAP4 sequence), residing within proline-directed consensus sites for cyclin B-cdc2 kinase phosphorylation, highlighted in boxes. Identical amino acids among species are represented by bars and deletions by asterisks. Note that the region surrounding ser-696 is more highly conserved than that around ser-787.

View larger version (63K): [in a new window]   Fig. 2. Mutation of ser-696 or ser-787 to alanine blocks their phosphorylation by cyclin B-cdc2 kinase. 2D phosphopeptide mapping of recombinant MAP4 bacterial proteins, showing wild-type (A) and wild-type mutated singly (B,C) or doubly (D) at residues 696 and 787 from serines to alanines and subjected to cyclin B-cdc2 kinase phosphorylation as described previously (Ookata et al., 1997).

View larger version (56K): [in a new window]   Fig. 3. Wild-type and mutant MAP4 colocalize with MTs in mouse Ltk- cells. (A) GFP fluorescence and anti-tubulin-stained images of Ltk- cells stably expressing WT-, KK-, AA-, or EE-MAP4 mutants. Cells were induced with dex for 36 hours, fixed in methanol, and stained with 3F3, a monoclonal tubulin antibody. Note that, because fixation decreases the brightness of GFP fluorescence, the micrographs shown accentuate images of bright, clustered or bundled GFP-EE-MAP4 MTs, compared with single MTs. By contrast, in live cell imaging, GFP fluorescence of single MTs was easily detected (data not shown), confirming that the MT distribution in GFP-MAP4 cells was indistinguishable from that of Ltk- cells. (B) Immunoblots of Ltk- cell extracts containing transfected MAP4 constructs. Two clones each of transfectants expressing wild-type (WT) and mutant MAP4 form were isolated, and 50 µg of each cell extract was immunoblotted with an antibody against human MAP4 (top lanes) and 3F3 tubulin antibody (bottom lanes). Live GFP fluorescence of EE-MAP4 from the last clone was visible under microscopy at low levels, but EE-MAP4 was not readily detected by western blotting. This clone of cells showed a very low level of expression (see Table 1 for details).

View larger version (35K): [in a new window]   Fig. 4. Cell cycle progression in Ltk- cells expressing WT and mutant MAP4 constructs. Cells grown on coverslips were fixed and expressing cells were quantified by scoring GFP-MAP4 fluorescence. Cells were scored for presence of: (1) two centrosomes with an intact nucleus (late G2 or G2/M transition); (2) mitotic spindle (M); or (3) midbody separating two daughter cells (early G1), as illustrated in the schematic diagrams and fluorescence images. Cells expressing WT- and mutant MAP4 species (2 clones for each) were scored for the proportion of the total GFP-MAP4-expressing cells that were in late G2, M, or early G1 phase.

View larger version (32K): [in a new window]   Fig. 5. Binding of mutant MAP4 forms to MTs. (A) WT-MAP4-expressing Ltk- cells before (ext) and after (H1P) a single step of Taxol-dependent MT polymerization (Chapin et al., 1991) The Coomassie-stained electropherogram shows an enrichment of the tubulin band at 50 kDa in the H1P fraction. (B) H1P fractions from WT- and KK-MAP4 cells (AA- and EE-MAP4 are not shown) were eluted with 0-0.6 M NaCl, and the eluted material was assayed by western blotting with an antibody against human MAP4 (top two panels) and an antibody against endogenous mouse MAP4 (bottom panel). Essentially all MAP4 was released with 0.6 M NaCl. (C) Quantification of MAP4 eluted from crude MTs (H1P) prepared from cells expressing transfected WT- and mutant MAP4 forms. Western blots such as those shown in Fig. 5B were scanned and the percentage of MAP4 released from H1P fraction at each salt concentration is shown. The MAP4 in each eluted sample was quantified and normalized to the total MAP4 that was released by elution with 0.6 M NaCl. nd indicates that no MAP4 was detected in this sample; error bars show ±s.d.

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