Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells

Abstract

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.

Figure 1

Co-localization of MEK1 and tubulin in various stages of the cell cycle in MCH603MEK1^act and MCH603 cells. (A) MCH603MEK1^act cells and B, MCH603 cells were stained for MEK1 (green), tubulin (red), and nuclei were stained with DAPI (blue). Bars, 5 μm. C, Co-localization coefficients in MCH603MEK1^act and MCH603 cells. *, p < 0.0087.

Figure 2

Co-immunoprecipitation, GST pull down studies and kinase assay of MEK1 and tubulin. A, Total protein lysates from sub-confluent MCH603MEK1^act cells were co-immunoprecipitated with MEK1, or tubulin, rabbit antibodies and then tested by immunoblotting with tubulin, and MEK1, mouse monoclonal antibodies. The input proteins were included as controls. B, Mapping of the tubulin-binding region in MEK1^act by deletion analysis. C, Characterization of ERK-tubulin interaction using GST pull down studies. D, In vitro kinase assay of GST-MEK^act using tubulin and ERK as substrates.

Figure 3

Peptide array binding analysis of MEK1^act to tubulin. A, Tubulin binding to MEK1^act peptide arrays. Aligned sequences of peptides 32-35, 41-43, and 65-69 (with MEK1^act residue numbers in parentheses) are also shown. B, Binding of tubulin to MEK1^act peptides having single alanine replacements. *, parent unsubstituted peptide. Arrows pointed to reduced binding. C, Location of tubulin-binding peptide regions on MEK1. The backbone structure of an N-truncated form of MEK1 [residues 61-382; (; PDB 1s9j)] is shown as a yellow ribbon with bound ATP (red) and the inhibitor PD184352 (magenta) included as stick models. Side chains of residues common to peptides 32-35, 41-43, and 65-69 are shown as space-filling spheres (white) with arginine and lysine residues colored cyan (carbon) and blue (nitrogen). The view in panel a is similar to that in Figure 4 of (41); the view in panel b is rotated 90° about the Y-axis. The figure was generated using VMD (42). D, Mutation of the key residues in the tubulin-binding site of MEK^act results in the loss of tubulin binding. Lanes 1 and 2, the input was 5 and 10 μg of GST-MEK^act, respectively. Lanes 3 and 4, the input was 5 and 10 μg of GST-MEK1^act-Q, respectively. Lane 5, the input was 10 μg of GST while in lane 6, the input was purified tubulin.

Figure 4

MEK1^act increased mitotic spindle defects and genomic instability in MCH603 cells. A, Flow cytometry analysis of cell DNA content. B, Chromosome ploidy analysis of MCH603 and MCH603MEK1^act cells. Bars indicate 2 μM. a, Pseudodiploid M-phase spread of an MCH603 cell; b, Polyploid and metaphase spreads of MCH603MEK1^act cells, with one showing an anaphase bridge (arrow head); and c, lack of cytokinesis in MCH603MEK1^act cell. C, Ploidy variations in mitotic cells of HT1080 and its variants. Values represent the percentages of diploid and polyploid cells in 400 total cells of each cell line.

Figure 5

The effect of R108A, R113A double mutation (MEK1^act-D) on phospho-ERK levels, kinase activity, interaction with tubulin and ploidy. A,a, Phospho-ERK (pERK) in MCH603-FLAG-MEK1^act and FLAG-MEK1^act-D cells was assayed by immunoblotting with pERK antibody. Fold difference indicates pERK levels relative to MCH603 cells. b, Total protein lysates from 293 HEK cells transiently transfected with FLAG-MEK1^act and FLAG-MEK1^act-D were immunoprecipitated with anti-FLAG M2-Affinity gel and their kinase activity was assayed with the MEK Activity Assay Kit. c, Total protein lysates from sub-confluent transfectants were co-immunoprecipitated with anti-FLAG M2 Affinity gel. The immune complexes were tested by immunoblotting with anti-tubulin and anti-MEK1 mouse monoclonal antibodies. Fold difference indicates the level of co-immunoprecipitated tubulin level relative to the FLAG-MEK1^act-D in IP sample. B, Ploidy of the indicated cell lines was measured by flow cytometry analysis of DNA content.

Figure 6

Effect of MEK1^act and MEK1^act-D on actin stress fibers. Actin stress fiber organization in indicated cell lines. Bar indicates 2 μM.