A new identity for MLK3 as an NIMA-related, cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization

Abstract

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.

Figure 1

MLK3 and NIMA are similar, especially in the C-terminal regions. (A) The amino acid sequence of NIMA's C-terminal extension (aa 281–699) was aligned pairwise with the corresponding regions, as indicated, of the NIMA-related kinases and with MLK3 using the Lipman-Pearson algorithm (DNASTAR software). The percent of identical and similar residues for each alignment is indicated. (B) The Lipman-Pearson alignment of NIMA (aa 281–699) with MLK3 (365–847). The N (MLK3 aa 365–401 and NIMA aa 281–324) and C (MLK3 aa 369–847 and NIMA aa 371–699) terminal sequences of these regions, although included in the alignment, were not of sufficient similarity, as determined by this program, to be aligned and so were omitted in the figure. Identical residues are indicated by black-shaded boxes, similar residues by gray-shaded boxes. (C) Schematic alignment of NIMA and MLK3 protein subsequences, drawn to scale (amino acid position).

Figure 2

Cell cycle stimulation of MLK3's phosphorylation state and kinase activity in the absence of JNK activation. HeLa cells were synchronized at G₁/S by a double-thymidine block (0) and then released into media lacking thymidine. Samples were collected at subsequent times for 14 h and analyzed as described. (A) Flow cytometric quantification of DNA content per cell (x-axis, propidium iodide stain) vs. cell number (y-axis). Distribution of DNA content of cells from an asynchronous culture is also shown (Asynch). Twenty micrograms of each sample was separated by SDS-PAGE and subjected to immunoblot analysis using anti-MLK3 (B) or antiphosphorylated histone H3 (D). (C) Full-length immunoblot of MLK3 from 0- and 10-h samples. Molecular weight markers are indicated on the left. (E) Anticyclin B1 immune precipitation/kinase assay analysis (100 μg/sample) using histone H1 as substrate. (F) Immunoblot of MLK3 in G1/S (0 h) and G2/M (10 h) samples that were incubated with λ-phosphatase in the presence or absence of the phosphatase inhibitor, EDTA, or were left untreated. (G, upper left panel) MLK3 kinase activity toward myelin basic protein (MBP) was determined for G₁/S (0 h) and G₂/M (9 h) cell extracts after immunoprecipitation with anti-MLK3 (I) or with nonimmune (N) antibodies. (G, lower left panel) Average values of MLK3 immune precipitation/kinase assays from six different synchronous cultures collected at G₁/S (0 h) and G2/M (9, 10, 11 or 12 h, depending on culture). Shown is the average fold stimulation of activity from G2/M samples (2.4 times) relative to G₁/S (set at 1) ± the SD (0.67). (G, right panels) Immunoblots of MLK3 and actin in G₁/S and G₂/M samples before (Tot) and after (Post IP) immunoprecipitation with anti-MLK3 (I) or with nonimmune (N) antibodies. (H) Immunoblot of samples from (A) reacted with anti-JNK antibodies. Twenty micrograms of a sample collected from an asynchronous HeLa culture treated with anisomycin for 20 min was also included (Anis). (I) Anti-JNK immune precipitation/kinase assay analysis (100 μg/sample) of the samples in (H) using GST-N-Jun as substrate. As part of the JNK assays, the anisomycin-treated sample also was subjected to immune precipitation with the anti-JNK antibody (I) or with a nonimmune control antibody (NI) followed by incubation in kinase assay buffer. Products of the kinase reactions (E and H) were separated by SDS-PAGE and substrate incorporation of ³²P was detected by phosphorimager exposure.

Figure 3

MLK3 is localized near the centrosome in a cell cycle–dependent manner. Asynchronous HeLa cells growing on coverslips were fixed with methanol and then triple stained for MLK3 (left panels; a, d, g, j, m, and p), γ-tubulin (middle panels; b, e, h, k, n, and q), and DNA (right panels; c, f, i, l, o, and r). Samples p, q, and r were stained in the presence of MLK3-blocking peptide. Representative cells from different cell cycle stages are shown: interphase (a–c); prophase (d–f); metaphase (g–i); anaphase (j–l); telophase (m–o). Scale bar, 10 μM, applies to all images of a given field.

Figure 4

Localization of the centrosome-associated MLK3 sprinkles is partially dependent on microtubules. Asynchronous HeLa cells growing on coverslips were either: 1) untreated (a and d); 2) incubated in the presence of nocodazole (6 μg/ml) first on ice for 45 min and then at 37°C for 1 h (b and e); or 3) treated with taxol (5 μM) for 4 h (c and f). Cells were fixed in methanol and stained for MLK3 (a–c) and α-tubulin (d–f). Arrowheads in e denote brighter spots of α-tubulin staining indicative of centrosomes. The reduced levels of MLK3 staining after treatment with cold/nocodazole or taxol required longer photographic exposures for imaging leading to a commiserate increase in the level of cytoplasmic staining seen in b and c relative to a. Scale bars, 10 μM, applies to all images of a given field.

Figure 5

MLK3 overexpression disrupts microtubule organization. HEK293 cells containing a stable, inducible, wild-type MLK3 expression plasmid grown on coverslips were incubated with tetracycline (1 μg/ml; Ac and d, Bc and d; Induced) or vehicle (ethanol) alone (0.1%; Aa and b, Ba and b; Uninduced) for 23 h. Cells were then fixed in methanol and stained for α-tubulin. (A) metaphase cells. To detect maximal immunofluorescence signal in the astral area, images were collected using conventional microscopy (Aa and c). For clarity, confocal images (antibody and d) were also collected. (B) interphase cells. Shown are representative single fields of both uninduced (Ba and b) and induced (Bc and d) cells from which confocal optical slices were captured at two different depths. Ba and c are images focused close to the topmost cellular peripheries, whereas Bb and d are images focused 2–3 μM more internally, toward the cell centers. Scale bars, 10 μM, applies to image pairs Aa and c, Ab and d and to all images of a given field in B.

Figure 6

Comparison of MLK3 properties with those of NIMA. (A) Homogenates made from HEK293 cells (Tot) were separated by centrifugation into a soluble (Sup) and insoluble fraction (Pel). The soluble fraction was incubated with either GST-Pin1 beads or GST-beads, which were then washed extensively before the addition of SDS sample buffer. Homogenate fractions as well as bead-bound material were subjected to immunoblot analysis using MLK3-specific antibodies. The bead-bound material shown (GST-Pin1 and GST) was derived from ∼10 times the volume of soluble extract shown in the Sup sample. (B) HEK293 cells were transfected with HA-tagged MLK3 (a and b) or NIMA (c and d) expression constructs for 9 h, fixed with methanol, and stained for HA (a and c) and γ-tubulin (b and d). The γ-tubulin–staining centrosomes are indicated by the closed arrowheads. (C) HEK293 cells were transfected with HA-tagged MLK3 (a–c) or NIMA (d–f) expression constructs for 33 h, fixed with methanol, and stained for DNA (a and d), HA (b and e), and phospho-histone H3 (c and f). Transfected cells are indicated by closed arrowheads, and untransfected prometaphase cells are indicated by open arrowheads. Scale bar, 10 μM.

Figure 7

siRNA depletion of MLK3 increases cell sensitivity to taxol. (A) Immunoblot of untreated SAOS2 cells (Untr.) and those transfected with MLK3 siRNA duplex or GL2 luciferase siRNA duplex (nonspecific siRNA control) and then incubated for 45 and 69 h. The top portion of the immunoblot was probed with anti-MLK3 antibody and the bottom portion with antiactin antibody to check for equal loading of total protein. (B) SAOS2 cells were transfected with MLK3 siRNA or GL2 luciferase siRNA for 54 h before the addition of taxol at increasing concentrations. After a further 16-h incubation, cells were fixed and prepared for immunofluorescence microscopy using antiphospho-histone H3 antibodies. The individual cells from three fields of each sample (125–530 cells/field) were examined under low power and the percentage of phospho-histone H3-positive cells determined, the average of which is plotted ± the SD.