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. 2005 Sep;25(17):7839-53.
doi: 10.1128/MCB.25.17.7839-7853.2005.

Loss of SMEK, a novel, conserved protein, suppresses MEK1 null cell polarity, chemotaxis, and gene expression defects

Michelle C Mendoza  1 , Fei Du, Negin Iranfar, Nan Tang, Hui Ma, William F Loomis, Richard A Firtel
Affiliations

Loss of SMEK, a novel, conserved protein, suppresses MEK1 null cell polarity, chemotaxis, and gene expression defects

Michelle C Mendoza et al. Mol Cell Biol. 2005 Sep.

Abstract

MEK/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1(-) (MEK1 null) and erk1(-) cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1(-) and erk1(-) chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene and protein (smkA and SMEK, respectively), whose loss partially suppresses the mek1(-) chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK, but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via a nuclear localization signal (NLS) at its C terminus. The importance of SMEK's nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1(-) and erk1(-) cells. The misexpression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner.

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Figures

FIG.1.
FIG.1.
Discovery and primary structure of SMEK (suppressor of mek1−). (A) Developmental morphology of KAx-3 (wild-type), mek1−, and mek1−/smkA− strains at the mound stage of development. (B) Comparison of full-length Dictyostelium (Dd) and human (Hs) (DictyBase gene DDB0188242 and GenBank gene ID 55671) SMEK domain structures. A/B indicates a conserved acidic/basic stretch. (C) Amino acid sequence alignment of SMEK homologs Dictyostelium discoideum SMEK (www.dictybase.org, DDB0188242), Homo sapiens SMEK-1 (GenBank gene ID 55671) (which is expressed as a full-length variant 1 and a C-terminally truncated variant 2), H. sapiens SMEK-2 (GenBank gene ID 57223), Xenopus laevis (Xl) SMEK (GenBank accession number XM 048092), Drosophila melanogaster (Dm) SMEK (GenBank gene ID 41675 and www.flybase.bio.indiana.edu falafel), and S. cerevisiae SMEK (GenBank gene ID 213192). The red box shows the N-terminal EVH1 domain, the blue box shows the DUF-625 domain, and the black box shows the C-terminal conserved acidic/basic stretch. Asterisks indicate the conserved Y and W found in other type I EVH1 domains. Residues identical between the homologs are highlighted in yellow, incompletely conserved residues are in blue, and similar residues are in light green.
FIG. 2.
FIG. 2.
smkA− and Ax3/SMEKOE development and chemotaxis profiles. (A) Developmental morphology of KAx-3, mek1−, smkA−, mek1−/smkA−, smkA−/SMEKOE, KAx-3/SMEKOE, and mek1−/SMEKOE strains. Development at 9 h (wild-type mound stage), 16 h (wild-type slug stage), and 24 h (wild-type stalk and fruiting body stage) is shown. A 36-h time point is shown for the smkA−strain, rather than 24 h, because the smkA− cells do not form fruiting bodies until that time. (B) Representative traces of wild-type KAx-3, mek1−, smek−, mek1−/smek−, and SMEKOE strains moving up a gradient towards a pipette filled with 150 μM cAMP. Time-lapse recordings were taken at 6-s intervals; superimposed images show the cell shape, direction, and length of path at 1-min intervals.
FIG. 3.
FIG. 3.
smkA− and mek1−/smkA− cells exhibit defects in cytokinesis and myosin II assembly. (A) Quantitation of the percentage of cells with 1, 2, 3 or 4, 5 to 9, and 10 or more nuclei after growth in shaking culture for 1 and 5 days for KAx-3, smkA−, mek1−, and mek1−/smkA− strains. Error bars represent standard errors of the means for multiple experiments. (B) Kinetics of myosin II accumulation in the Triton X-100-insoluble fraction in response to cAMP stimulation. All values depicted are relative to the amount of myosin II at time zero for the wild-type KAx-3 reference cells. Error bars represent standard errors from multiple experiments.
FIG. 4.
FIG. 4.
SMEK localizes to the cell cortex in vegetative cells and translocates to the nucleus during starvation. Localization of full-length HA-SMEK and myosin II in vegetative and chemotaxing (cAMP-pulsed) KAx-3 cells is shown. Cells were labeled with DAPI (4′,6′-diamidino-2-phenylindole) stain (blue) to demarcate the nuclei and with HA (red) and myosin II (green) antibodies. Each image represents a deconvolved integration of multiple optical sections through the given cell sample. TRITC, tetramethyl rhodamine isocyanate; FITC, fluorescein isothiocyanate.
FIG. 5.
FIG. 5.
SMEK localization is regulated by its EVH1 domain and an NLS at the C terminus. (A) SMEK deletion constructs. All constructs are HA tagged. (B) Confocal images show the localization of SMEKΔEVH1 (red) in vegetative KAx-3 cells and cells pulsed with cAMP for 6 h. The merged vegetative cell image is superimposed on a phase-contrast image; the merged pulsed-cell image is superimposed on a myosin II-stained image (green) to demarcate the cell bodies. TRITC, tetramethyl rhodamine isocyanate; FITC, fluorescein isothiocyanate. DAPI, 4′,6′-diamidino-2-phenylindole. (C to E) Confocal images show KAx-3 cells expressing EVH1 (C), SMEKΔNLS (D), and SMEKΔC (E).
FIG. 6.
FIG. 6.
SMEKΔEVH1, SMEKΔNLS, and SMEKΔC development and chemotaxis profiles. (A) Developmental morphology of KAx-3 and mek1−/smkA− strains expressing SMEK, SMEKΔEVH1, SMEKΔNLS, and SMEKΔC. (B) Representative traces of strains moving up a gradient towards a pipette filled with 150 μM cAMP. Time-lapse recordings were taken at 6-s intervals; superimposed images show the cell shape, direction, and length of path at 1-min intervals. The KAx-3/SMEKOE trace is taken from Fig. 2B.
FIG. 7.
FIG. 7.
Expression profiles of 20 genes during early development. (A) Bar graphs for the expression profile of each gene in KAx-3, mek1−, erk1−, smkA−, and mek1−/smkA− cells represent the average change (n-fold). Absolute values for each target are at http://www.biology.ucsd.edu/loomis-cgi/microarray/Smek-array.html. The first gene listed, pdiA, is an early gene with SMEK-dependent expression after 4 h of development. The following genes are expressed precociously in mek1− and erk1− cells. Loss of SMEK reduces the level of precocious expression of 16 of the 19 genes but does not affect the remaining 3 out of 19 genes. (B) Northern analysis of pdiA, tagB, and tipB transcripts during development of KAx-3, mek1−, erk1−, smkA−, and mek1−/smkA− cells.
FIG. 8.
FIG. 8.
Model of the regulation of Dictyostelium chemotaxis by MEK1 and SMEK. MEK1 and SMEK are in separate signaling pathways that converge on chemotaxis function through gene regulation in the nucleus or unknown cytoplasmic targets.
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