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. 2007 May;27(10):3817-27.
doi: 10.1128/MCB.02194-06. Epub 2007 Mar 12.

MEK1 and protein phosphatase 4 coordinate Dictyostelium development and chemotaxis

Michelle C Mendoza  1 , Ezgi O Booth, Gad Shaulsky, Richard A Firtel
Affiliations

MEK1 and protein phosphatase 4 coordinate Dictyostelium development and chemotaxis

Michelle C Mendoza et al. Mol Cell Biol. 2007 May.

Abstract

The MEK and extracellular signal-regulated kinase/mitogen-activated protein kinase proteins are established regulators of multicellular development and cell movement. By combining traditional genetic and biochemical assays with a statistical analysis of global gene expression profiles, we discerned a genetic interaction between Dictyostelium discoideum mek1, smkA (named for its role in the suppression of the mek1(-) mutation), and pppC (the protein phosphatase 4 catalytic subunit gene). We found that during development and chemotaxis, both mek1 and smkA regulate pppC function. In other organisms, the protein phosphatase 4 catalytic subunit, PP4C, functions in a complex with the regulatory subunits PP4R2 and PP4R3 to control recovery from DNA damage. Here, we show that catalytically active PP4C is also required for development, chemotaxis, and the expression of numerous genes. The product of smkA (SMEK) functions as the Dictyostelium PP4R3 homolog and positively regulates a subset of PP4C's functions: PP4C-mediated developmental progression, chemotaxis, and the expression of genes specifically involved in cell stress responses and cell movement. We also demonstrate that SMEK does not control the absolute level of PP4C activity and suggest that SMEK regulates PP4C by controlling its localization to the nucleus. These data define a novel genetic pathway in which mek1 functions upstream of pppC-smkA to control multicellular development and chemotaxis.

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Figures

FIG. 1.
FIG. 1.
Protein-protein interactions in the PP4 complex. Cells expressing various combinations of FLAG-PP4C, HA-SMEK, HA-SMEKΔEVH1, and/or HA-EVH1 as indicated (+, present) were lysed, and the proteins were immunoprecipitated with anti-FLAG (A) or anti-HA (B) antibodies. The immunoprecipitates were analyzed by Western blotting with the indicated antibodies. V, vegetative cells; P, cells pulsed with 30 nM cAMP for 5 h; IP, immunoprecipitation; IB, immunoblotting; WCL, whole-cell lysate.
FIG. 2.
FIG. 2.
The PP4 complex affects developmental morphology. Cells were developed on 12 mM Na+-K+ phosphate agar plates for the indicated times, and photographs were taken (bars = 1 mm). Developing wild-type cells form aggregates (9 h), protrude a mound tip (12 h), transform into a migrating slug (16 h), and differentiate into stalks and fruiting bodies (24 h).
FIG. 3.
FIG. 3.
The PP4 complex regulates chemotaxis. Images show representative tracings of cells moving towards a micropipette filled with 150 μM cAMP. Time lapse recordings were taken at 6-s intervals; superimposed images show the cell shapes and directions and the length of the path at 1-min intervals for 11 min.
FIG. 4.
FIG. 4.
Quantitative epistasis analysis. Quantitative gene expression and chemotaxis phenotypes of smkA−, pppC−, mek1−, mek1− smkA− double null, mek1− pppC− double null, and wild-type (KAx-3) cells during development were analyzed. (A) Hierarchical clustering dendrogram based on the differences (Euclidian distances) between the mutants’ microarray phenotypes. Each value at the branch splitting points is the P value calculated by multiscale bootstrap resampling. (B) Euclidian distances between microarray phenotypes of smkA−, mek1−, and mek1− smkA− double null cells were calculated and are presented as the edges of a triangle. Numbers indicate lengths (distances). P values for distances were calculated by random sampling (n = 1,000). (C) Euclidian distances between microarray phenotypes of pppC−, mek1−, and mek1− pppC− double null cells were calculated and are presented as the edges of a triangle. Numbers indicate lengths (distances). P values for distances were calculated by random sampling (n = 1,000). (D) Hierarchical clustering dendrogram based on the differences (Euclidian distances) between the mutants’ chemotaxis phenotypes (cell speed, directionality, and roundness and PP4C activity during chemotaxis). Values at the branch splitting points are the P values calculated by multiscale bootstrap resampling. (E) Euclidian distances between the morphology phenotypes of smkA−, mek1−, and mek1− smkA− double null cells were calculated and are presented as the edges of a triangle. Numbers indicate lengths (distances). P values were calculated by random sampling (n = 1,000). (F) Euclidian distances between morphology phenotypes of pppC−, mek1−, and mek1− pppC− double null cells were calculated and are presented as described in the legend to panel B. (G) A genetic pathway was inferred from the epistatic relationships.
FIG. 5.
FIG. 5.
Gene ontology analysis of 498 differentially expressed genes. We examined the 498 genes differentially expressed among the six mutants for gene ontology annotation enrichment. Bar lengths indicate the levels of enrichment (n-fold) for gene ontology terms (scales below graphs), and the gene ontology levels are indicated by the numbers to the left. The branching patterns indicate derivative annotations (e.g., the dityrosine layer of the spore wall is a specific case of spore wall [sensu Fungi]). The numbers under the heading “list” indicate the numbers of genes in the list of 498 genes with the respective annotations, the numbers under the heading “total” indicate the total numbers of genes found on the entire microarray with the respective annotations, and P values indicate the statistical significance of the enrichment. Enrichments for gene ontology annotations indicating cellular components (A) and biological processes (B) are shown. Annotations such as “embryonic development” are derived from sequence homology analysis and indicate that the Dictyostelium genes are homologous to genes in other organisms that have the indicated annotations.
FIG. 6.
FIG. 6.
SMEK regulates PP4C subcellular localization, and PP4C is epistatic to SMEK. (A) Cells were developed on 12 mM Na+-K+ phosphate agar plates for the indicated times, and photographs were taken. Multicellular developmental morphology at 9 h (wild-type mound stage), 16 h (wild-type slug stage), and 24 h (wild-type stalk and fruiting body stage) is depicted. SMEKOE, cells overexpressing SMEK; pppC−/SMEKOE, pppC− cells overexpressing SMEK. (B) Serine/threonine phosphatase activity in T7-PP4R2 immunoprecipitates as a function of the protein input (number of cells lysed and used in immunoprecipitation) and the length of time of the assay. (C) HA-PP4C localization in vegetative KAx-3 HA-PP4C-expressing cells and in KAx-3 HA-PP4C-expressing cells moving by chemotaxis. (D) HA-PP4C and V5-SMEK localization in KAx-3 HA-PP4C- and V5-SMEK-expressing cells moving by chemotaxis. FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isocyanate; DAPI, 4′,6′-diamidino-2-phenylindole.
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