Identification and functional analysis of a developmentally regulated extracellular signal-regulated kinase gene in Dictyostelium discoideum.
We have cloned a developmentally regulated mitogen-activated protein kinase (extracellular signal-regulated kinase) from Dictyostelium discoideum designated ERK1. Using anti-pTyr antibodies, we show that ERK1 is phosphorylated on tyrosine in vivo and that it will phosphorylate myelin basic protein. The gene expresses two transcripts, one that is preferentially expressed during vegetative growth and early development and one that is induced during the multicellular stages. Developmental Western blots (immunoblots) using anti-ERK1 antibodies indicate that ERK1 is present throughout development. ERK1/lacZ reporter constructs suggest that, in the multicellular stages, the gene is preferentially expressed in a subpopulation of cells scattered throughout the organism, similar to the pattern seen with anterior-like cell markers. Antisense mutagenesis from a derepressible promoter indicates that ERK1 is essential for vegetative growth. Overexpression of ERK1 from either the Actin 15 promoter or the ERK1 promoter results in abnormal morphogenesis starting at the slug stage. Overexpression of ERK1 in null mutants of the phosphotyrosine phosphatase PTP2 results in the production of large aggregation streams and subsequent abnormal morphogenesis that indicate a genetic interaction between ERK1 and PTP2. These cells produce very large aggregation streams that break up into very small mounds that undergo abnormal morphogenesis. The genetic interaction between ERK1 and PTP2 appears to be specific since overexpression of ERK1 in a ptp1- null mutant does not produce the same phenotype. Our results indicate that ERK1 plays an essential role during the growth and differentiation of D. discoideum.
Gaskins
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C
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Maeda
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M
Firtel
R A
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D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development*; D005786:Gene Expression Regulation; D017125:Genes, Protozoan / Q000235:genetics*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D000913:Antibodies, Protozoan; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases; D001616:beta-Galactosidase