ABSTRACT
In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity we investigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 among Kek family members and that this function is not ligand or tissue specific. Finally, we have identified a unique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V.
Subject(s)
Drosophila Proteins/chemistry , ErbB Receptors/antagonists & inhibitors , Membrane Proteins/chemistry , Protein Tyrosine Phosphatases/chemistry , Alleles , Amino Acid Sequence , Animals , Cell Membrane/metabolism , Cloning, Molecular , Dimerization , Drosophila melanogaster , ErbB Receptors/metabolism , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation , Ligands , Models, Biological , Molecular Sequence Data , Mutation , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Signal Transduction , Wings, Animal/metabolismABSTRACT
Throughout development, cells utilize feedback inhibition of receptor tyrosine kinase (RTK) signaling as an important means to direct cellular fates. In Drosophila, epidermal growth factor receptor (EGFR) activity is tightly regulated by a complex array of autoregulatory loops, involving an assortment of inhibitory proteins. One inhibitor, the transmembrane protein Kekkon1 (Kek1) functions during oogenesis in a negative feedback loop to directly attenuate EGFR activity. Kek1 contains both leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain, two of the most prevalent motifs found within metazoan genomes. Here we demonstrate that Kek1 inhibits EGFR activity during eye development and use this role to identify kek1 loss-of-function mutations that implicate the LRRs in directing receptor inhibition. Using a GMR-GAL4, UAS kek1-GFP misexpression phenotype we isolated missense mutations in the kek1 transgene affecting its ability to inhibit EGFR signaling. Genetic, molecular, and biochemical characterization of these alleles indicated that they represent two functionally distinct classes. Class I alleles directly diminish Kek1's affinity for EGFR, while class II alleles disrupt Kek1's subcellular localization, thereby indirectly affecting its ability to associate with and inhibit the receptor. All class I alleles map to the first and second LRRs of Kek1, suggesting a primary role for these two repeats in specifying association with and inhibition of EGFR. Last, our analysis implicates glycine 160 of the second LRR in regulating EGFR binding.
Subject(s)
Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila/genetics , Drosophila/metabolism , ErbB Receptors/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Alleles , Amino Acid Sequence , Animals , Animals, Genetically Modified , Drosophila/growth & development , Drosophila Proteins/chemistry , ErbB Receptors/genetics , ErbB Receptors/metabolism , Eye/growth & development , Female , Gene Expression Regulation, Developmental , Genes, Insect , Male , Membrane Proteins/chemistry , Molecular Sequence Data , Mutation, Missense , Phenotype , Protein Tyrosine Phosphatases/chemistry , Sequence Homology, Amino Acid , Suppression, GeneticABSTRACT
The net ecosystem exchange of carbon dioxide was measured by eddy covariance methods for 3 years in two old-growth forest sites near Santarém, Brazil. Carbon was lost in the wet season and gained in the dry season, which was opposite to the seasonal cycles of both tree growth and model predictions. The 3-year average carbon loss was 1.3 (confidence interval: 0.0 to 2.0) megagrams of carbon per hectare per year. Biometric observations confirmed the net loss but imply that it is a transient effect of recent disturbance superimposed on long-term balance. Given that episodic disturbances are characteristic of old-growth forests, it is likely that carbon sequestration is lower than has been inferred from recent eddy covariance studies at undisturbed sites.
