LKB1 is mutated in both familial and spontaneous tumors, and acts as a master kinase that activates the PAR-1 polarity kinase and the adenosine 5′monophosphate–activated kinase (AMPK). This has led to the hypothesis that LKB1 acts as a tumor suppressor because it is required to maintain cell polarity and growth control through PAR-1 and AMPK, respectively. However, the genetic analysis of LKB1–AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. We describe the identification of mutations in the single Drosophila melanogaster AMPK catalytic subunit AMPKα. Surprisingly, ampkα mutant epithelial cells lose their polarity and overproliferate under energetic stress. LKB1 is required in vivo for AMPK activation, and lkb1 mutations cause similar energetic stress–dependent phenotypes to ampkα mutations. Furthermore, lkb1 phenotypes are rescued by a phosphomimetic version of AMPKα. Thus, LKB1 signals through AMPK to coordinate epithelial polarity and proliferation with cellular energy status, and this might underlie the tumor suppressor function of LKB1.
The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5¢-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal a-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal a-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal a-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.
Fig. 4 Human GTPCH promoter activity is stimulated by 8Br-cAMP treatment. (a) GTPCH 5¢-flanking sequence showing restriction sites used for generating the luciferase reporter gene deletion constructs 1171GTPCHluc (DraI), 613GTPCHluc (HindIII), and 313GTPCHluc (AccIII). Also shown are putative Sp1, CRE, CCAAT-box and TATAbox like sequences located within the 313GTPCHluc construct. (b) SK-N-BE(2)M17 cells were transfected with GTPCHluc constructs and pCMV-b-gal DNA. Then, 5 mM 8Br-cAMP was added 18 h later and the cultures continued for another 8 h, when cells were lysed and assayed for luciferase and b-galactosidase activities. Luciferase activity was divided by b-galactosidase activity to correct for transfection efficiency and was expressed as relative luciferase activity. Data are the mean ± SE of three independent experiments each determined in triplicate.
Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actinTGFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of .4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actinTGFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.
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