Regulation of cell proliferation and adhesion by means of a novel region of drosophila merlin interacting with Sip1.

BACKGROUND: The tumor suppressor protein merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of merlin is associated with Neurofibromatosis Type 2 (NF2) tumors. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. We investigate how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. RESULTS: We identify two conserved arginine residues (R325 and R335) in Merlin which, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over-expression of Merlin(R325A) and/or Merlin(R335L) in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co-over-expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. CONCLUSIONS: Sip1 facilitates the activation of Merlin as a tumor suppressor protein. Thus, our work provides insight into how Merlin functions as a tumor suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis.

The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila.

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin/Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. We find that Drosophila melanogaster protein phosphatase type 1 ß (flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by over expression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. Our results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization.

Dnr1-dependent regulation of the Drosophila immune deficiency signaling pathway.

Innate immunity is a critical metazoan defense strategy that rapidly detects and neutralizes invading microbes. As the signaling pathways that drive innate immune responses are evolutionarily conserved, there is considerable interest in the characterization of innate immune signaling in genetically tractable models, such as Drosophila melanogaster. Drosophila responds to detection of diamonopimelic-type microbial peptidoglycan through activation of the immune deficiency (Imd) pathway, a signaling pathway with numerous similarities to the mammalian pro-inflammatory TNF pathway. In this manuscript, we focus on a molecular and in vivo characterization of Dnr1, a putative regulator of Imd pathway activity. A previous cell culture RNAi screen indicated that Dnr1 may serve as a negative regulator of the Imd pathway. However, there are no in vivo data to validate this hypothesis and there are scant molecular data to identify the mechanism by which Dnr1 may inhibit the Imd pathway. In this manuscript, we present in vivo data that are consistent with a negative regulatory role for Dnr1 in the Imd pathway. Additionally, we provide molecular data to indicate that Dnr1 inhibits the Imd pathway at the level of the initiator caspase Dredd.

A member of the p38 mitogen-activated protein kinase family is responsible for transcriptional induction of Dopa decarboxylase in the epidermis of Drosophila melanogaster during the innate immune response.

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria.

Quantitative evaluation of signaling events in Drosophila S2 cells.

Drosophila activates a robust defense response to gram-negative bacteria through the Immune deficiency (Imd) pathway. Imd signaling proceeds through c-Jun N-terminal Kinase (JNK), NF-kB and caspase modules. The individual signaling modules act in a highly coordinated manner to yield a stereotypical response to infection. While considerable attention has focused on NF-kB-mediated antimicrobial activities, more recent studies have highlighted the involvement of JNK signaling in the Imd pathway response. JNK signaling occurs in a transitory burst and drives the expression of a number of gene products through the AP-1 transcription factor. In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates.

A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster.

A neuropeptide hormone-signalling pathway controls events surrounding eclosion in Drosophila melanogaster. Ecdysis-triggering hormone, eclosion hormone and crustacean cardioactive peptide (CCAP) together control pre-eclosion and eclosion events, whereas bursicon, through its receptor rickets (RK), controls post-eclosion development. Cuticular tanning is a convenient visible marker of the temporally precise post-eclosion developmental progression, and we investigated how it is controlled by the ecdysis neuropeptide cascade. Together, two enzymes, tyrosine hydroxylase (TH, encoded by ple) and dopa decarboxylase (DDC, encoded by Ddc), produce the dopamine that is required for tanning. Levels of both the ple and Ddc transcripts begin to accumulate before eclosion, coincident with the onset of pigmentation of the pharate adult bristles and epidermis. Since DDC activity is high before the post-eclosion onset of tanning, a different factor must be regulated to switch on tanning. Transcriptional control of ple does not regulate the onset of tanning because ple transcript levels remain unchanged from 24 hours before to 12 hours after eclosion. TH protein present before eclosion is degraded, and no TH activity can be detected at eclosion. However, TH protein rapidly accumulates within an hour of eclosion and we provide evidence that CCAP controls this process. Furthermore, we show that TH is transiently activated during tanning by phosphorylation at Ser32, as a result of bursicon signalling. We conclude that the ecdysis hormone cascade acts as a regulatory switch to control the precise onset of tanning by both translational and activational control of TH.

Interactions of DNR1 with the apoptotic machinery of Drosophila melanogaster.

Caspases are crucial activators of apoptosis and NF-kappaB signaling in vertebrates and invertebrates. In Drosophila, the caspase-9 counterpart Dronc is essential for most apoptotic death, whereas the caspase-8 homolog Dredd activates NF-kappaB signaling in response to gram-negative bacterial infection. The mechanics of caspase regulation are conserved and include the activities of a family of inhibitor of apoptosis (IAP) proteins. The RING-domain-bearing protein Defense repressor 1 (Dnr1), blocks ectopic Dredd-mediated induction of an NF-kappaB reporter in the Drosophila S2 cell line. In this study, we present novel data indicating that Dnr1 impacts on Dronc-dependent regulation of the apoptotic program. We show that depletion of Dnr1 results in elevated Dronc protein levels, which translates to increased caspase activation and activity upon induction of apoptosis. Conversely, we demonstrate that overexpression of Dnr1 blocks apoptotic caspase activity and prevents induction of apoptosis in tissue culture assays. Furthermore, we show that Dnr1 overexpression significantly reduces Dronc protein levels and identify the domains of Dnr1 necessary for these effects. From these data, we propose that Dnr1 inhibits initiator caspases in S2 cells.