Wrinkled/head involution defective: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Wrinkled

Synonyms - head involution defective

Cytological map position - 75C1--75C2

Function - programmed cell death

Keywords - programmed cell death, wing, CNS, head involution

Symbol - W

FlyBase ID:FBgn0003997

Genetic map position - 3-[45]

Classification - Death domain protein

Cellular location - unknown



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

The Wrinkled (W) gene (more often referred to as head involution defective or hid) was described by V. Jollos in a 1936 paper entitled "Mutations observed in Drosophila stocks taken up into the stratosphere." The originally described mutation is dominant. Wings remain small and unexpanded. The phenotype of heterozygotes is not as severe as that of homozygotes. Wings of heterozygotes are often expanded but wrinkled, blistered and the surface finely pebbled and grayish. From the prepupal stage through to the adult, wing bases are abnormally narrow, possibly preventing the flow of body fluids in sufficient quantities to expand the wings (Waddington, 1940, as described in FlyBase). There is a general decrease in apoptosis, or programmed cell death (PCD) throughout the recessive hid mutant embryo. This phenotype is most noticeable in the head region prior to completion of head involution. Striking defects in head morphogenesis occur, in part, from a failure of the dorsal fold to migrate to the anterior in hid mutants (Abbott, 1991). It is possible that these morphogenetic defects result from the decrease of PCD in this region (Grether, 1995). Since the Drosophila literature invariable refers to Wrinkled as hid, this essay will conform to that convention, and not use the more proper name Wrinkled, which is the one recommended by FlyBase.

When hid is expressed from a heat shock promoter, high levels of PCD are observed within 2 hours of heat shock (hs) induction. The hs-hid transgene induces ectopic cell death in wild-type embryos following heat shock. The cell death induced by the hs-hid transgene is lethal to wild-type embryos, and a single heat pulse during embryogenesis kills all flies bearing the hs-hid construct. The ability of hid to kill is not significantly augmented by the presence of an endogenous reaper gene. The embryonic pattern or RPR mRNA is not significantly affected by ectopic expression of hid, even under conditions where large numbers of cells are dying. It is concluded that the induction of PCD by hid occurs independently of rpr (Grether, 1995).

Transformants carrying a single copy of hid expressed from a synthetic glass promoter display a dramatic eye ablation phenotype. Normally, compound eyes consist of about 800 regular units, called ommatidia, each of which consists of several distinct cell types. In hid transformants, only undifferentiated cuticle and a dense band of bristles remain in the places normally occupied by the compound eyes. It appears that these bristles represent the mechanosensory bristles normally found at the corner of each ommatidium. Apparently, these cells are less susceptible to hid-induced death. However, the number of these cells is severely reduced in transformants that are homozygous for the hid transgene, indicating that their survival is sensitive to the dose of hid expression. It is possible that the hid transgene is expressed only weakly in bristle precursors. Alternatively, bristle cells may be better protected against hid-induced PCD. Interestingly, a very similar phenotype is obtained from expression of reaper in the developing retina. The hid-induced eye phenotype is completely suppressed by coexpression of the baculovirus p35 gene (Grether, 1995).

A number of peptide factors including the neurotrophins, insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF), and epidermal growth factor (EGF) promote mammalian cell survival by suppressing the intrinsic cell death program. The mechanisms by which survival factors inactivate the intrinsic cell death program are currently the subject of intensive investigation. The growth factors listed above bind to and activate receptor tyrosine kinases (RTKs) at the cell surface; in turn, these factors stimulate the antiapoptotic activity of the proto-oncogene ras (reviewed by Downward, 1998). Ras (Drosophila homolog: Ras oncogene at 85D) controls the activity of a number of effector pathways, two of which result in activation of protein kinases known to mediate its antiapoptotic effect: the mitogen-activated protein kinase p42/44 (MAPK: Drosophila homolog Rolled) of the ERK-type (extracellular signal-related kinase) via Raf and the Akt kinase via Phosphoinositide 3-kinase (PI3-K). Recently, it has been shown that activation of the antiapoptotic PI3-K/Akt-kinase pathway leads to phosphorylation of Bad, a proapoptotic member of the Bcl-2 family (see death executioner Bcl-2 homologue), resulting in its binding to 14-3-3 (Drosophila homolog: Leonardo) as an inactive complex. Activation of the Erk-type MAPK is required to protect PC-12 cells from apoptosis induced by NGF withdrawal. However, a direct mechanistic link between the Raf/MAPK survival pathway and the cell death machinery has not been demonstrated thus far (Bergmann, 1998 and references).

The strong eye ablation phenotype in Drosophila, caused by expressing hid under the control of an eye-specific promoter, was used to perform a genetic screen aimed at identifying components that regulate and mediate Hid activity. Mutations in genes that regulate the EGF receptor (EGFR)/Ras1 (Ras oncogene at 85D) pathway were recovered as strong suppressors of Hid-induced apoptosis. The survival effect of the EGFR/Ras1 pathway is specific for Hid-induced apoptosis, since neither Reaper- nor Grim-induced apoptosis is affected by the EGFR/Ras1 pathway. The Ras1 pathway has been shown to inhibit Hid activity apparently by the direct phosphorylation of Hid by MAPK (Rolled). Alteration of the MAPK phosphorylation sites within the HID sequence blocks the survival signals generated by constitutively activate Ras1 and constitutively active MAPK. It is concluded that the hid gene in Drosophila provides a mechanistic link between the survival activity of Ras1 and the apoptotic machinery. Post-translational modification of Hid is a survival signal regulating Hid activity (Bergmann, 1998).

In addition to a post-translational regulation of Hid, the Ras/MAPK pathway promotes cell survival in Drosophila by downregulating the expression of hid. Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid expression. Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).

In the embryo, it is likely that Ras protection also acts through non-hid pathways. Even the complete removal of hid, in hid null embryos, results in only a mild decrease in apoptosis (Grether, 1995). This indicates that Ras downregulation of hid cannot account for all of the protection afforded by activated Ras and that Ras must have additional antiapoptotic targets in the embryo. An alternative pathway may be Ras activation of phosphoinositide 3-kinase (PI3-K) and its target Akt/PKB, which have been shown to protect mammalian cells from apoptosis. Drosophila PI3-K isoforms have been cloned and characterized, yet the role of this gene in Drosophila apoptosis is not well understood. Eye-specific expression of a Ras binding PI3-K isoform, Dp110, gives rise to flies with larger eyes (Leevers, 1996). This effect of PI3-K overexpression, however, is not due to inhibiting cell death, but rather due to increased cell size. Nonetheless, recent evidence suggests that the PI3-K target Akt/PKB may produce antiapoptotic activity in the embryo (Staveley, 1998).

We are left with trying to evaluate the relative importance of post-transcriptional modification vs. transcriptional regulation. Perhaps the former response provides a first line of protection against apoptosis, while the transcriptional regulation makes this protection permanent. Transcriptional regulation might also ensure, in instances where the surviving cell generates progeny, that the decisions made in the parent cell are passed on as permanent regulatory changes in the progeny.


GENE STRUCTURE

cDNA clone length - 3902

Bases in 5' UTR - 399

Exons - 4

Bases in 3' UTR - 2272


PROTEIN STRUCTURE

Amino Acids - 411

Structural Domains

Hid is a novel protein. There are five potential MAP kinase consensus phosphorylation sites. The predicted protein sequences for Hid and Reaper have some limited sequence similarity at their amino termini that is not shared with any other proteins in the data bases (Grether, 1995)

The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases. Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs. The crystal structure of Smac/DIABLO at 2.2 Å resolution reveals that it homodimerizes through an extensive hydrophobic interface. Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO. As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro. These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO (Chai, 2000).

Apoptosis is an essential process in the development and homeostasis of all metazoans. The inhibitor-of-apoptosis (IAP) proteins suppress cell death by inhibiting the activity of caspases; this inhibition is performed by the zinc-binding BIR domains of the IAP proteins. The mitochondrial protein Smac/DIABLO promotes apoptosis by eliminating the inhibitory effect of IAPs through physical interactions. Amino-terminal sequences in Smac/DIABLO are required for this function, because mutation of the very first amino acid leads to loss of interaction with IAPs and the concomitant loss of Smac/DIABLO function. The high-resolution crystal structure of Smac/DIABLO complexed with the third BIR domain (BIR3) of XIAP is reported in this study. These results show that the N-terminal four residues (Ala-Val-Pro-Ile) in Smac/DIABLO recognize a surface groove on BIR3, with the first residue Ala binding a hydrophobic pocket and making five hydrogen bonds to neighboring residues on BIR3. These observations provide a structural explanation for the roles of the Smac N terminus as well as the conserved N-terminal sequences in the Drosophila proteins Hid/Grim/Reaper. In conjunction with other observations, these results reveal how Smac may relieve IAP inhibition of caspase-9 activity. In addition to explaining a number of biological observations, this structural analysis identifies potential targets for drug screening (Wu, 2000).

X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspase-9 and inhibits its activity, whereas Smac (also known as DIABLO) relieves this inhibition through interaction with XIAP. XIAP associates with the active caspase-9-Apaf-1 (see Drosophila Apaf-1-related-killer) holoenzyme complex through binding to the amino terminus of the linker peptide on the small subunit of caspase-9, which becomes exposed after proteolytic processing of procaspase-9 at Asp315. Supporting this observation, point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9, or deletion of the linker peptide, prevents caspase-9 association with XIAP and its concomitant inhibition. The N-terminal four residues of caspase-9 linker peptide share significant homology with the N-terminal tetra-peptide in mature Smac and in the Drosophila proteins Hid/Grim/Reaper, defining a conserved class of IAP-binding motifs. Consistent with this finding, binding of the caspase-9 linker peptide and Smac to the BIR3 domain of XIAP is mutually exclusive, suggesting that Smac potentiates caspase-9 activity by disrupting the interaction of the linker peptide of caspase-9 with BIR3. These studies reveal a mechanism in which binding to the BIR3 domain by two conserved peptides, one from Smac and the other one from caspase-9, has opposing effects on caspase activity and apoptosis (Srinivasula, 2001).


Wrinkled/head involution defective: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 21 November 98

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