Gene name - small wing
Synonyms - PLC-gamma
Cytological map position - 14B15--14B15
Function - Phospholipase C
Symbol - sl
FlyBase ID: FBgn0003416
Genetic map position - 1-53.5
Classification - Phosphatidylinositol-specific phospholipase, Src homology 2 and 3 domain protein
Cellular location - cytoplasmic
The enzyme Phospholipase C plays a crucial role in signal transduction in the cell. The enzyme cleaves a membrane lipid into two products. In turn, each of these products, described below, activates other proteins that play crucial roles in signaling inside the cell. The small wing (sl) gene, originally identified by Bridges in 1915 (Morgan, 1925; Sivertzev-Dobzhansky and Dobzhansky, 1933), encodes a Phospholipase C-gamma (PLC-gamma) involved in eye and wing morphogenesis and is also likely to play other essential roles in fly biology. PLC-gamma is only one of several PLC proteins known to be present in the fly. Another PLC gamma is encoded by the no receptor potential A (norpA) gene, and is involved in signal transduction downstream of light receptors in the eye.
During cell growth and differentiation, growth factor receptors with tyrosine kinase activity trigger signal transduction cascades, which ultimately lead to profound changes in cell behavior. PLC-gamma is an intracellular enzyme that is activated by many such receptor tyrosine kinases (RTKs), via an interaction between one of two SH2 (Src-homology 2) domains in PLC-gamma with a specific phosphotyrosine on the intracellular part of the activated receptor. This association results in the phosphorylation of PLC-gamma and an increase in its catalytic activity. PLC-gamma catalyzes the hydrolysis of a lipid [phosphatidylinositol (4,5) bisphosphate] into two second messengers, a phosphosugar termed inositol 1,4,5- trisphosphate (IP3) and a lipid, diacylglycerol (DAG). IP3 stimulates release of Ca 2+ from internal stores (see Inositol 1,4,5,-tris-phosphate receptor), thereby mediating a variety of cellular processes, including fertilization and cell growth. DAG is an activator of Protein kinase C (Pkc), a serine/threonine kinase involved in a wide range of cellular activities, including responses to hormones, neurotransmitters and growth factors. Binding of different ligands to their respective receptor tyrosine kinases results in the phosphorylation of multiple tyrosines on the RTK's intracellular domain; each phosphotyrosine is recognized by one of several distinct SH2-containing proteins. Simultaneous activation of several proteins by the same RTK suggests that multiple signals might be sent, which may explain some of the complexity in the role of PLC-gamma in RTK-signaling. The emerging picture is one of a multiprotein complex being recruited to the activated receptor, from which multiple signaling pathways radiate into the cytoplasm. Furthermore, crosstalk between the branches also occurs, with the effect that the cellular response to ligand binding is actually integrated from the competing effects of many proteins (Thackeray, 1998 and references).
Genetic studies of RTK pathways that control cell fate determination in invertebrates have led to major advances in understanding RTK-mediated signaling. Three RTK pathways in Drosophila have received particular attention: Sevenless (Sev), which controls R7 photoreceptor cell development in the eye; the Drosophila EGF receptor homolog (Egfr), which is required during development of the oocyte and embryo, during wing vein differentiation and in all photoreceptor cells (R1-R8) of the eye, and Torso, which is involved in embryonic development. Although each pathway uses a different RTK, all three employ the highly conserved Ras/Raf/MEK/MAPK cassette of proteins to reach their nuclear targets. Activation of each receptor triggers a relay of signals, first through the adaptor protein Drk, then the guanine exchange factor Sos, which activates Ras1; this, in turn, results in the sequential phosphorylation of the serine/threonine kinases Raf-1, MEK and MAP kinase (MAPK). MAPK then transmits the signal into the nucleus by phosphorylating a variety of nuclear proteins, including transcription factors. Genetic evidence has suggested that activation of any of these three Drosophila RTKs may result in signaling through additional pathways. For example, a Ras-independent route to Raf activation from Torso has been demonstrated (Hou, 1995), and a biochemical study in mammals implicates PLC-gamma in such a role (Huang et al., 1995a). Using clones of mutant tissue in an otherwise wild-type Drosophila wing, another study has shown that removal of Drk or Sos function has a less severe effect on Egfr-mediated phenotypes than removal of the receptor itself (Diaz-Benjumea, 1994), again implying that multiple pathways are activated by Egfr (Thackeray, 1998 and references).
Examination of the eye morphology of sl mutants reveals the presence of extra R7 photoreceptors. In wild-type, the presumptive R7 cell is recruited from a pool of five sevenless (sev)-expressing cells known as the R7 equivalence group. After the R7 cell is recruited, the remaining four cells adopt a cone cell fate. sl mutant homozygotes contain one, two or sometimes as many as three more cone cells than wild-type; in addition, an extra primary pigment cell is sometimes present. These phenotypes are remarkably similar to those seen in mutations that increase the number of R7 cells, such as sevenlessS11, rolledSem, Gap1 and yan. These data suggest that the eye phenotype results from overactivation of the Ras/Raf/MEK/MAPK cassette, implying that the PLC-gamma encoded by sl normally acts as a negative regulator of Ras-mediated signaling during photoreceptor development in vivo (Thackeray, 1998 and references).
To determine whether the effect of the sl mutations on cell fate determination in the eye is via the Ras/MAPK pathway, a series of double mutants was made. Signaling via the Ras/MAPK cascade occurs in the developing R7 cell from at least two RTKs: Sev and Egfr. It was asked whether sl affects Egfr signaling, by examining the effect of reduced Egfr dosage in a background that is homozygous for sl. Reducing the dosage of Egfr using either one of two independent null mutations of Egfr (flb1k35 or flb1P02) in an sl background, almost completely rescues the eye phenotype. For example, whereas approximately half of the ommatidia contained an extra R7 cell in sl;+/+ males, this is reduced to 2% of ommatidia in sl; +/flb1k35. This result shows that the supernumerary R7 cells seen in the sl mutants depend on Egfr; even a halving of Egfr+ dosage is sufficient to almost completely suppress the eye phenotype. It was also asked whether sl affects Sevenless signaling. Null mutations of sev result in the loss of the R7 cell from all ommatidia. Double mutants of sl with either sev1 or sevd2 (a null allele) have an eye phenotype that is intermediate between the two single mutants: 35%-49% of ommatidia have one R7 cell; an additional 6%-12% contain two R7 cells, and the remaining ommatidia lack R7 cells. This partial suppression of the sl mutant phenotype shows that production of the extra R7 cells in the sl homozygotes is not completely dependent on sev+ activity, and confirms the result of Freeman (1996) that R7 cells can be produced in a sev-independent manner (Thackeray, 1998).
To determine whether there are interactions between sl and other genes of the Ras pathway, a mutation of MAP kinase, rolled (rl1) was used. This mutation is viable and thus its effects can easily be examined in the adult eye (homozygous loss-of-function mutations of most known components acting downstream of the Sev and Egfr RTKs are embryonic lethal). The partial loss-of-function mutation of MAPK, rl1, has a mild impact on R7 formation: 22% of the ommatidia lack R7 cells. Both sl;rl1 double mutants have phenotypes comparable to the rl1 single mutant: 36% and 27%, respectively, are missing R7 cells and <1% contain extra R7 cells. Thus, whereas in sl mutants 51% of ommatidia have extra R7 cells, this is reduced to <1% in a rl1 background. The phenotype of the sl;rl double mutants suggests that sl is acting upstream of rl. To confirm this result, interaction was tested between sl and a gene downstream of rl, seven in absentia (sina), which encodes a nuclear protein and for which a viable loss-of-function allele, sina2, is also available. In sina2, the proportion of ommatidia with an R7 cell is less than 5% . Adding either an sl mutation into a sina2 background causes no increase in the proportion of ommatidia with R7 cells. Thus the results with sina are consistent with those from rl, indicating that sl affects Ras signaling upstream from the rl MAP kinase. The reduced wing length phenotype observed in the sl single mutants is unaffected in any of the homozygous double mutant combinations. In contrast, the ectopic wing vein phenotypes of sl mutants are strongly suppressed by rl1 : in sl, 49% of wings (n=43) have ectopic veins, compared to only 7% in the double mutant sl;rl 1 (Thackeray, 1998).
These data suggest that Small wing PLC-gamma normally has a role as a negative regulator in the pathways leading to R7 development. When this down-regulatory role is lost in the mutants, the increased and/or prolonged signal presumably results in additional cells being recruited to an R7 cell fate. If Sl is playing a negative role in R7 development, how might this be carried out? The principal catalytic function of PLC-gamma is its hydrolysis of PIP2 into DAG and IP3. DAG is known to activate Protein kinase C, which has been shown to have a role as an activator of RTK-signaling in some contexts (for example by phosphorylation of Raf) and as an inhibitor in others. Another study shows that sustained stimulation of PKCalpha by a phorbol ester in NIH3T3 cells leads to an association of PKC with the EGF receptor, followed by phosphorylation of the RTK and subsequent internalization and/or degradation of the receptor (Seedorf, 1995). Interestingly, overexpression of PLC-gamma enhances this down-regulatory effect, consistent with a role for PLC-gamma as a negative feedback regulator of signaling in this pathway. The results described by Thackeray (1998) are consistent with such a role for Sl, acting either as a direct or indirect inhibitor/attenuator of the Egfr signal. It may be that Sl is both activated by Egfr and then later required to attenuate the Egfr signal. When the Egfr signal is allowed to persist due to the absence of Sl activity, extra R7 cells are produced (Thackeray, 1998).
Most models of PLC-gamma activation propose that one of its SH2 domains binds to specific phosphotyrosines on the activated RTK. However, there is no consensus site for mammalian PLC-gamma SH2 binding in the intracellular domain of Egfr. It may be that Sl does not bind directly to Egfr, or it might do so at a different sequence. If it does not bind to Egfr it is possible that it is activated by binding to another RTK, or that it interacts with another protein which is itself activated by Egfr. One such intermediary protein might be Daughter of sevenless (Dos), which is required for Sev signaling (Raabe, 1996; Herbst, 1996). Dos is proposed to act as an adaptor protein that brings together a multiprotein complex at an activated RTK (Raabe, 1996). The Dos sequence contains consensus sites for binding mammalian PLC-gamma SH2 domains and a polyproline domain that might bind to the SH3 domain of PLC-gamma. Another protein that is likely to interact with Sl is the membrane protein PI3K. A recent biochemical study has shown that an adaptor protein for a Drosophila PI3K also binds to the Drosophila PLC-gamma (Weinkove, 1997). This interaction might be involved in targeting Sl to the membrane, as has been demonstrated for mammalian PLC-gamma (Falasca, 1998). Once at the membrane and activated by association with the RTK (presumably Egfr), PLC-gamma induced activation of PKC could result in phosphorylation either of one or more components of the multiprotein complex assembled at the RTK, or the RTK itself - this phosphorylation being required to terminate the signal correctly. Whatever the mechanism, it will be of interest to see what interactions exist between PI3K, Dos and Sl. The reduced wing-length phenotype observed in all three sl alleles could be due to a reduction in cell number, reduced cell size or both. In wing tissue homozygous for mutations in any of six different genes that reduce signaling in the Egfr pathway, including Egfr, drk, sos, Ras1, Raf and rl, cell density is higher than in surrounding wild-type wing tissue (Diaz-Benjumea, 1994). In addition, where such mutant clones overlap a vein, no vein is produced. This latter finding is consistent with the interpretation of Sl as a negative regulator of RTK signaling, in that the sl mutations result in the opposite phenotype: extra wing veins. The effect of the sl mutations on wing size appears to be mediated by a pathway different from that used in wing vein development, because the sl mutations in this case show a similar phenotype to those referred to above that reduce Egfr function. The fact that rolled suppresses the extra wing vein phenotype but not the wing size phenotype of sl, is also consistent with a role for Sl in two different pathways governing wing development (Thackeray, 1998).
Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. This study shows through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by small wing (sl) acts as such a link between growth and patterning/differentiation by modulating some MAPK outputs once activated by the insulin pathway; particularly, sl promotes growth and suppresses ectopic differentiation in the developing eye and wing, allowing cells to attain a normal size and differentiate properly. sl mutants have previously been shown to have a combination of both growth and patterning/differentiation phenotypes: small wings, ectopic wing veins, and extra R7 photoreceptor cells. This study shows that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, these results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes (Murillo-Maldonado, 2011).
By measuring cell density, this study shows that sl mutant wings have a reduction in cell growth but not cell proliferation. This defect is qualitatively similar to mutations in MAPK signaling; cells with homozygous mutations for members of this pathway have higher cell densities, suggesting smaller cells. Of the several signaling pathways known to be involved in Drosophila wing growth, only the MAPK and insulin pathways are triggered by tyrosine kinase receptors that are likely to activate Sl. The results show that indeed both pathways are genetically linked to Sl in promoting cell growth, probably acting in a concerted fashion; further molecular studies will be required to reveal the molecular mechanisms and physical interactions that allow this link. Sl signaling thus provides a means for coordinating growth by forming a regulatory link between the MAPK and insulin pathways. In this scenario, Sl activated by the insulin pathway would function by modulating MAPK output; that is to say, to reduce somewhat the levels of MAPK activity, but not to stop it, as no MAPK activity leads to no growth and cell death, and too much MAPK activity leads to ectopic differentiation and reduced growth (Murillo-Maldonado, 2011).
Sl regulates cellular growth in the eye. Whole eyes are smaller, and the difference in size can be largely explained by the presence of fewer ommatidia. This means that sl mutant eyes very likely contain fewer cells, despite the fact that some ommatidia sport one or two extra R7 cells, as the number of cells missing due to reduced numbers of ommatidia is bigger than the number of extra R7 cells present. This suggests either reduced proliferation or increased cell death in differentiating sl mutant eyes, and is different from the growth defect found in wings, yet consistent with a moderate requirement of MAPK output to promote growth and cellular survival (Murillo-Maldonado, 2011).
Not only is cell size reduced to a similar extent in both the eye and wing of sl homozygotes; the adult animal as a whole has reduced mass. Given that the reduction in mass (8%) is of a similar magnitude to the reduction in cell size in the eye (15%) and wing (20%), the most parsimonious explanation for this change in mass is that the same Sl functions found in the eye and wing are required more generally throughout the animal, suggesting that cell size may be reduced in many tissues. However, it was found that the reduced growth observed in the adult was not reflected by a reduction in length of sl mutant pupae. This is in contrast to mutations of other genes involved in growth control, such as the neurofibromin 1 gene, which shows a significant reduction in pupal length. This might be because sl has a relatively small effect on growth, varying between 5% and 20% in different contexts, so this sample may not have been large enough to observe a small change in mean length. Given that Sl does not appear to affect the length of appendages other than the wing, it may be that there are other compensatory effects resulting from lost Sl function that maintain the pupal case at an approximately wild-type length (Murillo-Maldonado, 2011).
Another complementary explanation for the reduction in adult mass is via a role for Sl on nutrient sensing. As Sl is clearly involved in insulin signaling, and as insulin is required for integrating nutrient sensation in Drosophila, the effect on mass might be a combination of impacts on both growth signaling and nutrient sensing (Murillo-Maldonado, 2011).
It is proposed that the overall role for Sl is to act as a pro-growth agent, allowing cells and tissues to attain normal numbers and sizes. This is achieved by dampening MAPK output in growth control in a non-cell autonomous manner, by restricting processing of EGFR ligand(s), as shown previously for R7 cell differentiation. Since both the MAPK and insulin pathways initially act to favor proliferation and growth, it is proposed that Sl functions here under insulin pathway control, allowing growth to continue, preventing ectopic differentiation. There are several ways in which it could do so: by directing activated MAPK to a different cellular compartment (cytosolic versus nuclear or by controlling overall strength and duration of signaling, examples of which have been shown to elicit such changes in developing wing cells in both Drosophila and PC12 cells (Murillo-Maldonado, 2011).
A central function of all phospholipase C enzymes is hydrolysis of PIP2. In this study has shown that regulation of growth and differentiation by Sl must depend on PIP2 hydrolysis to some extent, because of the interaction between sl and mutations in IP3R, PKC53E and Rack1. Also, by means of genetic tests, it was found that Sl requires the Spi processing machinery (S, Rho) to regulate growth and differentiation. It has previously been shown that Sl acts on Spi processing during R7 differentiation, by favoring Spi retention in the endoplasmic reticulum. In order to rationalize Sl function in all the phenotypes studied, it was reasoned that by inhibiting Spitz processing, Sl could delay initiation of differentiation, allowing still undifferentiated cells to grow and attain a normal size before the onset of differentiation. Sl modes of action in growth and differentiation may be different; sl alleles affecting the wing but not the eye is strong evidence for this assertion (Murillo-Maldonado, 2011).
In general, during growth, Sl activated by the insulin pathway acts as a liaison regulating MAPK pathway ligand processing, to promote MAPK activation to a level permitting growth. In agreement with a well-characterized case in mammalian cells, it is proposed that this level of activity of MAPK is different from the level required for differentiation; either it is of a different duration, or of an overall different stimulation level, or happening at a different time. Alternatively it occurs in a different subcellular compartment from that required for differentiation, acting thru Sl regulation of Spi processing. This scenario also requires both the MAPK and the insulin pathways to be active for cellular growth. Conversely, for differentiation, reduced insulin receptor signaling leads to altered (lower) levels of Sl activation and augmented Spi processing, and this in turn allows MAPK activation in a manner consistent with promotion of differentiation. This could either be caused by longer or stronger MAPK stimulation, as documented for PC12 cells, since lower Sl activity now allows higher levels of MAPK ligand processing, and/or by compartmentalization of the activated MAPK pathway, as shown for the Drosophila wing, besides happening at different times during development. In this second case, only the MAPK pathway is required to be fully active. Finally, loss-of-function mutant conditions for sl lead to ectopic differentiation at the expense of growth (Murillo-Maldonado, 2011).
Taken together, these results indicate that Sl participates in fine coordination of growth and differentiation during development. Although Sl is not essential for wing or eye growth and development, it is necessary to achieve appropriate final structure and size. In the absence of Sl function, these tissues arrest growth prematurely and probably initiate differentiation earlier, resulting in ectopic differentiation while attaining smaller cellular sizes. As such, Sl can be seen as exerting a kind of 'parental control' that protects cells from differentiating before attaining a normal size. This function requires Sl to change cellular behavior from growth (or possibly inhibition of differentiation) to differentiation in a short period of time (Murillo-Maldonado, 2011).
PLC-γ1 has been demonstrated to be a phosphorylation target of MAPK, and some PKC isoforms can phosphorylate PLC-γ without affecting PIP2 hydrolysis so it is clear that there is a complex interplay of signaling among this set of molecules following RTK activation. Further study of the dynamics of Sl-regulated EGF/MAPK signaling in space and time during wing and eye development in Drosophila may help to expose more of this network (Murillo-Maldonado, 2011).
Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), the PLC-gamma homologs were cloned and sequenced from Drosophila pseudoobscura and D. virilis and their gene structure and predicted amino acid sequences were compared with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution (Manning, 2003).
Exons - 4
A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion is observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved (Emori, 1994 and Thackeray, 1998).
date revised: 15 November 2012
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