InteractiveFly: GeneBrief

small wing : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - small wing

Synonyms - PLC-gamma

Cytological map position - 14B15--14B15

Function - Phospholipase C

Keywords - eye, wing, Egfr pathway, Insulin pathway

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



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

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).

Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila.

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).


DEVELOPMENTAL BIOLOGY

Embryonic

The mRNA of Small wing, Drosophila Plc gamma, is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole mount in situ hybridization, reveals that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes, mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation (Emori, 1994).

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. This study used insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. A Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, this study describes mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection (Tsuzuki, 2014).


EFFECTS OF MUTATION

All three sl alleles are recessive, homozygous viable mutations that are classically described as having a modest reduction in wing length and a mildly 'rough' eye (disturbance to the crystalline array of facets in the compound eye) (Lindsley, 1992). In addition to these phenotypes, animals homozygous for any of the three sl alleles have ectopic wing veins, most frequently adjacent to vein L2, but sometimes also connected or adjacent to the posterior crossvein or within the posterior cell. Heteroallelic combinations of all three alleles fail to complement with respect to the rough eye or wing phenotypes. This combination of ectopic wing veins and rough eyes resembles defects previously observed in mutations that activate the Ras/MAPK cassette, such as the Ellipse alleles of Epidermal growth factor receptor (DERElpB1), the Sevenmaker alleles of rolled (rlSem) and mutations of Gap1. Because these mutations disrupt photoreceptor cell fate, the compound eye was examined from each of the sl alleles for photoreceptor defects that might underlie the observed roughness in the eye (Thackeray, 1998).

The wild-type Drosophila compound eye consists of about 800 hexagonal units, or ommatidia, packed in a crystalline array. In flies homozygous for any of the three sl alleles, this regular array is disrupted and many ommatidia are abnormally shaped. In addition, the sl eyes have interommatidial bristle defects: some bristles are duplicated, some are missing and some appear at additional vertices. Each wild-type ommatidium contains a precise assembly of eight photoreceptor cells (R1-R8) capped by four lens-secreting cone cells and surrounded by a sheath of pigment and bristle cells. The rhabdomeres (photosensitive organelles) of each of the six outer photoreceptor cells (R1-R6) can be seen in tangential sections, arranged in a trapezoidal ring around a central, smaller rhabdomere. In the distal part of the retina, the central rhabdomere corresponds to photoreceptor R7; in more proximal parts, it corresponds to the R8 cell. Tangential sections through the distal part of sl homozygous mutant eyes indicate that many ommatidia (51%) contain extra inner photoreceptors (i.e. with centrally located rhabdomeres). In addition, a small fraction of mutant ommatidia have one or more extra (2%) or missing (10%) outer photoreceptors. The distal position, central location and small rhabdomere size of the extra inner photoreceptors in the mutants suggests that they are R7 cells. To confirm their identity, the expression of an Rh4-lacZ fusion construct was examined in an sl mutant background. In wild-type, expression of the Rh4 opsin is limited to a random subset (~70%) of R7 cells. In sl mutant eyes, the majority of the supernumerary cells express the Rh4- lacZ gene, demonstrating that they have both the molecular and morphological characteristics of R7 photoreceptors (Thackeray, 1998).

Since the C2 and PH domains, which constitute potential regions of interaction with Ca2 + and IP4, are required for GTPase-activating protein 1 in vivo activity, it became of interest to see whether genes involved in Ca2+ and phosphoinositide signaling regulate Gap1 function in vivo. One such candidate is the Drosophila gene small wing, which encodes a phospholipaseCgamma (PLCgamma) homolog. PLCgamma comprises a catalytic core, two PH, two SH2, and one SH3 domains and a conserved tyrosine phosphorylation site, all of which act as signal-dependent regulators of enzymatic activity. Subsequent to RTK stimulation, PLCgamma is recruited to the receptor through its SH2 domains and then itself activated by tyrosine phosphorylation. Upon activation, PLCgamma converts phosphatidylinositol-4,5-bisphosphate into diacylglycerol and IP3; IP3, in turn, serves as a precursor for IP4, both of which act as signals for mobilization of intracellular Ca2+. Loss-of-function mutations in the sl locus display a phenotype that is consistent with PLCgamma playing a role as a negative regulator of Egfr signaling, similar to Gap1 (Thackeray, 1998). In the eye, sl complete loss-of-function mutations cause a supernumerary R7 phenotype in approximately 30% of ommatidia; this is qualitatively similar to the mutation caused by Gap1, but much less severe. In hemizygous sl1 mutants, the average number of R7s is 1.3. Thus, the sl complete loss-of-function phenotype is extremely mild compared to that of Gap1 partial or complete loss-of-function mutations. To probe the relationship between PLCgamma and Gap1, a test was performed to determine whether they genetically interact. In a wildtype background, Gap1/+ flies are phenotypically wildtype, i.e. all ommatidia have only one R7 cell (Gaul, 1992; Lai, 1992), while flies hemizygous for sl1 and heterozygous for Gap1 have a supernumerary R7 phenotype greatly enhanced over that seen in hemizygous sl1 flies; greater than 80% of ommatidia have multiple R7 cells. Thus, the phenotype of mutants hemizygous for sl and heterozygous for Gap1 closely resembles that of the partial loss-of-function Gap1S1-11 allele, demonstrating a synergistic positive genetic interaction between sl and Gap1. Given PLCgamma’s role in Ca2+ and phosphoinositide signaling and Gap1’s requirement for its Ca 2+ and IP4-sensitive domains, this result suggests that Gap1 activity is positively regulated by PLCgamma (Powe, 1999).

Calcineurin is a Ca2+-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca2+ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca2+-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca2+ levels that can result from the opening of intracellular Ca2+ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate (InsP3), which then activates the InsP3 receptor Ca2+ channel (Sullivan, 2002).

GPCRs and RTKs activate an integrated signaling network that includes the Ras/mitogen-activated protein (MAP) kinase cascade, PI3-kinase, and the small GTPase Rho. Depending upon the cellular context, these pathways can either antagonize or cooperate with each other and with PI signaling. For example, T-cell activation requires the activation of both NFAT, which is transduced to the nucleus upon dephosphorylation by calcineurin, and AP1, which acts downstream of Ras and MAP kinase. Conversely, PI signaling has been found to antagonize the Ras pathway in Drosophila. The Egf receptor and Ras/MAP kinase cascade are essential for formation of wing veins and photoreceptor (R) cells in the eye. Mutations in the single phospholipase Cgamma gene, small wing (sl), cause the formation of extra R7 cells and wing vein material and also genetically interact with Egf-receptor-signaling components. A recently proposed model for sl-mediated repression of Egf receptor signaling was based on the identification of the GTPase-activating protein Gap1 as an InsP4 receptor. PLCgamma-generated InsP3 is converted to InsP4, which then activates Gap. Gap converts the active form of Ras, Ras-GTP, to the inactive form, Ras-GDP (Sullivan, 2002 and references therein).

An activated form of Pp2B-14D, canAact, was made by deleting the autoinhibitory and calmodulin-binding domains. The canAact construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canAact.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

The canAact.gl screen yielded 11 complementation groups, 9 of which failed to modify rough eyes caused by other glass-induced transgenes. This demonstrates that the majority of the modifier groups do not act through the glass enhancer. The nine specific modifiers were then divided into class I genes, which act downstream of calcineurin, and class II genes, which act at the level of CanB (Sullivan, 2002).

The hypermorphic allele EgfrE1 inhibits Ras signaling; thus it might be expected to enhance the effects of activated calcineurin. However, low levels of inappropriate Egf receptor activity in eye development are thought to increase secretion of the Egf receptor antagonist Argos. The Argos protein inhibits subsequent Egf receptor signaling that is required for photoreceptor determination. Thus, suppression of the EgfrE1 rough eye by canAact.gl may be the result of activated calcineurin inhibiting inappropriate Egf receptor signaling (Sullivan, 2002).

Consistent with these findings, PLCgamma is a negative regulator of Egf receptor/Ras signaling in eye and wing development. However, PLCgamma was identified in this study as a strong suppressor of activated calcineurin, although biochemically PLCgamma has been placed upstream of calcineurin in the PI signaling pathway. One explanation is that PLCgamma acts on one of the other canA genes. Another possibility is that the signaling pathways activated by PLCgamma parallel to calcineurin are required for calcineurin function (Sullivan, 2002).

In a recent model, PLCgamma has been proposed to inhibit Egf receptor/Ras signaling via the activation of Gap1 by InsP4. The results presented in this study suggest that PLCgamma is also acting through calcineurin. The genetic evidence presented indicates that calcineurin intersects with the Ras pathway at roughly the same point that PLCgamma does, and thus a modified model is proposed for the function of PI signaling in Drosophila development. Additionally, the fact that calcineurin can be activated by any sustained Ca2+ flux suggests a mechanism by which other signaling pathways, such as GPCRs acting via PLCß, can modulate Egf receptor signaling (Sullivan, 2002).

A simplified schematic is presented that illustrates upstream Egf receptor signaling components in an eye disc cell. PLCgamma is activated by the Egf receptor and cleaves PIP2 to yield InsP3. PLCgamma is proposed to negatively regulate Egf receptor signaling through InsP4, which is generated from InsP3 by an InsP3-3 kinase. Gap1 is then activated by InsP4, which results in the inhibition of Ras. Sprouty, which may be linked to the Egf receptor by the adaptor protein Drk, may facilitate the inactivation of Ras by Gap. In this model, it is proposed that PLCgamma also acts via Ca2+ and calcineurin. Genetic evidence suggests that calcineurin acts at the level of sty and Gap1, although it should be noted that calcineurin may act further upstream, e.g., at the level of InsP4. In addition, it is possible that calcineurin is activated by other Ca2+ signaling pathways (Sullivan, 2002).

The Drosophila genome contains a single phospholipase C-gamma (PLC-gamma) homolog, encoded by small wing (sl), that acts as an inhibitor of receptor tyrosine kinase (RTK) signaling during photoreceptor R7 development. Although the existing sl alleles behave genetically as nulls, they may still produce truncated Sl products that could in theory still provide limited PLC-gamma function. Both to identify a true null allele and to probe structure-function relationships in Sl, an F1 screen was carried out for new sl mutations and seven new alleles were identified. Flies homozygous for any of these alleles are viable, with the same short-wing phenotype described previously; however, two of the alleles differ from any of those previously isolated in the severity of the eye phenotype: sl9 homozygotes have a slightly more extreme extra-R7 phenotype, whereas sl7 homozygotes have an almost wild-type eye. The mutant defect was determined in all seven alleles, revealing that sl9 is a molecular null due to a very early stop codon, while sl7 has a missense mutation in the highly conserved Y catalytic domain. Together with in vitro mutagenesis of the residue affected by the sl7 mutation, these results confirm the role of Sl in RTK signaling and provide evidence for two genetically separable PLC-gamma-dependent pathways affecting the development of the eye and the wing (Mankidy, 2003).

One of the major goals of this study was to determine whether the previously characterized sl alleles indeed represent the loss-of-function phenotype, because the three previously characterized alleles could produce a truncated protein with several domains intact. Five of the seven new alleles showed a homozygous phenotype indistinguishable from that previously described for sl1, sl2, or sl3 homozygotes. The molecular defects in the new alleles vary widely in location within the protein, occurring at both N and C termini with different degrees of truncation and including an in-frame deletion and a single amino acid substitution among them. The most telling allele of all in this regard is sl9, which contains a nonsense codon at amino acid 54. Because mRNAs containing premature stop codons tend to be degraded by the nonsense-mediated decay pathway, it is unlikely that many sl9 transcripts would survive to be translated; however, even if some sl9 mRNAs escaped degradation, Sl products translated from them would have no recognized domains remaining. Therefore, the sl9 mutation clearly represents a true null allele. The sl9 homozygous phenotype is qualitatively the same as the previously characterized sl alleles (i.e., with extra-R7 cells and ectopic wing veins), which confirms previous results showing that Sl is a negative regulator of the RTK pathways involved in cell-fate decisions during ommatidial and wing-vein development (Mankidy, 2003).

The phenotype of sl9 homozygotes is identical to that of sl1,2,3,4,5,6,8,10, except that it is slightly more extreme in the eye. Why sl9 homozygotes should show a more extreme phenotype than that of the other alleles is uncertain; one possibility that cannot be ruled out is that a closely linked enhancer of the extra-R7 phenotype is present in the sl9 background. However, this allele is the only one unlikely to produce any protein; every other allele could produce either a mutated full-length protein or a truncated protein containing intact copies of the N-terminal PH domain, EF hand region, region X, and the N-terminal SH2 domain. Because mammalian PLC-gamma does not depend on its lipase function for its role in mitogenesis, truncated Sl proteins lacking intact X and Y catalytic domains (such as those that could be generated by the alleles other than sl9) might still be able to participate at some level in certain aspects of PLC-gamma function. For example, the intact N-terminal SH2 domain in each of the truncated Sl proteins would likely still be able to bind to an activated RTK at the membrane, where either its N-terminal PH domain or its EF hands might have an effect on signaling, perhaps by binding phospholipids or Ca2+ (Mankidy, 2003).

Three of the new alleles, sl5, sl7, and sl8, contain either a missense mutation or an in-frame deletion, identifying functionally important residues. In the case of sl5, a five-amino-acid deletion at the N-terminal end of the C2 domain removes five conserved residues within ß-strand 2/1 of the eight ß-strand C2 structure. C2 motifs typically bind calcium and phospholipids, but have proven to be capable of interacting with a wide range of other molecules, sometimes independently of calcium binding. The C2 domains of PLC-gamma proteins appear to lack several residues shown to be necessary for calcium binding in the PLC-delta1 C2 domain, suggesting that the PLC-gamma C2 domain has another (as yet unidentified) function. Combined with the fact that the C2 domain is well conserved in all PLC-gamma proteins, including within the genus Drosophila, the near-null phenotype observed in sl5 homozygotes indicates that the C2 domain does retain an indispensible function in Sl. The sl8 mutation replaces Gly385 with aspartate within the region X catalytic domain. There is a glycine at the homologous position in all of the described PLC proteins, and the immediately adjacent histidine on the N-terminal side of this glycine is required for PIP2 hydrolysis in both PLC-delta and PLC-gamma, playing a direct role in catalysis by acting as a proton donor. It is therefore not surprising that the introduction of an aspartate at this critical location would disable the phospholipase activity of the enzyme (Mankidy, 2003).

By far the most intriguing of the new alleles is sl7, because flies homozygous for this allele show the null wing-length phenotype, but have an almost wild-type eye. A trivial explanation of this phenotype might be the presence of a closely linked modifier that suppresses the extra-R7 phenotype, but not the wing-length phenotype. However, the fact that the X10-P1035A construct produced an almost identical phenotype in the eye, in an unmutagenized background, suggests that the sl7 phenotype is a genuine reflection of altered PLC-gamma activity rather than an artifact produced by an interacting mutation. Furthermore, there is a precedent for an sl allele with an sl7-like phenotype: sl34 was isolated by Gottschewski in 1934 (cited in Mankidy, 2003) and was described as having short wings, but normal eyes; unfortunately, sl34 has been lost. The fact that an independently isolated allele shows this same combination of eye and wing phenotypes tends to suggest that the sl7 mutation is not simply a rare hypomorph with threshold activity, but may in fact be a member of a class of sl mutations that are able to separate the extra-R7/ectopic wing-vein and wing-length phenotypes. This model is also consistent with evidence for two distinct Sl-mediated pathways in the eye and wing: a partial loss-of-function mutation of the rl-encoded MAPK, rl1, is able to suppress both the extra-R7 and ectopic wing-vein phenotypes of sl homozygotes, but does not suppress the short-wing phenotype (Mankidy, 2003).

The sl7 mutation is a replacement of Pro1035 by leucine within the region Y catalytic domain. An alanine at the same position recapitulates the phenotype of sl7 in the eye, but not in the wing, in which P1035A rescues the sl wing phenotype. By contrast, an sl construct containing valine at position 1035 is equivalent to one containing the wild-type proline, rescuing both the eye and wing defects of either sl1 or sl9. First of all, the ability of the X10-P1035V construct to rescue flies lacking sl function is very surprising, because this proline is one of a small number of absolutely conserved sites across all PLC isoforms in region Y. In PLC-delta1 the homologous proline is predicted to form a turn leading into one loop of a hydrophobic ridge that lines the active site, a role that might be expected to depend on a proline at this position. A possible explanation is that because valine has a side chain slightly more hydrophobic and compact than that of leucine it is drawn into the ridge by hydrophobic clustering, thereby partially overcoming the loss of proline. The slightly lower hydrophobicity and longer side chain of leucine may be unable to achieve this effect, possibly because of steric clashes due to its length. In contrast, alanine is much less hydrophobic than either valine or leucine and so may be unable to overcome the loss of the turn resulting from replacement of the proline. In any event, the fact that three different amino acids at position 1035 can produce three different combinations of eye and wing phenotypes clearly indicates that Pro1035 has a key modulatory role in Sl signaling (Mankidy, 2003).

Pro1035 of Sl is homologous to Pro552 of human PLC-delta in a region close to the active site where several mutations have been made and tested in vitro. One of the most interesting of these PLC-delta mutations was a replacement of Arg549 by alanine; this change dramatically reduces PIP2 hydrolysis, but has little effect on hydrolysis of PI, demonstrating that changes in this part of region Y can alter the substrate specificity of PLC-delta. All PLC enzymes are thought to be able to hydrolyze PIP2, PIP, and PI, although the relative physiological importance of the different substrates is unknown for any PLC. If the sl7 mutation has an effect similar to the Arg549 mutation of human PLC-delta1, hydrolysis of one substrate necessary for Sl-mediated signaling in the wing might be reduced, whereas the hydrolysis of a different substrate more important for signaling during photoreceptor development is less affected. An alternative explanation is that in sl7 homozygotes Sl is partially functional at a threshold of activity that is almost sufficient for wild-type function in the eye, but is not quite enough in the wing. However, the phenotype of sl7/sl8 and sl7/sl9 trans-heterozygotes suggests that there is a qualitative difference between these alleles, consistent with the sl7-encoded PLC-gamma protein lacking a function needed in the wing, but retaining a function needed in the eye. Direct assays of phospholipid hydrolysis will be required to determine whether differential substrate use by Sl occurs in different physiological contexts (Mankidy, 2003).

Small Wing PLCγ is required for ER retention of cleaved Spitz during eye development in Drosophila

The Drosophila EGF receptor ligand Spitz is cleaved by Rhomboid to generate an active secreted molecule. Surprisingly, when a cleaved variant of Spitz (cSpi) was expressed, it accumulated in the ER, both in embryos and in cell culture. A cell-based RNAi screen for loss-of-function phenotypes that alleviate ER accumulation of cSpi identified several genes, including the small wing (sl) gene encoding a PLCsγ. sl mutants compromised ER accumulation of cSpi in embryos, yet they exhibit EGFR hyperactivation phenotypes predominantly in the eye. Spi processing in the eye is carried out primarily by Rhomboid-3/Roughoid, which cleaves Spi in the ER, en route to the Golgi. The sl mutant phenotype is consistent with decreased cSpi retention in the R8 cells. Retention of cSpi in the ER provides a novel mechanism for restricting active ligand levels and hence the range of EGFR activation in the developing eye (Schlesinger, 2004).

The accumulation of cSpi in the ER appears to reflect a novel mechanism for ER retention. In contrast to ER retention of the full-length Spi precursor, cSpi remains in the ER when the retrograde trafficking machinery from the Golgi to the ER is compromised following incubation with dsRNA for COPI. Utilization of a novel ER retention and export machinery has been identified for the SREBP protein-regulating cholesterol synthesis (Schlesinger, 2004).

To identify the mechanism responsible for cSpi retention and assess its biological significance, a screen was conducted for dsRNAs that would compromise this property. The analysis focussed on the sl gene, in view of previous observations demonstrating that it is a negative regulator of EGFR signaling in the eye. Sl is broadly expressed. Compromising the levels of Sl in embryos, either by dsRNA injection or in a sl mutant background, led to efficient release of cSpi. Thus, Sl is also required in vivo for the retention of cSpi. The actual retention mechanism remains unknown. Sl is a cytoplasmic protein, while cSpi is a secreted protein that is retained within the ER lumen. Additional proteins must participate to form a physical link. While sl encodes a PLCγ, it is believed that its enzymatic activity is not necessary for the retention process. sl interacts genetically with EGFR signaling only in the eye. sl mutants that were defective in the catalytic domain did not give rise to an eye phenotype. In addition to the catalytic domain, Sl also contains several motifs that may mediate protein-protein interactions, including SH2, SH3, and PH domains. It is thus possible that in addition to its enzymatic role, Sl serves as a scaffold protein in other contexts. In mammalian cells, PLCγ has been shown to function in the cells receiving the signal, downstream to receptor-tyrosine kinases (Rhee, 2001). The implicated role of Sl/PLCγ in the cells producing the signal points to a novel function of this protein (Schlesinger, 2004).

While Sl is broadly expressed, the EGFR hyperactivation phenotype of sl null flies is manifested only in the eye. This phenotype entails recruitment of extra R7 photoreceptor cells and misrotation of ommatidia. The restricted effect led to an examination of the possibility that cSpi is normally generated in the ER only in the eye. The cleavage of EGFR ligands depends upon a distinct family of serine proteases that carry out intramembrane proteolysis. Rhomboid-1 is the primary player, and hence mutations in this gene give rise to embryonic phenotypes that are similar to spi or Star (Schlesinger, 2004).

Two additional members of the family, Rhomboid-2/Brho and Rhomboid-3/Roughoid (Ru), are expressed in the oocyte and the eye, respectively. Recently, expression of Ru was also detected in the embryonic VUM neurons, where it plays a role in guidance of tracheal migration in the CNS. Homozygous null mutations for ru demonstrated that it is essential for normal eye development, but its role is partially redundant with Rhomboid-1, since some photoreceptor cells are recruited in ru mutants. The question is whether there are properties of Ru that are distinct from those of Rhomboid-1 and may account for the generation of cSpi in the ER during eye development (Schlesinger, 2004).

As far as substrate specificity is concerned, the Rhomboid 1-3 proteins are all capable of cleaving the membrane precursors of the EGFR ligands Spi, Keren, and Gurken. In addition, all three proteins are enriched in the Golgi when expressed in mammalian cells. However, functional assays in cell culture, including both Drosophila and mammalian cells, suggest that in contrast to Rhomboid-1, Ru may be capable of cleaving the Spi precursor already in the ER. However, cSpi is secreted only upon coexpression of Star. While Ru is located primarily in the Golgi, this cleavage may take place en route to the Golgi. The failure to secrete cSpi in the absence of Star likely represents the property of ER retention that was uncovered in this work (Schlesinger, 2004).

ru and sl give rise to opposite phenotypes in the eye. It is assumed that they act in a sequential manner, i.e., Ru generates cSpi in the ER and Sl mediates the retention of this ligand, to avoid excessive secretion. The genetic interaction experiments between ru and sl can be interpreted within this context. Indeed, the eye phenotype of hypomorphic mutations in ru could be efficiently rescued by mutations in sl. While the level of cSpi in the ER was compromised, more efficient secretion was facilitated in the sl background, thus compensating for the initial defect. Surprisingly, even null mutations in ru were partially rescued by sl mutants. In a ru null background, Rhomboid-1 is the only other known Rhomboid family protease that is functional in the eye. It is suggested that residual levels of cSpi may also be generated in the ER by Rhomboid-1. An efficient release of these low levels in the absence of Sl may lead to the partial rescue that was observed in ru null mutants (Schlesinger, 2004).

Finally, the requirement for Sl specifically in the cells processing the ligand was demonstrated by the capacity to rescue sl null flies by expressing Sl in the R8 cells. Normally, expression of Rhomboid-1 and Ru in the differentiating photoreceptor cells is induced by EGFR activation, thus making these cells a source for subsequent rounds of photoreceptor cell recruitment. Incomplete rescue by expression of Sl in R8 cells may be explained by the failure to restore ER retention of cSpi in the other photoreceptor cells expressing Ru (Schlesinger, 2004).

In conclusion, it has been demonstrated that the cleaved form of Spi is efficiently retained in the ER through a novel mechanism. This retention is significant only in the developing eye, where the Rhomboid-3/Ru protein may normally generate the cleaved ligand in the ER. Thus, in spite of efficient cleavage of mSpi in the ER, only the molecules that will overcome retention by association with Star will be secreted to activate EGFR in the neighboring cells. small wing, encoding a PLCγ, provides a link to the retention mechanism, and sl mutants exhibit EGFR hyperactivation phenotypes mainly in the eye. The eye is a tissue where the restricted range of EGFR activation is particularly crucial. The number of undifferentiated precursor cells is limited. EGFR activation is responsible for sequential inductions of the different cell types comprising the mature ommatidia. It is thus imperative to restrict the number of cells that are induced at every cycle. Negative feedback loops that are transcriptionally induced by EGFR activation in the cells receiving the signal were previously shown to be central to this restriction. This study demonstrates that fine tuning the level of ligand that is released by the cells providing the signal represents another cardinal tier of regulation (Schlesinger, 2004).


EVOLUTIONARY HOMOLOGS
PLC activation by Epidermal growth factor receptor

EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation. This suggests that the immediate downstream effector molecule contains an src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. Signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells were used to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF augments PLC activity in all five EGFR mutant cell lines which also respond by increased cell movement. Basal phosphoinositide turnover is not affected by EGF in the lines that do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggests, but does not prove, a causal link. A specific inhibitor of PLC, U73122, diminishes both the EGF-induced motility and PLC responses, while its inactive analog U73343 has no effect on these responses. Both the PLC and motility responses are decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Anti-sense oligonucleotides to PLC gamma-1 were found to reduce both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induce the mitogenic response while only those that are autophosphorylated induce PLC activity. U73122 does not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment does not diminish EGF-induced thymidine incorporation. All kinase active EGFRs stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induce cell movement; this EGF-induced MAP kinase activity is not affected by U73122 at concentrations that depress the motility response. Thus, the signaling pathways that lead to motility and cell proliferation diverge at the immediate post-receptor stage, and it is suggested that this is accomplished by differential activation of effector molecules (Chen, 1994).

Phospholipase C-gamma (PLC gamma) is required for EGF-induced motility; however, the molecular basis of how PLC gamma modulates the actin filament network underlying cell motility remains undetermined. It is propose that one connection to the actin cytoskeleton is direct hydrolysis of PIP2, with subsequent mobilization of membrane-associated actin modifying proteins. Signaling-restricted EGFR mutants expressed in receptor-devoid NR6 fibroblast cells were used to investigate whether EGFR activation of PLC causes gelsolin mobilization from the cell membrane in vivo and whether this translocation facilitates cell movement. Gelsolin anti-sense oligonucleotide treatment of NR6 cells expressing the motogenic full-length (WT) and truncated c'1000 EGFR decrease endogenous gelsolin by 30%-60%; this results in preferential reduction of EGF-induced cell movement by > 50%, with little effect on the basal motility. Since 14 h of EGF stimulation of cells does not increase total cell gelsolin content, it was necessary to determine whether EGF induces redistribution of gelsolin from the membrane fraction. EGF treatment decreases the gelsolin mass associated with the membrane fraction in motogenic WT and c'1000 EGFR NR6 cells but not in cells expressing the fully mitogenic, but nonmotogenic c'973 EGFR. Blocking PLC activity with the pharmacologic agent U73122 diminishes both this mobilization of gelsolin and EGF-induced motility, suggesting that gelsolin mobilization is downstream of PLC. Reorganization of submembranous actin filaments is concomitantly observed, correlating directly with PLC activation and gelsolin mobilization. In vivo expression of a peptide that is reported to compete in vitro with gelsolin in binding to PIP2 dramatically increases basal cell motility in NR6 cells expressing either motogenic (WT and c'1000) or nonmotogenic (c'973) EGFR; EGF does not further augment cell motility and gelsolin mobilization. Cells expressing this peptide demonstrate actin reorganization similar to that observed in EGF-treated control cells; the peptide-induced changes are unaffected by U73122. These data suggest that much of the EGF-induced motility and cytoskeletal alterations can be reproduced by displacement of select actin-modifying proteins from a PIP2-bound state. This provides a signaling mechanism for translating cell surface receptor-mediated biochemical reactions to the cell movement machinery (Chen, 1996a).

Having determined that the Epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level (Chen, 1994), the question arises as to how these two mutually exclusive cell responses cross-communicate. A possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis was investigated. Inhibition of PLC gamma activation by U73122 augments the EGF-induced [3H]thymidine incorporation by 23%-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis is observed in parallel. The time dependence of this increase reveals that it is due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrate augmented mitogenic responses by 25%-68%, when compared with control cells. PLCz- or U73122-augmented mitogenesis is not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) results in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrate EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells are treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response (Chen, 1996b).

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. The localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor has been investigated. Addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrate that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies it is found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signaling complex of its receptor, phospholipase C-gamma 1 and filamentous actin, which is primarily localized at membrane ruffles (Diakonova, 1995).

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites results in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhances its inositol trisphosphate (IP3) and calcium signals and results in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibits a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impairs the IP3/calcium signal and attenuates the MAP kinase activation and receptor dephosphorylation, but results in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. These findings establish the significance of PLC gamma affinity for signal definition; the role of this receptor tyrosine kinase substrate as a negative feedback regulator, and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations (Obermeier, 1996).

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, an investigation was carried out to determine how epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFRs) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30%, within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility (0.1 nM EGF). EGFR kinase activity is required because cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. Known motility-associated pathways were examined to determine whether these contribute to EGF-induced effects. Phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973), which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, it became necessary to explore the possible involvement of this signaling pathway, one not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration, has been revealed for the first time. Furthermore, a model of EGF-induced motogenic cell responses is proposed in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion (Xie, 1998).

The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-gamma1 is regarded to be critical for EGF-induced mitogenesis. In this study, a phospholipase complex composed of PLC-gamma1 and phospholipase D2 (PLD2) is reported. PLC-gamma1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation analysis in COS-7 cells show that the Src homology (SH) 3 domain of PLC-gamma1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-gamma1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol 1,4,5-trisphosphate [IP(3)] formation and Ca(2+) increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-gamma1 and PLD2 and fails to potentiate EGF-induced IP(3) formation and Ca(2+) increase. However, neither PLD2 wild type nor PLD2 mutant affects the EGF-induced tyrosine phosphorylation of PLC-gamma1. These findings suggest that, upon EGF stimulation, PLC-gamma1 directly interacts with PLD2 and this interaction is important for PLC-gamma1 activity (Jang, 2003).

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis Cbl has been identified as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 is induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 are recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogates the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuates EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation (Choi, 2003).

Phospholipase C-gamma couples receptor signaling to Ras activation

Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, Sos1/Sos2 double- or RasGRP3-deficient B cell lines were generated and their ability to mediate Ras activation upon B cell receptor (BCR) stimulation was determined. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation (Oh-hora, 2003).

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. In response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocates to the Golgi where it activates Ras. Whereas Ca(2+) positively regulates Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, is limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induces differentiation of PC12 cells, transformed fibroblasts and mediates radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane (Bivona, 2003).

Phospholipase C-gamma1 (PLC-gamma1), which interacts with a variety of signaling molecules through its two Src homology (SH) 2 domains and a single SH3 domain has been implicated in the regulation of many cellular functions. PLC-gamma1 acts as a guanine nucleotide exchange factor (GEF) of dynamin-1, a 100 kDa GTPase protein, which is involved in clathrin-mediated endocytosis of epidermal growth factor (EGF) receptor. Overexpression of PLC-gamma1 increases endocytosis of the EGF receptor by increasing guanine nucleotide exchange activity of dynamin-1. The GEF activity of PLC-gamma1 is mediated by the direct interaction of its SH3 domain with dynamin-1. EGF-dependent activation of ERK and serum response element (SRE) are both up-regulated in PC12 cells stably overexpressing PLC-gamma1, but knockdown of PLC-gamma1 by siRNA significantly reduces ERK activation. These results establish a new role for PLC-gamma1 in the regulation of endocytosis and suggest that endocytosis of activated EGF receptors may mediate PLC-gamma1-dependent proliferation (Choi, 2004).

Interaction of Phospholipase C gamma with growth factor receptors and other tyrosine kinases

Binding of macrophage colony stimulating factor (M-CSF) to its receptor (Fms) induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins that relay growth and development signals. To determine whether a distinct signaling pathway is responsible for the Fms differentiation signal versus the growth signal, a search for new molecules involved in Fms signaling was carried out employing a two-hybrid screen in yeast, using the autophosphorylated cytoplasmic domain of the wild-type Fms receptor as bait. Clones containing SH2 domains of phospholipase C-gamma2 (PLC-gamma2) are frequently isolated and have been shown to interact with the phosphorylated Tyr721 of the Fms receptor, which is also the binding site of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase). At variance with previous reports, M-CSF induces rapid and transient tyrosine phosphorylation of PLC-gamma2 in myeloid FDC-P1 cells; this activation requires the activity of the PI3-kinase pathway. The Fms Y721F mutation strongly decreases this activation. Moreover, the Fms Y807F mutation decreases both binding and phosphorylation of PLC-gamma2 but not that of p85. Since the Fms Y807F mutation abrogates the differentiation signal when expressed in FDC-P1 cells and since this phenotype could be reproduced by a specific inhibitor of PLC-gamma, it is proposed that a balance between the activities of PLC-gamma2 and PI3-kinase in response to M-CSF is required for cell differentiation (Bourette, 1997).

The neuregulins comprise a subfamily of epidermal growth factor (EGF)-like growth factors that elicit diverse cellular responses by activating members of the ErbB family of receptor tyrosine kinases. Although neuregulin-1 and neuregulin-2 are both binding ligands for the ErbB3 and ErbB4 receptors, they exhibit distinct biological activities depending on cellular context. In MDA-MB-468 human mammary tumor cells, neuregulin-2beta (NRG2beta) inhibits cell growth, whereas neuregulin-1beta (NRG1beta) does not. In these cells, NRG2beta appears to preferentially act through the EGF receptor, stimulating receptor tyrosine phosphorylation and the recruitment of phospholipase C-gamma, Cbl, SHP2, and Shc to that receptor. NRG1beta preferentially acts through ErbB3 in these cells by stimulating the tyrosine phosphorylation and recruitment of phosphatidylinositol 3-kinase and Shc to that receptor. In MDA-MB-453 cells, both NRG1beta and NRG2beta stimulate the tyrosine phosphorylation of the ErbB2 and ErbB3 receptors to similar extents, but only NRG1beta potently stimulates morphological changes consistent with cellular differentiation. The profiles of SH2 domain-containing proteins that are efficiently recruited to activated receptors differ for the two factors. These observations indicate that despite their overlapping receptor specificity, the neuregulins exhibit distinct biological and biochemical properties. Since both of these cell lines express only two of the known ErbB receptors, these results imply that EGF-like ligands might elicit differential signaling within the context of a single receptor heterodimer (Crovello, 1998).

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains that promote protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain [SH2(N) domain] results in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutants fail to associate with either Grb2 or a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain [SH2(C) domain] does not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 is not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase (Stoica, 1998).

Interactions and biological functions of Phospholipase C gamma's SH3 domains

Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit-induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit-induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1, with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation (Sette, 1998).

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Do sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) affect the binding to Cbl, a target used as a prototypical ligand? A weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain has been demonstrated but a high-avidity binding occurs to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increase binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl depends entirely on the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency (Graham, 1998).

Phospholipase C gamma 1 (PLC-gamma 1) hydrolyses phosphatidylinositol-4,5-bisphosphate to the second messengers inositol-1,4,5-trisphosphate and diacylglycerol. PLC-gamma 1 also has mitogenic activity upon growth-factor-dependent tyrosine phosphorylation; however, this activity is not dependent on the phospholipase activity of PLC-gamma 1, but requires an SH3 domain. PLC-gamma 1 acts as a guanine nucleotide exchange factor (GEF) for PIKE (phosphatidylinositol-3-OH kinase [PI(3)K] enhancer). PIKE is a nuclear GTPase that activates nuclear PI(3)K activity, and mediates the physiological activation by nerve growth factor (NGF) of nuclear PI(3)K activity. This enzymatic activity accounts for the mitogenic properties of PLC-gamma 1 (Ye, 2002).

Other interactions of PLCgamma

Epidermal growth factor (EGF)-induced autophosphorylation of the EGF receptor results in high-affinity binding of the adaptor protein GRB2, which serves as a convergence point for multiple signaling pathways. Present studies demonstrate that in WB cells EGF induces the co-immunoprecipitation of phospholipase C (PLC)-gamma1 with the adaptor protein GRB2 and the guanine nucleotide exchange factor Sos, but not with the adaptor protein SHC. Inhibition of PLC-gamma1 tyrosine phosphorylation by phenylarsine oxide reduces the co-immunoprecipitation of PLC-gamma1 with GRB2. Furthermore, angiotensin II, a G protein-coupled receptor agonist, also induces the tyrosine phosphorylation of PLC-gamma1 and its co-immunoprecipitation with GRB2 in WB cells. Interestingly, angiotensin II stimulation also causes tyrosine phosphorylation of the EGF receptor, suggesting that angiotensin II-induced PLC-gamma1 tyrosine phosphorylation in WB cells may be via EGF receptor tyrosine kinase activation. In addition, there is some level of association between PLC-gamma1 and GRB2 that is independent of the tyrosine phosphorylation of PLC-gamma1 in both in vivo and in vitro studies. In vitro studies further demonstrate that the Tyr771 and Tyr783 region of PLC-gamma1 and the SH2 domain of GRB2 are potentially involved in the tyrosine phosphorylation-dependent association between PLC-gamma1 and GRB2. The association of PLC-gamma1 with GRB2 and Sos suggests that PLC-gamma1 may be directly involved in the Ras signaling pathway and that GRB2 may be involved in the translocation of PLC-gamma1 from cytosol to the plasma membrane as a necessary step for its effect on inositol lipid hydrolysis (Pei, 1997).

Tyrosine phosphorylation of cellular proteins mediates the assembly and localization of effector proteins through interactions facilitated by modular Src homology 2 (SH2) and phosphotyrosine binding domains. Two tyrosine-phosphorylated proteins with Mr values of 70,000 and 68,000 are described that have been found to interact with Grb2, phospholipase C (PLCgamma1 and PLCgamma2), and Vav -- this occurs after B cell receptor cross-linking. The interaction of pp70 and pp68 with PLC and Vav is mediated by the carboxyl-terminal SH2 domain of PLC and the SH2 domain of Vav. In contrast, the interaction of pp70 and pp68 with Grb2 requires cooperative binding of the SH2 and SH3 domains of Grb2. Western blot analysis demonstrates that neither pp70 nor pp68 represent the recently described linker protein SLP-76, which binds Grb2, PLC, and Vav in T cells after T cell receptor activation. Moreover, SLP-76 protein is not detected in a number of B cell lines or in normal mouse B cells. Hence, it is proposed that pp70 and pp68 likely represent B cell homologs of SLP-76 which facilitate and coordinate B cell activation (Fu, 1997).

The role of the phospholipase C gamma 1 (PLC gamma 1) in signal transduction was investigated by characterizing its interactions with proteins that may represent components of a novel signaling pathway. A 145-kDa protein that binds SH2 domain of PLC gamma 1 was purified from rat brain. The sequence of peptide derived from the purified binding protein now identifies it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Stable association of PLC gamma 1 with synaptojanin has been demonstrated. Synaptojanin is a protein that not only binds the carboxyl terminal SH2 domain of PLC gamma 1, but also inhibits PLC gamma 1 activity (Ahn, 1998).

PLC gamma membrane targeting by interaction with inositol phospholipids

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. The inositol phospholipid known as phosphatidylinositol 3,4, 5-trisphosphate (PtdIns-3,4,5-P3) has been shown to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K result in an approximately 40% increase in both intracellular IP3 generation and intracellular calcium release, as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibits the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3 production and intracellular calcium release, potentially via enhanced PtdIns-4, 5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCgamma to the lipid bilayer (Rameh, 1998).

Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase C gamma (PLC gamma) and phosphatidylinositol (PI) 3-kinase. While it is well established that PLC gamma activation requires tyrosine phosphorylation, additional regulation of PLC gamma is provided by the lipid products of PI 3-kinase. The pleckstrin homology (PH) domain of PLC gamma binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLC gamma PH domain-mediated membrane targeting and PLC gamma activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLC gamma membrane targeting by overexpression of the PLC gamma PH domain prevents growth factor-induced PLC gamma activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism (Falasca, 1998).

Phospholipase C gamma signaling and downstream functions

Platelet-derived growth factor (PDGF) induces the phosphorylation of phospholipase C (PLC) gamma1. Phosphorylation on tyrosine (Tyr) 783 of PLCgamma1 is essential for phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vivo, while phosphorylation does not affect the catalytic activity in vitro. To study the roles of Tyr-783 phosphorylation in vivo, a polyclonal antibody containing phosphotyrosine 783 (alpha-PLCgamma1 PY) was developed that recognizes PLCgamma1 . Tyr-783-phosphorylated PLCgamma1 is not detected in the absence of PDGF:it appears after stimulation, increases for 30 min, and then decreases to near the prestimulation level. Immunostaining of cells show that PDGF-produced Tyr-783-phosphorylated PLCgamma1 localizes predominantly at membrane ruffles and stress fibers, where it colocalizes with actin filaments within 30 min. Ninety minutes after PDGF stimulation, the actin filaments are disassembled to short fragments, and the levels of Tyr-783-phosphorylated PLCgamma1 are remarkably decreased in membrane ruffles and cytoskeleton. Furthermore, the depolymerization of actin filaments and membrane ruffling caused by PDGF stimulation are blocked by microinjecting alpha-PLCgamma1 PY, such as occurs following the microinjection of the PLCgamma1-2SH2 domain, which is expected to associate with phosphorylated PDGF receptors and to block PLCgamma1 binding. It is worth noting that the microinjection of tyrosine-phosphorylated peptide (consisting of 13 amino acids containing Tyr-783) induces the disassembly of actin filaments and membrane ruffling as observed in PDGF-stimulated cells, while nonphosphorylated peptide does not cause any effect. These data suggest that the phosphorylation of PLCgamma1 on tyrosine 783 by PDGF plays an important role in cytoskeletal reorganization in addition to mitogenesis (Yu, 1998).

Phospholipase C gamma1 (PLC-gamma1) is phosphorylated upon treatment of cells with nerve growth factor (NGF). To assess the role of PLC-gamma1 in mediating the neuronal differentiation induced by NGF treatment, PC12 cells were established that overexpress whole PLC-gamma (PLC-gamma1PC12); the SH2-SH2-SH3 domain (PLC-gamma1SH223PC12); SH2-SH2-deleted mutants (PLC-gamma1deltaSH22PC12), and SH3-deleted mutants (PLC-gamma1deltaSH3PC12). Overexpressed whole PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 stimulates cell growth and inhibits NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-gamma1 that lack the SH2-SH2 domain or the SH3 domain have no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-gamma1 results in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-gamma1 does not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-gamma1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-gamma1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-gamma1 as well as intact PLC-gamma1 is tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-gamma1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-gamma1 but to the SH2-SH2-SH3 domain of PLC-gamma1 (Bae, 1998).

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. Evidence is presented for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma are observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, a specific inhibitor of PLC, U73122, was used to block insulin-stimulated GLUT4 translocation. Microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma blocks insulin-stimulated GLUT4 translocation. Inhibition of 2-deoxyglucose uptake is demonstrated in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. It is concluded that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes (Kayali, 1998).

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways; release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM; maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacities to release Ca2+ when a subsequent stimulus results in production of InsP3 (Wilson, 1998).

The cytoplasmic regions of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) bind and activate phospholipase C-gamma1 (PLC-gamma1) and other signaling proteins in response to ligand binding outside the cell. Receptor binding by PLC-gamma1 is a function of its SH2 domains and is required for growth factor-induced cell cycle progression into the S phase. Microinjection into MDCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide corresponding to the noncatalytic SH2-SH2-SH3 domains of PLC-gamma1 (PLC-gamma1 SH2-SH2-SH3) block growth factor-induced S-phase entry. Treatment of cells with diacylglycerol (DAG) alone, or just DAG and microinjected inositol-1,4,5-triphosphate (IP3), both products of activated PLC-gamma1, is not sufficient to stimulate cellular DNA synthesis, although such treatment does suppress the inhibitory effects of the PLC-gamma1 SH2-SH2-SH3 polypeptide (but not the cell cycle block imposed by inhibition of the adapter protein Grb2 or p21 Ras). Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, as well as a wild-type version (wtSRE-CAT) and a mutant (pm18) were used to investigate the function of PLC-gamma1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to both protein kinase C (PKC)-dependent and -independent signals, while the mutant, pm18, responds only to PKC-independent signals. Microinjection of the dominant-negative PLC-gamma1 SH2-SH2-SH3 polypeptide greatly reduces the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but has no effect on the responses of mutant pm18. These results indicate that in addition to the Grb2-mediated activation of Ras, PLC-gamma1-mediated DAG production is required for EGF- and PDGF-induced S-phase entry and gene expression, possibly through activation of PKC (Wang, 1998).

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. The FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, advantage was taken of this difference: receptor chimeras were prepared containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 have been identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction reveals that the JM region of FGFR-1 confers strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein is one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involves the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functions primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis reveals that the JM region does not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR (Lin, 1998).

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), targeted disruption of the Plcg1 gene in the mouse was used to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF show a time- and dose-dependent increase in the production of total inositol phosphates that is substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity, which has a similar dose dependence to the PLC response but is down-regulated after 15 min. Phospholipase D activity, however, is markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remain in the Plcg1(-/-) cells can be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells is phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells show Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells is activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells is not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin (to mimic the effect of PLC-gamma1) PLD activity is restored. The targeted disruption of Plcg1 does not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase is similar in Plcg1(+/+) and Plcg1(-/-) cells. Because an increase in plasma membrane ruffles occurs in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible that neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs (Hess, 1998).

Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. The phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, it is shown that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs (Rong, 2004).

Mutation of Phospholipase C gamma

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E 9.0. These results clearly demonstrate that PLC-gamma1 with (by inference) its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes (Ji, 1997).

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-gamma1, a ubiquitous tyrosine kinase substrate. Plcg1(-/-) embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1(+/+) embryos. The null cells do grow to a higher saturation density in serum-containing media, since their capacity to spread out is decreased when compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1(-/-) cells respond equivalently to PLcg1(+/+) cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-gamma1 for many responses to growth factors (Ji, 1998).


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