BIOLOGICAL OVERVIEW

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, the only protein phosphatase regulated by both Ca²⁺ and calmodulin, is a key player in Ca²⁺ signal transduction from yeast to humans and has been implicated in a wide array of processes, from disease progression to development (for a general review, see Rusnak, 2000). In the mammalian immune system, calcineurin is essential for T-cell activation and is the target of immunosuppressant drugs such as cyclosporin (Liu, 1991; Clipstone, 1992). An abundant neuronal protein, calcineurin has been implicated in various forms of synaptic plasticity (reviewed in Yakel, 1997). In addition, calcineurin is involved both in the development of cardiac valves (Ranger, 1998) and in hypertrophy of cardiac muscle (Molkentin, 1998) following disease or injury (Sullivan, 2002 and references therein).

The enzyme consists of an ~60-kD catalytic subunit, calcineurin A (canA), bound to the regulatory subunit, calcineurin B (see Drosophila Calcineurin B), a 19-kD EF-hand Ca²⁺-binding protein (reviewed in Klee, 1998). CanB is essential for phosphatase activity and can be dissociated from canA only by denaturants. CanA has two variable regions at the N and C termini, a highly conserved catalytic domain, and a regulatory region. The regulatory region consists of a binding site for Ca²⁺-calmodulin and a short autoinhibitory domain that blocks substrate access to the active site in the absence of Ca²⁺-calmodulin (Sullivan, 2002).

A Ca²⁺-calmodulin-independent, constitutively active phosphatase is made by deleting the canA regulatory region (O'Keefe, 1992). Studies from a number of different organisms indicate that, aside from a small degree of Ca²⁺ sensitivity mediated by canB, activated calcineurin functions identically to the full-length, Ca²⁺-calmodulin-activated form (Mendoza, 1996; Shibasaki, 1996; Winder, 1998; Sullivan, 2002 and references therein).

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 (reviewed in Berridge 1993). 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 (Crabtree, 1999) 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 (Sullivan, 2002).

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).

The Drosophila genome contains three canA genes and two canB genes that are 75% and 88% similar to the vertebrate genes, respectively. To date, no mutants have been described for any of the five genes. To study calcineurin function in Drosophila, a constitutively active form of CanA1 was expressed during imaginal development and the resulting phenotypes were examined. The activated calcineurin rough eye phenotype was used to perform a genetic modifier screen. Specific enhancers and suppressors were successfully isolated and characterized and two suppressors were identified as canB2 and sprouty. The activated calcineurin rough eye was also tested extensively for genetic interactions with an array of signaling cascades. Taken together, the genetic evidence is consistent with calcineurin functioning as a negative regulator of Egf receptor/Ras signaling during imaginal development, possibly in the same pathway as PLCgamma (Sullivan, 2002).

ThecanA gene Pp2B-14D was used for these experiments because it is expressed throughout development, including in eye discs (Brown, 1994). The adjacent canA gene at 14F1 (gene designation CG9819) encodes a protein that is 83% identical to Pp2B-14D; however, canA-14F is not represented in the expressed sequence tag collection and the expression pattern has not been characterized. The Calcineurin A1 gene at 100B4, which was incorrectly localized to 21B, is undetectable by Northern analysis (Guernini, 1992) and appears to have a highly restricted expression pattern (Sullivan, 2002).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains (O'Keefe, 1992; Mendoza, 1996). The canA^act 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 canA^act.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).

Removing one copy of glass by introducing the null allele gl^60J strongly suppresses the canA^act.gl rough eye phenotype. This demonstrates that the canA^act.gl rough eye phenotype is dependent on glass and is not caused by the insertion site of the transgene or by some other factor (Sullivan, 2002).

Reducing the dosage of canB-4F or canB2 by introducing deficiencies that uncover the 4F or 43E genomic region results in suppression of the canA^act.gl rough eye. Western blots confirmed that CanB protein is present in the eye disc; however, it is not known whether the protein is derived from one or both canB genes. Consistent with the effect of reduced CanB levels, canB4F^gl, which alone has no phenotype, increases the severity of the canA^act.gl rough eye (Sullivan, 2002).

Because expression occurs throughout the later stages of eye development, glass-dependent transgenes can affect many different processes. On the basis of these observations, activated calcineurin may have multiple effects on the differentiation and morphology of photoreceptor and other cell types. However, no effect of canA^act.gl on cell proliferation or cell death was observed (Sullivan, 2002).

Several lines of evidence demonstrate that phosphatase activity is required for ectopic canA^act phenotypes in Drosophila. Genetically raising or lowering the level of CanB, which is essential for activity, respectively enhances or suppresses the phenotype of canA^act. Transgenic flies expressing a form of canA^act that lacks an intact CanB-binding site are indistinguishable from wild-type flies. Finally, the full-length phosphatase, which is inactive in the absence of Ca²⁺-calmodulin, does not have a detectable phenotype when overexpressed throughout development (Sullivan, 2002).

Activated calcineurin has been used reliably to identify physiologically relevant functions of calcineurin in a number of systems (Fruman, 1995; Mendoza, 1996; Molkentin, 1998; Winder, 1998). However, it remains formally possible that calcineurin does not normally function in the cells or at the stage of development in which canA^act has a detectable phenotype, even though Pp2B-14D appears to be ubiquitously expressed. The role of calcineurin must be confirmed by mutational analysis of the canA and canB genes. Despite this caveat, the genetic screen presented in this study can still be used to identify physiological targets of calcineurin in Drosophila, as well as to provide insight into the roles calcineurin may play in development (Sullivan, 2002).

The canA^act.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).

Consistent with this classification, the class II group CS2-1 is CanB2. The allele CS2-1⁸⁷ has an inversion that breaks within 400 bp of the CanB2 start of transcription. CS2-1⁸⁷ and CS2-1¹⁸⁰ have decreased protein levels compared to similarly staged controls. Finally, the lethality of CS2-1¹⁸⁰ is partially rescued by CanB-4F. The ability of CanB-4F to rescue the CanB2 lesion suggests that the CanB-4F protein can at least partially substitute for CanB2. More importantly, isolation of the calcineurin regulatory subunit in the canA^act.gl modifier screen demonstrates that the screen is capable of identifying genes that are required for calcineurin function (Sullivan, 2002).

The class I modifier group CS3-3 failed to complement the hypomorphic sprouty alleles sty^Delta5 and sty^Delta64; both sty^Delta5 and sty^Delta64 also suppressed canA^act.gl, and the sty gene from CS3-3⁵¹⁸ harbored a nonsense mutation (Q250Stop). Therefore, it is concluded that the CS3-3 complementation group is sprouty. The fact that sty falls into the class II group suggests that sprouty functions downstream of calcineurin and/or in a parallel pathway (Sullivan, 2002).

Two lines of evidence suggest that calcineurin is a negative regulator of Egf receptor/Ras signaling. First, a negative regulator of RTK signaling, sprouty, was isolated as a suppressor of the canA^act.gl rough eye phenotype in the dominant modifier screen. Both sprouty and canA^act suppress wing vein formation and reduce the number of photoreceptor cells per ommatidium. Egf receptor/Ras signaling is essential for both wing vein and R-cell formation (Sullivan, 2002).

A thorough examination of genetic interactions between canA^act and components of RTK and other signaling pathways has confirmed that canA^act specifically represses the Egf receptor/Ras pathway and that it acts upstream in the pathway. The lack of convincing genetic interactions with other signaling pathways in the imaginal eye disc does not rule out a role for calcineurin in these pathways in other developmental contexts. With the exception of pnt, activated calcineurin was not modified by components downstream of Ras and was modified only by a subset of genes that act between the Egf receptor and Ras. While Gap1 and sty alleles modify the effects of activated calcineurin, drk and cbl do not. Thus calcineurin may act downstream of, or parallel to, drk and cbl. The more downstream components of the Ras/MAP kinase pathway may not interact with activated calcineurin because they are too far removed from the point(s) of intersection between calcineurin and the pathway. Alternatively, these components may not be limiting, so that reduction of gene dose, which is the basis of a dominant modifier screen, would have no appreciable effect (Sullivan, 2002).

The hypermorphic allele Egfr^E1 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 Egfr^E1 rough eye by canA^act.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).

In conclusion, it has been demonstrated that a dominant modifier screen can be used successfully to isolate mutations in genes involved in calcineurin function. The mutations in the calcineurin B gene that was isolated in the screen will help determine the roles of calcineurin in Drosophila development. In addition, compelling genetic evidence was presented that calcineurin negatively regulates the Egf receptor/Ras signaling pathway at the level of Gap1 and sprouty. Calcineurin may act directly by dephosphorylating one or more signaling components, or it may target a transcription factor and act indirectly through changes in gene expression. More work will be needed to elucidate the molecular mechanism, and the modifiers isolated in the canA^act.gl screen should prove valuable in this endeavor. Furthermore, given the conservation of signal transduction between fruit flies and vertebrates, it is likely that the signaling network that was identified is employed in other organisms (Sullivan, 2002).

GENE STRUCTURE

cDNA clone length - 2542

Bases in 5' UTR - 327

Exons - 12

Bases in 3' UTR - 446

PROTEIN STRUCTURE

Amino Acids - 568

Structural Domains

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of Calcineurin A, the catalytic subunit of calcineurin, were used to isolate Calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67% and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man (Guerini, 1992).

A 3.3 kb cDNA encoding the complete amino acid sequence of a calcium/calmodulin regulated protein phosphatase has been isolated from a Drosophila eye disc cDNA library. The predicted protein of 560 amino acids (molecular mass 62 kDa) is 73%-78% identical to human PP2B isoforms. The cDNA hybridized to the X-chromosome at cytological position 14D1-4. Two transcripts of 3.5 kb and 3.0 kb were expressed during embryonic development, their levels being highest in the early embryo. The larger transcript was also clearly present in adult females (Brown, 1994).

date revised: 10 August 2002

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