InteractiveFly: GeneBrief

flap wing: Biological Overview | References

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. Drosophila PP1β, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1β. Loss of PP1β leads to increased levels of phosphorylated nonmuscle MRLC (Squash) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1β, and activates PP1β's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1β and MYPT genes suggests this role may be conserved (Vereshchagina, 2004).

Nonmuscle myosin II, a molecular motor closely related to vertebrate smooth muscle myosin, powers the actomyosin cytoskeleton. It is required for the coordinated changes in the shape and position of individual cells during morphogenesis as well as for cytokinesis and other cell movements. Nonmuscle myosin II activity is also modulated by metastasis-related and tumor suppressor genes (reviewed Bresnick, 1999: Vereshchagina, 2004).

The regulation of nonmuscle myosin is thought to be broadly similar to that of vertebrate smooth muscle myosin (Bresnick, 1999). Contraction and relaxation of vertebrate smooth muscle are regulated by the reversible phosphorylation of myosin regulatory light chain (MRLC), principally on Ser-19. The motor activity of smooth muscle myosin is regulated by the balance of activatory phosphorylation, leading to muscle contraction, and inhibitory dephosphorylation, leading to relaxation. The spectrum of stimulating kinases includes myosin light-chain kinase (MLCK), Rho-associated protein kinase (ROK), p21-associated kinase (PAK), integrin-linked kinase (ILK) and leucine zipper-interacting protein kinase (Dlk/ZIP kinase). The antagonistic protein phosphatase is the catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) in association with its myosin phosphatase targeting subunit MYPT1 or MYPT2, and a small subunit of unknown function (reviewed by Hartshorne, 1998). These kinases and phosphatases are themselves subject to regulation by reversible phosphorylation, for example ROK not only phosphorylates and activates MRLC, but also phosphorylates MYPT1 and inhibits MRLC dephosphorylation (reviewed Kaibuchi, 1999; Somlyo, 2000). The nonmuscle roles of these myosin-regulating kinases are less clear, though at least one (ROK) also regulates non-muscle myosin II in both mammals and Drosophila. Similarly, though PP1 is often assumed to be the major non-muscle MRLC phosphatase, PP2A has also been implicated. The various phosphorylation events have been investigated biochemically, but little is known about their physiological significance, particularly in nonmuscle cells (Vereshchagina, 2004).

Drosophila nonmuscle myosin II heavy chain zipper (zip) and regulatory light chain spaghetti squash (sqh) are essential for the normal development of a very wide range of cells and tissues. Drosophila Rho-kinase (Drok) phosphorylates both Sqh and DMBS (the single Drosophila homolog of MYPT1/2; Mizuno, 2002; Tan, 2003). By analogy to the vertebrate smooth muscle system it was proposed that this phosphorylation activates myosin and inhibits myosin phosphatase (Vereshchagina, 2004).

PP1 is involved in the regulation of many cellular functions including glycogen metabolism, muscle contraction, and mitosis (reviewed Bollen, 2001; Cohen, 2002). In Drosophila, the four genes encoding isoforms of PP1c are named by their chromosome location and subtype: PP1β9C, PP1α13C, PP1α87B, and PP1α96A (Dombrádi, 1990b, Dombrádi, 1993). Of these, PP1α87B contributes 80% of the total PP1 activity, therefore the phenotypes of PP1α87B loss of function mutants (Axton, 1990; Dombrádi, 1990a; Baksa, 1993) may be due to a loss of overall PP1 activity, rather than identifying specific functions unique to the PP1α87B protein. Mice and humans have three PP1 genes: PP1α and PP1γ are related to the fly PP1α genes, although PP1δ (also known as PP1β) corresponds to fly PP1β. Of the mammalian genes, functional analysis by gene knockout in mice has so far only been performed for PP1γ (Varmuza, 1999). This knockout eliminates both the widely expressed PP1γ 1 and the testis-specific PP1γ 2. Homozygous mutant female mice are viable and fertile; homozygous mutant males are viable but sterile, with defects in spermatogenesis. Presumably the somatic and female germline functions of PP1γ are redundant with PP1α and/or PP1δ (Vereshchagina, 2004).

The in vitro biochemical activities of the PP1c isoforms are very similar. However, genetic analysis provides a powerful approach to analyze the specific, nonredundant functions of each isoform. The Drosophila PP1β catalytic subunit gene PP1β9C corresponds to flapwing (flw), weak alleles of which are viable but flightless (Raghavan, 2000). The semilethality of a strong allele, flw⁶, demonstrated that PP1β is essential in flies. flw⁶ larval body wall muscles appeared to form normally, but then detached and degenerated, leading to a semiparalyzed larva that could not feed properly (Raghavan, 2000). In addition to muscle defects, the occasional male flw⁶ survivors were sterile and had blistered wings, indicating a nonredundant role for PP1β9C in nonmuscle cells as well as in muscles (Vereshchagina, 2004).

This study shows that the essential role of PP1β in flies is to regulate nonmuscle actomyosin. The lethality of strong flw (PP1β) mutants is suppressed by reducing the level of phospho-Sqh (MRLC), either using nonphosphorylatable point mutants of sqh or by reducing the gene dosage of key regulators such as Rho1 or RhoGEF2. flw mutants are also suppressed by reducing the gene dosage of nonmuscle myosin heavy chain (zipper). Clones of ovarian follicle cells mutant for flw⁶ have increased levels of phospho-Sqh, leading to disorganized or absent F-actin and to increased levels of myosin. Therefore, although PP1 isoforms collectively have many known roles, the essential, nonredundant role for PP1β in Drosophila is in the regulation of nonmuscle myosin activity and actin organization (Vereshchagina, 2004).

Drosophila has been reported to have only one MYPT homolog, named DMBS (Mizuno, 2002; also known as DMYPT, Tan, 2003). This study demonstrates that DMBS binds both α and β isoforms of PP1 and is therefore unlikely to mediate a PP1β-specific function. However a Drosophila PP1β-specific regulatory subunit, MYPT-75D, has been identified that is similar to mammalian MYPT3, a prenylated MYPT1/2 paralog (Skinner, 2001). MYPT-75D binds specifically to PP1β in vitro and the two proteins coimmunoprecipitate from fly extracts. MYPT-75D can stimulate PP1β's Sqh phosphatase activity in vitro and MYPT-75D, PP1β and Sqh proteins coimmunoprecipitate. Expression of a nonPP1 binding form of MYPT-75D in flies results in elevation of phospho-Sqh and phenotypic consequences that can be suppressed by reducing the level of Sqh phosphorylation. It is concluded that PP1β is targeted to Sqh by MYPT-75D, where it performs an essential role in the regulation of Sqh phosphorylation, and hence myosin activity, for which other PP1c isoforms cannot substitute. The conservation of all of these components, including the PP1α and β isoforms, suggests that regulation of nonmuscle myosin in mammals may also involve the activity of PP1β and an isoform-specific myosin targeting subunit (Vereshchagina, 2004).

This study shows that two semilethal mutant alleles of PP1β can be dominantly suppressed by loss-of-function extragenic mutations. The existence of single-gene extragenic suppressors indicates that PP1β has a single essential role, the identity of the suppressors indicates that this role is in the regulation of actin and/or myosin. Though the main defect observed in flw mutants is muscle detachment and degeneration, it is clear from these data that it is nonmuscle myosin, rather than muscle myosin, that is affected. Zipper and Sqh are components of nonmuscle myosin; the muscle version of Sqh, Mlc2, does not interact with flw (Raghavan, 2000). Similarly, Tm1, but not the muscle-specific Tm2, suppresses flw. Disruption of nonmuscle myosin in flw mutants may lead to disruption of the actin cytoskeleton and affect cell adhesion in many cell types, but seems to be most readily apparent in contractile muscle, particularly the highly specialized indirect flight muscles (Raghavan, 2000). Though not directly involved in generation of contractile force, nonmuscle myosin seems to be necessary for the correct development of striated myofibrils (Vereshchagina, 2004).

The dominant suppression of the lethality of flw⁶ and flw⁷ mutants by Sqh^A20A21, coupled with the enhancement of flw¹ by Sqh^E20E21, implies that the essential role of PP1β 9C is related to the regulation of the phosphorylation state of Sqh. To address whether this interaction is direct or indirect, it has been shown that PP1β can directly dephosphorylate phospho-Sqh in vitro and that the two proteins coimmunoprecipitate from Drosophila extracts. Furthermore, a new PP1β-specific MYPT has been identified, and this study shows that binding of MYPT-75D to PP1β stimulates dephosphorylation of nonmuscle MRLC both in vitro and in vivo. It is therefore concluded that the major or only essential role of PP1β in Drosophila is to dephosphorylate Sqh and that this role is mediated, at least in part, by association with a β-specific MYPT protein. Although flw⁶ behaves as a null allele by genetic tests (Raghavan, 2000), the possibility cannot be ruled out that it has some residual activity and that this is sufficient to perform one or more additional essential functions of PP1β, which for some reason require only a very low level of PP1β activity. PP1β, MRLC and MYPT proteins are highly conserved between flies and mammals, so it seems likely that dephosphorylation of MRLC is also an essential role of PP1β in humans (Vereshchagina, 2004).

Though PP1β can dephosphorylate Sqh directly and manipulating the phosphorylation state of Sqh is sufficient to suppress strong mutants of flw, the possibility that flw has other substrates in the same pathway cannot be excluded. For example, nonmuscle myosin heavy chain, which in mammals can be phosphorylated by PKC and CKII, could also be a substrate for PP1β (Vereshchagina, 2004).

What is the molecular basis of the suppression of flw? It is believed that the key defect, both in flw mutants and in flies expressing MYPT-75D^F117A, is the hyperphosphorylation of Sqh, particularly on Ser-21; this is directly suppressed by the nonphosphorylatable Sqh mutants. In these experiments a pool of normal Sqh remains, so essentially the ratio of phosphorylated and nonphosphorylated Sqh is being manipulated. Phosphorylation of Sqh leads to activation of the myosin motor; reduction in the amount of myosin heavy chain in zipper^+/- presumably reduces the amount of active motor. Sqh is known to be a substrate for Rho-kinase, itself activated by a pathway that includes two more suppressors: Rho1 and RhoGEF2. Rho-kinase itself is located on the X chromosome and was therefore not accessible to the genetic screen (Vereshchagina, 2004).

Tm1, a strong suppressor of flw⁶, is not a member of Rho-kinase pathway but a cytoskeletal actin-binding protein. Several functions have been ascribed to nonmuscle tropomyosin in mammals: modulation of myosin function, actin polymerization , regulating microfilament branching, and suppression of neoplastic transformation. Reduction in the amount of Tm1 appears to mitigate the consequences of hyper-phosphorylated Sqh; the obvious mechanism is by reducing the binding of active myosin to actin, though Tm1 could have its effect through regulation of actin structure and polymerization (Vereshchagina, 2004).

The phenotypes described for flw somewhat resemble those of DMBS, particularly in the female germ line (Tan, 2003) and in that they both lead to the accumulation of phospho-Sqh (Mizuno, 2002), though DMBS mutants do not show the accumulation of myosin aggregates (Tan, 2003). The differences in lethal phase (embryonic for DMBS, predominantly larval for flw) might be accounted for by maternal contribution and differences in protein stability; it was not possible to investigate this further since both DMBS and flw are required for oogenesis. Furthermore, the flw suppressors sqh^A20A21, Rho1 and zipper have been shown or deduced to modify at least some of the DMBS phenotypes. This might indicate that the critical role of flw is mediated by DMBS. However, DMBS is not specific for PP1β. PP1α87B is much more abundant than PP1β, so flw mutants should have little effect on the DMBS: PP1c complex. It is possible that DMBS:PP1β has a unique role not shared by DMBS:PP1α; it is also possible that DMBS, which is phosphorylated by Rho-kinase, is itself directly or indirectly activated by a PP1β-specific phosphatase complex. However, because an additional, PP1β-specific MYPT has been identified, it seems much more likely that this is the key targeting subunit that mediates the essential role of PP1β in vivo and that the suppression of flw by Rho and RhoGEF is through a decrease in phosphorylation of nonmuscle MRLC by Rho-kinase (Vereshchagina, 2004).

Why do flies have two MYPTs apparently doing the same job, one PP1β-specific and the other not? Clearly DMBS is not completely redundant with MYPT-75D, since DMBS mutants are lethal; mutants for MYPT-75D are not available to test the converse. One possible explanation for the presence of multiple myosin targeting subunits in mammals, flies and nematodes lies at the C-termini: MYPT-75D/MYPT3 have a CaaX prenylation motif, whereas DMBS/MYPT1/2 do not. MYPT-75D localizes to the cell periphery; this implies the existence of two different nonmuscle myosin phosphatases in different compartments of the cell: DMBS:PP1c (PP1α or PP1β) in the cytoplasm and MYPT-75D:PP1β at the plasma membrane. These myosin phosphatases have different roles and may be subject to different regulation. However, gross perturbation, such as complete removal of one complex in either DMBS or flw mutants, may lead to hyperphosphorylation of Sqh throughout the cell and hence to similar phenotypic consequences. Similarly, overexpression of the cytoplasmic form at a sufficiently high level may compensate for loss of the membrane-associated form: it was found that overexpression of a DMBS cDNA can suppress flw⁶, indicating that greatly increased levels of DMBS:PP1α87B can partially compensate for loss of functional MYPT-75D: PP1β9C complexes. A reduction in DMBS gene dose did not enhance flw¹, indicating that DMBS is not itself the key targeting subunit for PP1β. Overexpression of MYPT-75D did not suppress flw⁶, presumably because MYPT-75D is not limiting or because increased levels of a defective MYPT-75D:PP1β9C complex are not helpful. High-level overexpression of MYPT-75D is lethal to wild-type flies, and modest overexpression somewhat reduces the viability of flw¹ flies. Both of these results are interpreted as being due to excess MYPT-75D diverting some PP1β from its normal role or location. flw¹ flies, in which the MYPT-75D:PP1β9C myosin phosphatase is already somewhat defective, would be predicted to be more sensitive to this effect, as was observed (Vereshchagina, 2004).

In conclusion, PP1β has an essential role, which is in the regulation of nonmuscle myosin, and this can be entirely explained by its role as an MRLC phosphatase. It associates with two different myosin-targeting subunits, one of which is specific for PP1β. These two myosin phosphatases have different roles, though sufficiently high-level expression of the putative cytoplasmic form can partially compensate for loss of the putative membrane-associated form. Loss of PP1β, and hence the PP1β-specific myosin phosphatase, leads to cytoskeletal defects and death, as does loss of the other myosin phosphatase, indicating that each has an important, nonredundant role. All of the components of the system analyzed are well conserved between flies and humans, suggesting that the PP1β-specific myosin phosphatase may also be conserved (Vereshchagina, 2004).

The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1ß (PP1ß). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. This study shows genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251, and this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, this study shows that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs (Kirchner, 2007; full text of article).

This study shows that the nonmuscle myosin phosphatase flw interacts genetically with components of the JNK signaling pathway. The proteins JNK and PP1ß (Flw), as well as the mechanisms of JNK signal transduction and nonmuscle myosin activation, are highly conserved between Drosophila and humans. This suggests that findings of Drosophila PP1ß regulating JNK through nonmuscle myosin may be relevant for similar processes in human cells and tissues (Kirchner, 2007).

bsk (JNK) mutants suppressed the semilethality of the strong allele flw⁶, while puc and constitutively active hep^CA enhanced the weak viable allele flw¹, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw¹/Y ; puc^A251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw¹/Y ; puc^A251/+, implies that flw can act as a negative regulator of JNK. This was further supported by the fact that puc expression was not upregulated in flw¹/Y ; bsk¹/+ ; puc^A251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw¹/Y ; puc^A251/+. A possible difficulty with using puc^A251 (or puc^E69, another frequently used puc enhancer trap) as a reporter of puc expression is that both lines are also puc mutants, and puc probably regulates its own expression through a negative feedback loop involving JNK. However, it was confirmed in an independent assay with an anti-P-JNK antibody that JNK was indeed ectopically activated in flw¹/Y ; puc^A251/+. Wing imaginal disc extracts from both flw¹ and puc^A251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK. In flw¹/Y ; puc^A251/+, it is suggested that dephosphorylation of JNK fails to such an extent that JNK is substantially diphosphorylated and thereby activated. Since it has been shown that activation of JNK in wing imaginal discs induces apoptosis, it is likely that the flw¹/Y ; puc/+ wing phenotype is due to increased death of cells with aberrantly activated JNK (Kirchner, 2007).

A zip mutant suppresses the upregulation of puc in wing imaginal discs as well as the adult wing phenotype of flw¹/Y ; puc^A251/+. This shows that nonmuscle myosin acts upstream of JNK and mediates an activating signal on JNK in a flw¹/Y ; puc/+ mutant background; this is also consistent with the finding that a mutant in the myosin phosphatase-targeting subunit Mbs enhances flw¹/Y ; puc^A251/+ to lethality. Drosophila encodes two myosin phosphatase-targeting subunits, Mbs and MYPT-75D. Mbs binds both Flw and Pp1-87B, while MYPT-75D binds Flw specifically Vereshchagina, 2004). Unfortunately, it was not possible to test for genetic interaction between flw¹/Y ; puc^A251/+ and MYPT-75D because no MYPT-75D mutants have been described. It was also found that a Rho1 mutant abolishes ectopic lacZ staining in flw¹/Y ; puc^A251 wing imaginal discs. Rho1 is an activator of Rho-dependent kinase, which phosphorylates and activates myosin light chain, as well as phosphorylating and inhibiting Mbs. Furthermore, both zip and Rho1, in combination with other genetic interactors, have been reported to show a malformed third-leg phenotype that resembles that of flw¹/Y ; puc/+ (Kirchner, 2007).

Dorsal closure and wound healing depend on both nonmuscle myosin and JNK activity, but a clear genetic or molecular interaction between these pathways has not been previously demonstrated. This is the first study that shows that disruption of nonmuscle myosin can induce activation of JNK, although the mechanism of signal transduction is not clear. The single essential and nonredundant function of flw is the inhibition of nonmuscle myosin activity, presumably by dephosphorylating Sqh at T21 and S22. However, the results suggest that hyperphosphorylation of Sqh at these residues may not explain the elevated expression of puc in flw¹/Y ; puc^A251/+ flies. Additional mechanisms may exist to regulate nonmuscle myosin assembly and activity through phosphorylation, and the complex Flw/Mbs may have other targets in the actomyosin network in addition to Sqh. For example, moesin and focal adhesion kinase are potential targets for mammalian myosin phosphatase. Interestingly, overexpression of the focal adhesion protein tensin (blistery) in Drosophila wing imaginal discs activates JNK and induces apoptosis (Kirchner, 2007).

Two important questions remain unanswered regarding the results on the activation of JNK through nonmuscle myosin. (1) What is the molecular pathway from nonmuscle myosin to JNK? Several mechanisms have been identified that activate JNK and induce apoptosis in wing imaginal discs. For example, mutations in the caspase inhibitor DTraf1 (Drosophila tumor necrosis factor receptor-associated factor 1), as well as inhibition of DTraf1 by overexpression of Hid (head involution defective), Rpr (reaper), or Grim, induces JNK-mediated apoptosis, possibly through Msn (misshapen) or Ask1 (apoptosis signal-regulating kinase 1). Other factors involved in inducing apoptosis and activating JNK are Eiger (Drosophila tumor-necrosis factor superfamily ligand), the serine C-palmitoyltransferase Lace, Blistery (Drosophila Tensin), and Decapentaplegic and Wingless. It is not clear how these factors signal to JNK or whether they act in a single pathway, let alone whether any of them interact with nonmuscle myosin. It is likely that there are several independent ways of activating JNK, and it has been suggested that induction of apoptosis through JNK activation is a regulatory mechanism to eliminate abnormally developing cells in wing imaginal discs. (2)This leads directly to the second question: are the results of nonmuscle myosin signaling to JNK significant in a developmental context? The fact that bsk¹ suppresses the semilethality of flw⁶ indicates that the interactions that uncovered are not confined to the main experimental model of ectopic JNK activation in wing imaginal discs. The obvious system for studying possible interactions between nonmuscle myosin and JNK would be dorsal closure, which depends on both the coordinated assembly and the contraction of the actomyosin cytoskeleton and on activation of JNK. So far, there is no evidence that dorsal closure is affected in flw mutant embryos; however, there is a maternal contribution of flw that may conceal embryonic phenotypes. Actomyosin and JNK do not promote dorsal closure completely independently from each other; for example, the expression of many components of the actomyosin cytoskeleton is upregulated in response to JNK. Also, actin and myosin failed to accumulate along the leading edge of the epidermis in the puc mutant background. Both findings would place the actomyosin cytoskeleton downstream of JNK, whereas genetic data place flw and zip upstream of JNK and puc. It is possible, however, that actomyosin acts both upstream and downstream of JNK during dorsal closure. Because of the conserved nature of the components involved, it is likely that the finding that nonmuscle myosin can signal to and activate JNK is relevant to furthering the understanding of processes like dorsal closure and wound healing in Drosophila and humans (Kirchner, 2007).

Protein phosphatase 1β is required for the maintenance of muscle attachments

Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle. Drosophila and humans both have multiple genes encoding PP1 isoforms; each has one β and several α isoform genes (α1, α2, α3 in flies, α and γ in humans; mammalian PP1β is also known as PP1δ). The α/β subtype differences are highly conserved between flies and mammals. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, this study shows that the Drosophila β isoform has a distinct biological role. PP1β9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs). A new, semi-lethal flw allele has been islated that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate (Raghavan, 2000; full text of article).

Though Drosophila has four genes encoding PP1 isoforms, one of these (PP1α87B) encodes 80% of the total PP1 activity. Another PP1 gene, PP1β9C, is much more closely related (94% identity) to mammalian PP1β than it is to other PP1 genes from flies or mammals (85% identity), suggesting that it has a conserved function distinct from that of PP1α. This study analysed the intron/exon structure of PP1β9C and it was found that three of the four introns in its coding region correspond exactly to three of the seven introns in the human gene, confirming the ancestral origin of this isoform. The α and β isoforms therefore appear to have been preserved in each lineage since their divergence >500 million years ago, despite their extreme sequence identity (Raghavan, 2000).

The PP1 isoforms have indistinguishable activity in vitro, so attempts were made to identify the non-redundant role(s) of PP1β by mutational analysis. It was found that PP1β9C corresponds to the flightless mutant flapwing (flw), which was originally isolated in a screen for viable, flightless mutants. The conservation of PP1β between flies and mammals suggests an essential role. Therefore lethal alleles of flw were screened for using the chemical mutagen ethyl methane sulphonate (EMS). flw⁶, a new, semi-lethal allele of flw, was successfully isolated. This demonstrates for the first time that the α and β isoforms are both essential, despite their 85% sequence identity. The flw¹/flw⁶ flies are completely flightless and their muscle and wing phenotypes resemble combinations of flw¹ with deficiencies covering the flw region (flw¹/Df(1)N110 and flw¹/Df(1)Hk), so flw⁶ appears to act as an amorphic allele (Raghavan, 2000).

The IFMs comprise two sets of muscles, the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). Alternating contraction of these two sets of muscles resonates the thorax, which in turn drives the wings. The development of the IFMs has been analysed in some detail. They are constructed during pupation from myoblasts, previously sequestered in the wing imaginal disk. The DLMs are built on a template of the larval oblique muscles, which are spared from the general histolysis of larval muscles, whereas the DVMs are formed by de novo fusion of myoblasts. The IFM defect was investigated in flw mutants using polarised light microscopy, dissection and plastic sections. The flw¹ flies show a variable phenotype ranging from normal, through disorganised to absent IFMs. This phenotypic range was equally broad in an isogenic derivative line. IFMs were never present in flw¹/Df(1)N110, flw¹/Df(1)Hk or flw¹/flw⁶ flies, but the jump muscle (tergal depressor of trochanter muscle or TDT) appeared normal. In the few (<1%) flw⁶/Y males that eclosed, the IFMs were absent and the TDT disorganised. All aspects of the flw⁶/Y phenotype were fully rescued by a PP1βC cDNA expression construct, demonstrating that flapwing corresponds to PP1β9C (Raghavan, 2000).

IFM development was analyzed in flw¹/Df(1)Hk pupae. The early development of the IFMs appeared normal: the pre-templates on which the DLMs form were present, the DVMs also developed normally. Muscle defects became apparent at about 28 h after puparium formation. In the wild type, the muscles shorten and then elongate to form the final structure as they send out processes to their attachment sites. In the mutant, most of the muscles broke away from their posterior attachment sites and were found as ball-like structures at the anterior segment boundaries. These clumps were dissected and examined by electron microscopy. The sarcomere organisation appeared normal (Raghavan, 2000).

Less than 1% of flw⁶/Y animals survive to adult at 25°C. The period of lethality extends from the second larval instar onwards. Dying larvae are very sluggish, suggesting a possible defect in the larval body wall musculature. In the abdominal segments of the larva (A1-A7), each hemisegment contains a stereotyped set of 30 muscles. The musculature of flw⁶/Y was examined under polarised light and by using a muscle-specific LacZ marker. The muscle pattern in these larvae was clearly disrupted. Many muscles are missing, and others floated away during dissection, indicating very weak attachment. It is suggested that this muscle attachment defect leads directly to the semi-paralysed phenotype of the dying larvae and causes death by an inability to feed. Adult escapers also have very poor ambulatory ability, but the muscle attachments at this stage were not examined. Since the muscles appear to develop normally, but then detach, it is suggested that PP1β9C is required for the maintenance, rather than the formation of muscle attachments (Raghavan, 2000).

PP1β is the major isoform in rabbit skeletal muscle and so is the main isoform complexed to the myosin-targeting or M subunit of PP1. This complex is thought to dephosphorylate myosin regulatory light chain (MLC), antagonising myosin regulatory light chain kinase (MLCK). It was considered whether the phenotype of flw mutants might be due to a failure to dephosphorylate Mlc-2, but Mlc-2 mutants do not show defects in muscle attachment. A S66A/S67A non-phosphorylatable mutant of Mlc-2 does not suppress any flw mutant phenotypes (Raghavan, 2000).

Though PP1α87B is the major isoform overall, it might be that PP1β9C is the only or major isoform present in muscles. The flw phenotype might then be due to a reduction in the overall PP1 activity in the muscle, rather than any isoform-specific role of PP1β9C. Therefore the PP1 activity of muscle extracts were measured from various PP1 mutants. The total PP1 activity of larvae homozygous for Pp1-87B¹ (also known as Su-var(3)6⁰¹) is correspondingly reduced to about 20% of that of wild type and that of heterozygotes to about 50% of wild type. It was found that the PP1 activity from extracts of larval body wall muscles of these genotypes was reduced by a similar proportion, showing that PP1α87B is the major PP1 gene in these muscles. In contrast, the PP1 activity of equivalent extracts from flw¹/Y or flw⁶/Y larval body walls were only slightly lower than wild-type controls (92% and 84% of wild type). Similar results were obtained from IFM extracts from these genotypes. Pp1-87B¹ homozygous and heterozygous larvae do not show the muscle detachment phenotype of flw⁶/Y larvae, nor do the adults show the IFM loss of flw¹ and flw⁶ . These phenotypes are therefore due to a specific requirement for PP1β9C and not to loss of overall PP1 activity in the affected muscles (Raghavan, 2000).

PP1β9C is required for the maintenance of muscle attachments. Whether PP1β9C is required in the muscle, the epidermis or elsewhere, was investigated. Using the GAL4-UAS expression system, a PP1β9C cDNA was placed under the control of a muscle-specific or an epidermis-specific promoter (Gal4-24B and Gal4-69B respectively. Neither of these rescues flw⁶, but a combination of the two does. This suggests that PP1β9C is required on both sides of the muscle attachment site (Raghavan, 2000).

In addition to the muscle attachment defects, strong flw mutants show extensive blistering and/or crumpling of the wing. The wing forms from two sheets of cells; blistering is due to a failure of these two sheets to adhere to each other. PP1β is therefore required for cell adhesion in non-muscle tissues as well as in the maintenance of muscle attachments (Raghavan, 2000).

Normal levels of PP1β9C protein are produced in flw¹ and flw⁶. Therefore the organisation of the PP1β9C gene was analyzed, and the coding region and intron/exon boundaries in each mutant was sequenced. Each has a single amino acid change. Both mutations affect amino acids that are completely conserved across all four fly PP1 genes, and indeed through to mammals. Based on the published X-ray structure of PP1, Y133 forms a hydrogen bond to the peptide backbone of the substrate. Flw⁶ protein is therefore predicted to bind all substrates with reduced affinity. V284 is in a hydrophobic pocket adjacent to the binding site for a set of regulatory proteins. Flw¹ protein is therefore predicted to have altered affinity for these regulatory proteins (Raghavan, 2000).

The PP1 gene family encodes a set of closely related proteins. Despite their similarity in sequence and in vitro activity, subtle sequence differences between different isoforms are conserved between flies and humans. Of the mammalian genes, functional analysis by gene knockout has so far only been performed for one PP1 isoform, PP1γ. This knockout eliminated both the widely expressed PP1γ1 and the testis-specific PP1γ2. Homozygous mutant female mice were viable and fertile; homozygous mutant males were viable but sterile, with a range of defects in spermatogenesis. Presumably the somatic functions of PP1γ are redundant with PP1α and/or the less closely related PP1β. This study has shown an essential, in vivo role for a minor PP1 isoform. The recent demonstration of differential subcellular distribution of the mammalian isoforms suggests that this may be mediated by isoform-specific targeting and regulatory subunits, localising the isoforms to different subcellular compartments and substrates (Raghavan, 2000).

Search PubMed for articles about Drosophila Flap wing

Axton, J. M., Dombradi, V., Cohen, P. T., and Glover, D. M. (1990). One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63: 33-46. PubMed citation: 2170019

Baksa, K. et al., (1993). Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster. Genetics 135: 117-125. PubMed citation: 8224813

Bollen, M, (2001). Combinatorial control of protein phosphatase-1.Trends Biochem. Sci. 26: 426-431. PubMed citation: 11440854

Bresnick, A, (1999). Molecular mechanisms of nonmuscle myosin-II regulation. Curr. Opin. Cell Biol. 11: 26-33. PubMed citation: 10047526

Cohen, P, (2002). Protein phosphatase 1 - targeted in many directions. J. Cell Sci. 115: 241-256. PubMed citation: 11839776

Dombrádi, V., Axton, J. M., Barker, H. M., and Cohen, P. T. (1990a). Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation. FEBS Lett. 275: 39-43. PubMed citation: 2175717

Dombrádi, V., Axton, J. M., Brewis, N. D., da Cruz e Silva, E. F., Alphey, L., and Cohen, P. T. W. (1990b). Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1. Eur. J. Biochem. 194: 739-745. PubMed citation: 2176604

Dombrádi, V., Mann, D. J., Saunders, R. D. C., and Cohen, P. T. W. (1993). Cloning of the fourth functional gene for protein phosphatase 1 in Drosophila melanogaster from its chromosomal location. Eur. J. Biochem. 212: 177-183. PubMed citation: 8383037

Hartshorne, D, (1998). Myosin phosphatase: subunits and interactions. Acta Physiol. Scand. 164: 483-493. PubMed citation: 9887971

Kaibuchi, K., Kuroda, S., and Amano, M, (1999). Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68: 459-486. PubMed citation: 10872457

Kirchner, J., Gross, S., Bennett, D. and Alphey, L. (2007). The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-Terminal kinase in wing imaginal discs of Drosophila. Genetics 175(4): 1741-1749. PubMed citation: 17277363

Mizuno, T., Tsutsui, K., and Nishida, Y, (2002). Drosophila myosin phosphatase and its role in dorsal closure. Development 129: 1215-1223. PubMed citation: 11874917

Raghavan, S. et al, (2000). Protein phosphatase 1beta is required for the maintenance of muscle attachments. Curr. Biol. 10: 269-272. PubMed citation: 10712908

Skinner, J. and Saltiel, A. (2001). Cloning and identification of MYPT 3, a prenylatable myosin targetting subunit of protein phosphatase 1. Biochem. J. 356: 257-267. PubMed citation: 11336659

Somlyo, A. and Somlyo, A. (2000). Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J. Physiol. 522: 177-185. PubMed citation: 10639096

Tan, C., Stronach, B. and Perrimon, N. (2003). Roles of myosin phosphatase during Drosophila development. Development 130: 671-681. PubMed citation: 12505998

Varmuza, S., Jurisicova, A., Okano, K., Hudson, J., Boekelheide, K. and Shipp, E. B. (1999). Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1cγ gene. Dev. Biol. 205: 98-110

Vereshchagina, N., et al. (2004). The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin. Mol. Biol. Cell 15(10): 4395-405. PubMed citation: 15269282

date revised: 7 June 2008

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