InteractiveFly: GeneBrief

falafel: Biological Overview | References

BIOLOGICAL OVERVIEW

Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts, the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) was identified as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). These findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization (Sousa-Nunes, 2009).

Drosophila neuroblasts (NBs) are stem-cell-like neural progenitors, which undergo repeated asymmetric divisions to self-renew and generate neurons and/or glia. During each round of division the cell fate determinants Pros (a homeodomain-containing transcription regulator), Numb (a negative regulator of Notch signaling), as well as Brain Tumor (Brat, whose mechanism of action in cell fate specification is unclear) are asymmetrically localized as protein crescents on the NB cortex. In the embryo, the NB mitotic spindle is oriented along the apicobasal axis, the cell fate determinants and their adapter proteins localize to the NB basal cortex and segregate exclusively to the smaller basal daughter, called ganglion mother cell (GMC). The GMC divides terminally to produce two neurons or glial cells. The coordination between the basal localization of the cell fate determinants and the apicobasal orientation of the spindle during mitosis is mediated by several evolutionarily conserved proteins that localize to the apical NB cortex during the G2 stage of the cell cycle. These comprise [1] the Drosophila homologs of the Par3/Par6/aPKC protein cassette, [2] several proteins involved in heterotrimeric G protein signaling—Gαi/Partner of Inscuteable (Pins)/Locomotion defects (Loco), [3] as well as Inscuteable (Insc). In contrast to the embryo, NBs in the larval central brain divide without an apparent fixed orientation. Nevertheless the majority of central brain NBs appear to utilize the same molecular machinery as embryonic NBs, with the apical and basal molecules sharing similar hierarchical relationships and localizing to opposite sides of the NB cortex (Sousa-Nunes, 2009).

Asymmetric localization of Pros and Brat on the one hand and Numb on the other, is mediated through direct interactions with their respective adapters, the coil-coil proteins Miranda (Mira) and Partner of Numb (Pon). Although mutations affecting any of the apical proteins compromise asymmetric localization of basal proteins to varying extents, only in the case of aPKC has any mechanistic insight emerged. aPKC facilitates basal localization of cell fate determinants either through phosphorylation of the cytoskeletal protein Lgl and/or through direct phosphorylation of the determinant. Lgl is uniformly localized throughout the NB cortex, and is essential for cortical association and asymmetric localization of the cell fate determinants and their adapters. aPKC phosphorylates Lgl on three conserved serine residues and the triphosphorylated form appears to be inactive due to a conformational change. The proposed model is that unphosphorylated, active Lgl is restricted to the basal cortex because of apically localized aPKC. Consistent with this model, a nonphosphorylatable version of Lgl, Lgl^3A, in which the three target serines have been mutated to alanines, appears to be constitutively active and its expression leads to uniform cortical localization of the normally basally restricted cell fate determinants. Numb is a second protein that can be phosphorylated by aPKC and phosphorylation of three N-terminal serines causes it to become cytoplasmic (Sousa-Nunes, 2009).

How Lgl acts to facilitate the localization of cell fate determinants is less clear. Lgl can bind nonmuscle Myosin II (Zipper) and genetic experiments suggest that Myosin II and Lgl have antagonistic activities. Hence, one possible scenario would be that Myosin II is active at the apical cortex due to the presence of phosphorylated Lgl, which is incapable of binding to Myosin II. Myosin II can then act to exclude basal proteins from the apical cortex. Alternatively, since yeast Lgl orthologs function in exocytosis, it has been suggested that Lgl might act by regulating this process. It is possible that Lgl positively promotes delivery and cortical association of the basal molecules, and that this is antagonized by Myosin II apically. In this scenario, Lgl is inhibited apically both by aPKC and Myosin II, and only basal Lgl is active and able to promote cortical association of the basal proteins (Sousa-Nunes, 2009).

The unconventional Myosin VI (Jaguar, Jar) and Myosin II bind in a mutually exclusive manner to the basal adapter protein Mira. However, in contrast to Myosin II, which acts antagonistically to Lgl, Jar acts in a synergistical manner with Lgl to effect Mira basal localization. In mitotic NBs devoid of Jar, Mira is mislocalized to the cytoplasm. Jar possibly mediates association of Mira with the basal actin cytoskeleton (Sousa-Nunes, 2009).

In addition to aPKC, a few other serine/threonine protein kinases have been shown to play a role in facilitating asymmetric protein localization in NBs. These include Cdk1, required for the asymmetric localization of both apical and basal components during mitosis, Aurora A (AurA), and Polo, both of which mediate Numb and Pon asymmetric localization. With the exception of Polo kinase, which phosphorylates a serine residue within the Pon asymmetric localization domain, substrates for the other kinases have not been identified. The involvement of protein kinases in NB asymmetric divisions implies the involvement of protein phosphatases; however, to date, none have been implicated in the process (Sousa-Nunes, 2009).

In a clonal genetic screen designed to identify genes that mediate NB asymmetric divisions, multiple loss-of-function alleles of flfl. Falafel (Flfl) were identified as a regulatory subunit of the evolutionarily conserved Protein Phosphatase 4 (PP4) Phosphatase complex (Gingras, 2005). PP4 belongs to the best-studied family of cellular protein serine/threonine phosphatases, PP2A (the other major families being PP1, PP2B, and PP2C). Similarly to other PP2A-like phosphatases, PP4 functions as a heterotrimeric complex comprising of a catalytic subunit, PP4C, associated with two regulatory subunits, PP4R2 and PP4R3. PP4, or specifically PP4R3/Flfl, has been implicated in a variety of molecular and cellular processes including regulation of MEK/Erk (Yeh, 2004; Mendoza, 2005), insulin receptor substrate 4 (Mihindukulasuriya, 2004), Hematopoietic progenitor kinase 1 (Zhou, 2004), and Histone deacetylase 3 (Zhang, 2005) activities, centrosome maturation (Sumiyoshi, 2002), cell cycle progression (Kittler, 2004), apoptosis (Mourtada-Maarabouni, 2003), DNA repair (Gingras, 2005), cell morphology (Kiger, 2003), and lifespan control (Wolff, 2006; Samuelson, 2007; Sousa-Nunes, 2009 and references therein).

This study shows that loss of flfl, as well as attenuation of PP4C/Pp4-19C or PPR2/PPp4R2r function by RNAi specifically results in delocalization of Mira and its associated proteins throughout the cytoplasm in metaphase/anaphase NBs; in addition, both Mira and Pros localize to the NB nucleus prior to nuclear envelope breakdown. Excessive nuclear Mira is dependent on the presence of Pros. These results suggest that whereas cytoplasmic or membrane-associated PP4 is required for asymmetric cortical localization of Mira (and its associated proteins) during metaphase and anaphase, nuclear PP4 is required to exclude Pros (and as a consequence, Mira) from the NB nucleus prior to nuclear envelope breakdown. Moreover, Flfl can complex with Mira in vivo and directly interact with Mira, suggesting that Flfl targets PP4 activity to the Mira complex to facilitate its correct localization (Sousa-Nunes, 2009).

In a clonal screen on third-instar larval (L3) brains, designed to identify novel genes on chromosome arm 3R required for NB asymmetric division, a novel allele of flfl, flfl⁷⁹⁵ was isolated. In metaphase and anaphase flfl⁷⁹⁵ clone NBs, Mira displays weak cortical crescents but also a pronounced mislocalization throughout the cytoplasm, whereas in surrounding heterozygous NBs Mira is localized to a robust crescent like in wild type with little cytoplasmic accumulation. As with many mutations that disrupt NB asymmetry during metaphase and anaphase, flfl⁷⁹⁵ NBs display telophase rescue: The majority of the cytoplasmic Mira relocalizes asymmetrically to the NB cortex at telophase, resulting in asymmetric segregation of Mira into the GMC. Using the flfl⁷⁹⁵ allele, two additional alleles [flfl⁷⁹⁵⁽²⁾, flfl⁷⁹⁵⁽³⁾] were identified via complementation screening of an independent collection of ethylmethane sulfonate (EMS) mutant stocks. Sequencing of these three EMS-induced flfl alleles revealed single point mutations resulting in premature stop codons at positions 324 (flfl⁷⁹⁵) and 630 [flfl⁷⁹⁵⁽²⁾] of the longest isoform (980 amino acids) and a disruption to the splice acceptor site at the 3′ end of the fourth intron [flfl⁷⁹⁵⁽³⁾]. All three alleles display a mislocalization of Mira to the cytoplasm of metaphase NBs and form an allelic series in terms of phenotypic severity: flfl⁷⁹⁵ > flfl⁷⁹⁵⁽³⁾ > flfl⁷⁹⁵⁽²⁾ (Sousa-Nunes, 2009).

Homozygous flfl⁷⁹⁵ animals survive to pharate adults whereas hemizygous flfl⁷⁹⁵ animals [using Df(3R)Exel6170 to remove one copy of the flfl coding region] only survive until L3. Furthermore, although the cytoplasmic Mira phenotype of flfl⁷⁹⁵ homozygotes is highly penetrant, the majority of metaphase NBs still display weak Mira crescents, whereas the majority of metaphase NBs of flfl⁷⁹⁵ hemizygotes display no crescents. These results suggest that the strongest EMS allele (flfl⁷⁹⁵) is nevertheless a hypomorph. Therefore a flfl-null allele (flfl^N42) was generated by imprecise excision of the P-element P{EPgy2}flfl^EY03585, located ~1 kb upstream of the flfl translational start site. This allele was confirmed to be a genetic null by the similar expressivities of NB phenotypes in flfl^N42 homozygotes and flfl^N42 hemizygotes, as well as in flfl⁷⁹⁵/flfl^N42 and flfl⁷⁹⁵/Df(3R)Exel6170. Consistently, flfl^N42 NBs are antigen-minus (see below) and molecular analysis indicates that it is a deletion extending into the coding region, deleting the first 1075 base pairs of the coding sequence. Subsequent analyses of the phenotype were carried out using the flfl^N42 allele, hereafter referred to simply as flfl (Sousa-Nunes, 2009).

In addition to the mislocalization of Mira, the Mira-associated proteins Pros, Brat and Staufen (Stau), are similarly mislocalized to the cytoplasm of metaphase/anaphase flfl NBs. Pros mislocalization occurs in Asense (Ase)-positive NB lineages which comprise the majority of lineages in the central brain (Bowman, 2008); Ase-negative NBs are Pros-negative in flfl as well as in wild-type brains. In contrast, the localization of members of the other basal complex, Pon and Numb, and of apical complexes is unaffected. Hence, during NB division, flfl loss of function specifically affects the localization of the Mira complex (Sousa-Nunes, 2009).

Flfl homologs have been identified in several species, from yeast to humans. They all possess the same domain architecture: a Ran-binding domain (RanBD) at the N terminus, similar in three-dimensional structure to the Ena/VASP homology domain 1 (EVH1, which derives its name from the founding members Enabled and Vasodilator-stimulated phosphoprotein) and to the pleckstrin homology domain; followed by a conserved domain of unknown function (DUF625), a region containing armadillo/HEAT repeats, and a region of low complexity. Within the DUF625 domain, Flfl contains two putative NLSs (NLS1 and NLS2) as well as a nuclear export signal (NES); close to the C terminus Flfl contains a short conserved stretch of acidic and basic amino acid residues that has been shown to be required for nuclear localization of the Dictyostelium discoideum homolog, SMEK (NLS3) (Mendoza, 2005). Flfl contains many putative target sites for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in its C-terminal 300 amino acids and numerous putative phosphorylation sites throughout, some of which are predicted to be PKC targets (Sousa-Nunes, 2009).

In conclusion, loss of function or RNAi knockdown of the regulatory subunits flfl/PP4R3 or PPP4R2r/PP4R2 as well as knockdown of the catalytic subunit Pp4C-19C/PP4C of PP4 causes mislocalization of Mira/Pros/Brat/Stau to the cytoplasm of metaphase and anaphase NBs (Sousa-Nunes, 2009).

Attenuation of PP4 function above also causes increased frequency of nuclear Mira/Pros prior to nuclear envelope breakdown. The observation that depletion of the catalytic subunit of PP4 results in identical phenotypes to the depletion of its regulatory subunits, suggests that phosphatase activity plays a role in the localization of Mira/Pros throughout the NB cell cycle (Sousa-Nunes, 2009).

Nuclear mislocalization of Mira seen in flfl, jar, or mira^2L150 single-mutant NBs requires pros function. This suggests that, when transport of Mira toward or its tethering to the cortex is defective, Pros can take Mira into the nucleus. In this context, the normal relationship between Mira and Pros is reversed, with Pros instructing Mira localization rather than the converse. In the absence of pros, Mira is not localized to the nucleus, even when PP4 function is attenuated. Thus, the role of PP4 on these two temporally distinct localizations of Mira/Pros appears to involve distinct targets since one is a Mira-dependent localization and the other is Pros-dependent (Sousa-Nunes, 2009).

In contrast to serine-threonine kinases, substrate specificity for serine/threonine protein phosphatases is thought to be conferred not primarily by sequences adjacent to the target residues but rather by interaction between the substrate and regulatory subunits of the phosphatase complex. This is the case for the founding family member PP2A, whose variable subunit composition can also target the complex to distinct subcellular domains (for review, see Sontag 2001) and is thought to be the case also for PP4 (Cohen, 2005). Flfl, a regulatory subunit of PP4, is able to bind Mira and Flfl and Mira are found in a complex in vivo. No binding was detected between Flfl and Pros but since Mira and Pros still colocalize when PP4 function is attenuated, these results also suggest that PP4 function is not required for the Mira-Pros interaction. Therefore, Pros could be recruited to PP4 by its association with Mira, which in turn binds Flfl (Sousa-Nunes, 2009).

Flfl is nuclear before and cytoplasmic after nuclear envelope breakdown. The results from nuclear excluded and membrane targeted versions of Flfl suggest that nuclear Flfl is required to exclude Mira/Pros from the nucleus when inefficiently bound to the cytoskeleton/cortex, whereas cytosolic or membrane-associated Flfl is required for the cortical association and asymmetric localization of Mira/Pros/Brat/Stau at metaphase and anaphase. The localization of Mira/Pros prior to and after nuclear envelope breakdown by PP4 may involve different phosphatase substrates. It is tempting to entertain the possibility that Mira dephosphorylation by PP4 in the cytoplasm is required for its asymmetric cortical localization during mitosis, and that Pros dephosphorylation by PP4 in the nucleus is required for its nuclear exclusion/progression through prometaphase. Indeed, a previous study has shown that cortical Pros is highly phosphorylated relative to nuclear Pros. To test this hypothesis, attempts were made to detect enrichment of a lower mobility band of Mira::3GFP in flfl larval extracts compared with wild type but this it could not be detected, working at the limits of detectability (Sousa-Nunes, 2009).

Asymmetric cortical localization of proteins during NB asymmetric division is dependent on an intact actin cytoskeleton. Although flfl is required for Mira cortical association, at no point in the NB cell cycle does Flfl exhibit cortical enrichment. However, modified versions of Flfl that are either uniformly cytoplasmic or cortically enriched can both drive asymmetric cortical localization of Mira and its associated proteins. Moreover, the Mira mislocalization phenotypes of flfl are strikingly similar to those of Myo VI/jar. Both mutants exhibit nuclear Mira/Pros prior to and cytoplasmic Mira and associated proteins following NB nuclear envelope breakdown; both Flfl and Jar are cytoplasmic at metaphase/anaphase; and genetically, both Jar and Flfl act parallel to or downstream from Lgl. Further propelled by the presence of a putative actin-binding domain in Flfl (the RanBD domain, which is an EVH1-like domain), it was asked whether Flfl too might facilitate association of Miranda with the actin cytoskeleton either separately from or in association with Jar. However, in vitro assays clearly showed that Flfl does not bind F-actin, although Mira alone does, with comparable strength to that of α-Actinin and Jar, used as controls. Furthermore, Jar could not be detected in Flfl containing protein immunoprecipitates. Therefore, it seems unlikely that Flfl acts either directly or in a complex with Jar to facilitate Mira transport along or tethering to the actin cytoskeleton. Still, Flfl could act indirectly; for example, by stabilization of the Mira-Jar association. It is speculated that Flfl may act by targeting PP4 to the Mira complex and that the consequent dephosphorylation of a component of this complex facilitates Jar-Mira association (Sousa-Nunes, 2009).

In Dictyostelium, mutants in the flfl homolog, smkA, exhibit phenotypes similar to strains defective in Myo II assembly (Mendoza, 2005), suggesting that smkA may regulate Myo II function. However, in flfl NBs the Mira mislocalization phenotype does not resemble that of Myo II loss of function, which has been described to lead to Mira mislocalization to the mitotic spindle in embryonic NBs (Sousa-Nunes, 2009).

The reduced proliferation seen in flfl NBs correlates with nuclear localization of Pros/Mira. Nuclear Pros negatively regulates transcription of cell cycle genes and positively regulates differentiation genes, and has been shown to limit NB proliferation. Therefore, ectopic nuclear Pros is likely to be at least one cause of the NB underproliferation observed in flfl brains. Still, it is possible that flfl has additional functions in promoting proliferation, independent of its role in excluding Pros/Mira from the NB nucleus. Indeed, an excessive proportion of phospho-histone H3-positive flfl NBs was detected relative to wild type. These NBs typically had a nucleus but the cell morphology was not spheroid, as would be expected in prophase cells. This suggests that flfl NBs either have a block or delay in prometaphase or that PP4 may be required for dephosphorylation of Histone H3; in either case, it seems to be required for dephosphorylation of other proteins involved in cell cycle progression. Nonetheless, pros,flfl double-mutant NB clones are indistinguishable from those of pros single mutants, both showing extensive overproliferation, suggesting that the loss of flfl is unable to override the overproliferation induced by loss of pros (Sousa-Nunes, 2009).

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. This study identified Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. Evidence is provided to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. This work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression (Connell, 2024).

This study identified Kin17 as essential for the proper localization of Mira in Drosophila NBs. The localization of Mira during asymmetric division is essential to the proper segregation of the fate determinants to the daughter cell, and the regulation of its phosphorylation state via an interplay between kinases and phosphatases is essential to this localization. PP4 was demonstrated to be required for the dephosphorylation of Mira at S96 at the centrosome beginning in prophase and completing dephosphorylation by the end of mitosis. Dephosphorylation of Mira at the centrosome is essential for proper localization of Mira to the basal domain and prevents accumulation of Mira at the centrosome and in the cytoplasm. Knockdown of Kin17 leads to reduction in Flfl expression and PP4 activity; consequently, Mira cannot be fully dephosphorylated at S96 and accumulates in the cytoplasm and the centrosome instead of binding to the basal cortex, which leads to Pros being translocated to the nucleus. Further, similar mislocalization of Mira/Pros and reduction in flfl mRNAs observed in u6atac mutant and Kin17 knockdown NBs and binding of Kin17 to flfl mRNAs suggest that Kin17 is likely involved in flfl splicing (Connell, 2024).

Kin17 has not been well studied in Drosophila. In mammals, it is essential for DNA repair and replication. Kin17 has been shown to interact with the spliceosome and has very recently been identified as a splicing factor in C. elegans, where Kin17 is required to maintain the 5' splice site identity during spliceosome assembly. In this system, mutations in Kin17 led to changes in alternative 5' splice site usage. Kin17 may play a similar role in humans, as mass spectrometry has identified it to primarily interact with the spliceosome in B stage, which is the first formation of the entire spliceosome. This study shows that Kin17 binds to flfl mRNAs, that knockdown of Kin17 leads to reduction in flfl mRNAs, and that mutants for the minor spliceosome component u6atac phenocopy Kin17 knockdown, supporting that in Drosophila, Kin17 functions to regulate splicing of transcripts. In the mass spectrometry analysis, Kin17 was only found in complexes isolated from both HeLa and Drosophila Kc167 cell lines using fushi tarazu as bait but not zeste, suggesting that Kin17 likely regulates splicing of specific transcripts rather than acting as a general splicing factor. However, Kin17 may not just regulate the splicing offlfl pre-mRNAs in the NBs, as Kin17 also binds to aPKC and pros mRNAs, and Kin17 knockdown also leads to reduction in aPKC expression. It is possible that Kin17 may also regulate splicing of other transcripts such as aPKC transcripts. The findings that Kin17 is potentially involved in regulating splicing of particular transcripts could be helpful for investigating underlying mechanisms of pathogenesis of various cancers associated with increased Kin17 expression (Connell, 2024).

Mira localization to the mitotic spindle/centrosome has been observed in the syncytial embryo, and interactions between Mira and the microtubules have also been observed in the anterior pole of oocytes. Mutants in polarity proteins can lead to localization of Mira to the centrosome and mitotic spindle in NBs, raising the question of why Mira does not localize to the mitotic spindle in NBs and whether localization to the mitotic spindle during mitosis leads to consequences for asymmetric cell division. This work provides evidence that localization to the centrosome and potentially the mitotic spindle (based on the S96 alleles) does occur in WT NBs, but it is likely transient and appears to be cell-cycle dependent. This work also reveals that localization of Mira to the centrosome/spindle depends on the phosphorylation status of Mira at S96 and that localization of Mira to the centrosome is essential for dephosphorylation of S96 and cortical localization, as Sas-4 RNAi leads to defects in cortical localization and an increase in the levels of phosphorylated Mira. Although PP4 has been implicated in dephosphorylation of Mira at T591, this work identifies S96 as a dephosphorylation site of PP4 and shows that T591 phosphorylation does not contribute to the observed phenotype. In fact, a slight decrease was observed in T591D centrosomal localization. This may be due to the fact that T591 dephosphorylation is required prior to aPKC phosphorylation of Mira to ensure proper localization. Thus, Mira removal from the centrosome is specific to dephosphorylating S96. The finding of dephosphorylation of S96 by PP4 at the centrosome answers several outstanding questions regarding Mira localization after aPKC phosphorylation, including if PP4 dephosphorylates Mira, the function of centrosomal localization of Mira, and if dephosphorylation of S96 occurs prior to localization of Mira to the basal domain (Connell, 2024).

Interestingly, an increase was observed of Mira localization to the centrosome in PP4 and Flfl RNAi NBs with a minimal increase in cytoplasmic Mira. This suggests that centrosomal/spindle localization of Mira may be more sensitive to changes in the levels of PP4 activity. The phosphorylated Mira may preferentially localize to the centrosome/spindle and bring its cargo protein Pros to the centrosome as well, as indicated by the colocalization of Pros and Mira to the centrosome in Kin17 RNAi NBs. Additionally, in Kin17 RNAi NBs, defects were observed in Mira cortical localization in interphase, which is likely related to the localization of Pros to the nucleus. mira null mutants exhibit nuclear Pros localization, suggesting that Pros must be tethered to something to prevent nuclear localization and that defects in S96 dephosphorylation cause tethering to the cortex to be impaired, so Mira/Pros then localizes to the centrosome and nucleus. It is tempting to speculate that when the amount of phosphorylated Mira exceeds the binding capacity of the centrosome/spindle, it may "overflow" to the cytoplasm, leading to untethered Pros being transported into the nucleus (Connell, 2024).

In summary, these studies identify a factor, Kin17, that regulates the proper localization of Mira during asymmetric cell division potentially through regulation of the splicing of flfl transcripts and provides evidence to demonstrate that dephosphorylation of Mira at S96 by PP4 at the centrosome is essential for proper localization of Mira in NBs during asymmetric cell division (Connell, 2024).

There are several limitations to the study that prevent making further conclusions. First, while the data suggest that Kin17 functions in the splicing of the flfl transcript, this will have to be confirmed through further experiments that are able to capture the flfl pre-mRNA through sequencing or other methods that will detect the transcript. Second, the presence of redundant mechanisms, such as telophase rescue, may not allow seeing of the full effect of the loss of Kin17 in terms of asymmetric cell division and development. While these data suggest that when telophase rescue is also lost, Kin17 does affect cell polarity in telophase, indicating that Kin17 could potentially lead to defects in cell fate, further studies will have to be carried out to determine the effect this has on development. Thirdly, as RNAi was used, some of the phenotypes may not be fully penetrant, such as Flfl RNAi and PP4-19c RNAi, which may not lead to complete phenotypes and may prevent determining the full effect on cell polarity that these proteins have (Connell, 2024).

A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity

Using a combination of tandem affinity purification tagging and mass spectrometry, this study characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that was termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. This study demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally it was found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (Gingras, 2005. Full text of article).

Search PubMed for articles about Drosophila Falafel

Bowman, S. K., et al. (2008). The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila. Dev. Cell. 14: 535-546. PubMed ID: 18342578

Cohen, P. T., Philp, A. and Vazquez-Martin, C. (2005). Protein phosphatase 4 -- From obscurity to vital functions. FEBS Lett. 579: 3278-3286. PubMed ID: 15913612

Connell, M., Xie, Y., Deng, X., Chen, R., Zhu, S. (2024). Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression. Cell Rep, 43(3):113823 PubMed ID: 38386552

Gingras, A. C., et al. (2005). A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol. Cell Proteomics 4(11): 1725-40. PubMed ID: 16085932

Kiger, A. A., et al. (2003). functional genomic analysis of cell morphology using RNA interference. J. Biol. 2: 27. PubMed ID: 14527345

Kittler, R., et al. (2004). An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432: 1036-1040. PubMed ID: 15616564

Mendoza, M. C., et al. (2005). Loss of SMEK, a novel, conserved protein, suppresses MEK1 null cell polarity, chemotaxis, and gene expression defects. Mol. Cell. Biol. 25: 7839-7853. PubMed ID: 16107728

Mihindukulasuriya, K.. A., Zhou, G.., Qin, J. and Tan, T. H. (2004). Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation. J. Biol. Chem. 279: 46588-46594. PubMed ID: 15331607

Mourtada-Maarabouni, M., et al. (2003). Functional expression cloning reveals proapoptotic role for protein phosphatase 4. Cell Death Differ. 10: 1016-1024. PubMed ID: 12934076

Samuelson, A. V., Carr, C. E. and Ruvkun, G. (2007). Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 21: 2976-2994. PubMed ID: 18006689

Sontag, E. (2001). Protein phosphatase 2A: The Trojan Horse of cellular signaling. Cell. Signal. 13: 7-16. PubMed ID: 11257442

Sousa-Nunes, R., Chia, W. and Somers, W. G. (2009). Protein phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions. Genes Dev. 23(3): 359-72. PubMed ID: 19204120

Sumiyoshi, E., Sugimoto, A. and Yamamoto, M. (2002). Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C. elegans. J. Cell Sci. 115(Pt 7): 1403-10. PubMed ID: 11896188

Wolff, S., et al. (2006). SMK-1, an essential regulator of DAF-16-mediated longevity. Cell 124: 1039-1053. PubMed ID: 16530049

Yeh, P. Y., et al. (2004). Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation. J. Biol. Chem. 279: 26143-26148. PubMed ID: 15073167

Zhang, X., et al. (2005). Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev. 19: 827-839. PubMed ID: 15805470

Zhou G., Boomer J.S., Tan T.H. Protein phosphatase 4 is a positive regulator of hematopoietic progenitor kinase 1. J. Biol. Chem. 279: 49551-49561. PubMed ID: 15364934

date revised: 27 August 2025

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