InteractiveFly: GeneBrief

homeodomain interacting protein kinase: Biological Overview | References

BIOLOGICAL OVERVIEW

The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. This study provides genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhances Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. Similar molecular interactions were observed between Lef1/β-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling (Lee, 2009b).

Metazoan development is a highly dynamic and complex process that requires the action of several key signal transduction pathways. Their activity must be tightly regulated to ensure the proper patterning and growth of tissues. Regulation of a signaling pathway can occur at any level within the pathway, from perturbation of ligand-receptor interactions to regulation of the activity of transcription factors in the nucleus. Some regulators affect the on or off state of pathways, whereas others are involved in fine-tuning, an essential aspect that ensures that accurate and physiologically necessary levels of signaling are achieved without excessive signaling, which can have deleterious effects (Lee, 2009b).

The ability of the Wnt pathway to control different developmental events in a temporally and spatially specific manner requires coordination between numerous regulators. Canonical Wnt signaling controls cell fate by regulating transcription of target genes. Wingless (Wg), a secreted glycoprotein, is the best characterized of the seven Drosophila Wnt ligands, and initiates the canonical pathway by binding to the Frizzled2 (Fz2) and LRP5/6/Arrow co-receptors. This leads to the activation of Dishevelled, which then inhibits the activity of the destruction complex composed of Axin, glycogen synthase kinase 3β (GSK-3β)/Zw3 and adenomatous polyposis coli (APC). As a result, cytosolic Drosophila β-catenin called Armadillo (Arm) accumulates and enters the nucleus to interact with a Tcf/Lef (Drosophila Tcf) family transcription factor to promote target gene expression. In the absence of Wg signaling, the Axin/GSK-3β/APC complex promotes the proteolytic degradation of Arm, whereas transcriptional co-repressors bind to Tcf and repress transcription (Lee, 2009b).

The Nemo-like kinase family (Nlk) of protein kinases can regulate activation of Tcf/Lef target genes. In Drosophila, Nemo (Nmo) inhibits Drosophila Tcf activity, and is itself a transcriptional target of the Wg pathway. Recently Homeodomain-interacting protein kinase 2 (Hipk2) was proposed to participate in a kinase cascade to activate Nlk during the regulation of the Myb transcription factor (Kanei-Ishii, 2004). Therefore, attempts were made in this study to identify whether this regulation was perhaps more general and whether Drosophila Hipk played a role in regulating Nmo, and thus also Wg signaling. It was rapidly learned that Hipk exerts a positive effect on Wg signaling, distinct from Nmo, which has been more fully characterized using the developing wing as a model system (Lee, 2009b).

The patterning of the adult wing blade is a tightly regulated process involving numerous essential signaling pathways, including Wg, Notch, EGFR and TGFβ, making it an excellent tissue in which to examine regulatory and epistatic relationships between many genes involved in patterning. The adult wing blade possesses five longitudinal veins (LI-LV) that extend proximally to distally. These are connected by the anterior cross vein (ACV) and posterior crossvein (PCV). The Wg pathway acts at several stages of wing patterning and growth (reviewed by Martinez Arias, 2003). Wg is expressed along the dorsal/ventral boundary, which in imaginal discs is a stripe bisecting the wing imaginal disc, and in adult wings gives rise to the wing margin and bristles that surround the edge of the wing blade. Loss of wg can lead to loss of the entire wing, to wing to notum transformations, to wing notching or to loss of bristles along the entire wing margin. Wg also promotes proliferation in the wing disc and ectopic Wg can induce outgrowths from the ventral surface of the wing (Lee, 2009b).

Hipk2 is a member of a conserved family of serine/threonine kinases. Vertebrate species possess four Hipk proteins (Hipk1-4) that have evolved distinct functions (reviewed by Rinaldo, 2007). Singly mutant Hipk1 and Hipk2 mice are viable, whereas double mutant mice die before birth (Isono, 2006). Drosophila possesses a single Hipk ortholog (which has been referred to as both Hipk and Hipk2) (Choi, 2005; Link, 2007) that shares extensive sequence homology within the kinase domain with members of the vertebrate family (Lee, 2009b).

Vertebrate Hipk2 has been the most extensively studied member of the family (reviewed by Calzado, 2007; Rinaldo, 2007). Biochemical studies have identified a growing list of Hipk2 interactors, including proteins involved in transcriptional regulation, chromatin remodeling and key components of evolutionarily conserved signaling pathways. However, the biological investigation of these interactions in multicellular organisms has been minimal. In vivo examination of murine Hipk2 protein function has thus far revealed a role in neurogenesis and homeotic transformation of the skeleton (Isono, 2006; Wiggins, 2004; Zhang, 2007). Studies in Drosophila using transgenic flies expressing Hipk transgenes have uncovered a role for Hipk in regulating the global corepressor Groucho (Choi, 2005). Using loss-of-function mutant analyses, a role has been identified for Hipk in promoting Notch signaling during Drosophila eye development (Lee, 2009a; Lee, 2009b and references therein).

This study presents an analysis of the function of Hipk in Drosophila canonical Wg signaling. Genetic studies show that ectopic hipk can rescue phenotypes owing to loss-of-function wg alleles or inhibition of the pathway with a dominant-negative Fz2 receptor. Immunohistochemical studies show that hipk positively regulates expression of Wg targets, and that Hipk can act to stabilize cellular levels of the Arm protein in wing discs. Wnt reporter assays show that both Drosophila Hipk and mouse Hipk2 can promote the Wnt-responsive Topflash reporter. In addition, Hipk/Hipk2 can promote the stabilization of Arm/β-catenin in cell culture and in vivo. These results suggest that Hipk is a positive regulator of the Wg pathway that refines Wg activity during wing development. The findings suggest that these roles may be conserved across species (Lee, 2009b).

Loss of zygotic hipk results in pupal or larval lethality, a finding recently also described by Link (Link, 2007). Whole-mount in situ hybridization reveals that hipk is expressed broadly in a non-uniform pattern in multiple stages of development, including all imaginal discs. Removal of the maternal contribution caused embryonic lethality characterized by twisted embryos and head holes, showing that hipk is an essential gene for Drosophila development. These analyses have focused on the most severe allele, hipk⁴, which causes early larval lethality, or pupal lethality in trans to hipk³ (Lee, 2008). Given the embryonic lethality caused by loss of maternal Hipk, it is speculated that maternally contributed Hipk perdures and obscures its requirement at later stages and impacts the severity of mutant phenotypes. The FLP/FRT technique was used to generate mutant somatic clones to examine the requirements for Hipk in patterning adult structures (Lee, 2009b).

This study focused on the role of hipk in the development of the wing. Clones of cells mutant for hipk⁴ show ectopic veins in the anterior region of the wing blade along LII, loss of the PCV and occasional notches in the wing margin. Reducing hipk function by expression of two independent Gal4-responsive hipk-RNAi constructs in the wing pouch with sd-gal4 or vg-Gal4 also caused a wing notching phenotype reminiscent of those seen upon decreased Wg signaling (Lee, 2009b).

Next, the effects of ectopic expression of hipk was studied using the Gal4-UAS system. Phenotypes were observed that suggested that hipk is involved in promoting Wg signaling. Expression of one copy of hipk in the central domain of the imaginal wing discs with omb-Gal4 induced the formation of an additional wing margin and outgrowths emanating from the distal most tip of the ventral surface of the wing. Expression of two copies of UAS-hipk enhanced this phenotype and caused the outgrowths to extend further distally from the ventral plane of the wing. These effects phenocopied the effects observed in ectopic wg-expressing clones. Similarly, expression of UAS-hipk in the wing phenocopies the ectopic venation pattern seen both upon ectopic expression of activated Arm (Arm^S10) with bs-Gal4 and in nmo^DB24/nmo^adk2 mutants. Although control of wing vein patterning is not generally attributed to Wg signaling, ectopic venation has been observed upon elevated Wg signaling. For example, ectopic expression of constitutively active Arm by en-Gal4 in the posterior region of the wing, or ubiquitously with 69B-Gal4 or MS1096-Gal4, leads to disturbed and ectopic venation. Moreover, loss-of-function clones of sgg/zw3 (encoding the fly homolog of GSK3β, a component of the destruction complex) induce the formation of ectopic veins. The results of these phenotypic analyses of hipk are surprising because they demonstrated that Nmo and Hipk did not act in concert to inhibit Wg signaling. Rather, hipk mutant and gain-of-function phenotypes suggest a role in promoting the Wg pathway (Lee, 2009b).

This study has revealed that Hipk possesses an intrinsic ability to promote Wg pathway activity and this regulatory function for Hipk is conserved in both Drosophila and mammalian cells. Through a combination of genetic and biochemical analyses, the data reveal that Hipk proteins promote Tcf/Lef1-mediated transcription. Additionally, Hipks enhances the stabilization of Arm/β-catenin in several cell lines and hipk mutant clones in the wing disc have diminished Arm protein levels. Overexpression of Hipk induces a broader domain of stabilized Arm, suggesting Hipk is required to maintain the signaling pool of cytosolic Arm. A model is proposed in which Hipk promotes the Wnt/Wg signal via its regulation of Arm stabilization (Lee, 2009b).

Nlk is a conserved antagonist of the Lef1/β-catenin transcriptional complex. This research has shown that Nmo is also an inducible antagonist of the Wg signal in the developing Drosophila wing (Zeng, 2004). It was previously reported that Wnt1 induces the activation of a putative Tak1-Hipk2-Nlk kinase cascade to promote the degradation of the Myb transcription factor (Kanei-Ishii, 2004). The current study sought to delineate the physiological relevance of this potential kinase cascade; specifically, it was determined whether these interactions played a role in the regulation of the Wg pathway. The data reveal that in Drosophila Nmo and Hipk do not form a kinase cascade in this context, rather they exert opposing effects on the same pathway, probably through distinct mechanisms (Lee, 2009b).

The Wg morphogen can bring forth a spectrum of biological processes. Sustaining maximal levels of signaling could be accomplished through the amplification and enhancement of the signal in the wing margin. In support of this model, it was found that overexpression of Hipk expands the expression of Wg targets such as Dll, Sens and Ac. Transcriptional assays reveal that both Drosophila Hipk and mouse Hipk2 enhance the transcriptional activity of Tcf and Lef1, respectively, in a kinase-dependent manner. These findings strongly suggest that Hipk and Hipk2 function to enhance the activity of the transcriptional complex to promote the Wg/Wnt signal (Lee, 2009b).

Accumulation of stabilized Arm is paramount to effective Wg signaling. Failure to escape the destruction complex results in Arm degradation and inhibition of Tcf-mediated gene activation. Thus understanding the regulation of Arm is central to the global understanding of how the Wg signal is modulated. In these studies, a role was revealed for Hipk in Arm stabilization. This feature is highlighted by the loss of stabilized Arm in hipk mutant clones. Additionally, in hipk mutant discs, overexpressed wild-type Arm fails to accumulate, despite its expression in domains of high Wg signaling. These findings demonstrate that Hipk plays an important role in Arm stabilization. Hipk may reduce the ability of Arm to interact with destruction complex components or may increase the nuclear retention of Arm. In the absence of Hipk, either of these scenarios would give the destruction complex more access to Arm. In agreement with such a model, it was found that increasing Hipk activity in the wing surpasses the inhibitory effects of the degradation machinery and expands the perimeter of stabilized Arm. Furthermore, it was found that the presence of Hipk or Hipk2 in cell culture stabilizes Arm/β-catenin. Thus, the enhanced transcriptional activity is probably due to the elevated availability of Arm protein (Lee, 2009b).

It is crucial for normal development to maintain the proper amounts of β-catenin, since elevated levels of β-catenin can lead to cancer. Elaborate regulatory networks in the cytoplasm and nucleus are vital to maintaining appropriate levels of β-catenin. It is well documented that phosphorylation in the N terminus of β-catenin is crucial for its negative regulation. A chain of phosphorylation events begins when Casein Kinase I (CKI) primes β-catenin for successive modifications by GSK-3β. Central to this event is Axin, which provides a scaffold for APC, CK1, GSK-3β and β-catenin. N-terminally phosphorylated β-catenin is ubiquitinated by βTrCP ubiquitin ligase and targeted for degradation via the proteasome (Lee, 2009b).

Wnt signaling promotes the accumulation of β-catenin; however, some of the mechanisms governing this process remain enigmatic. Although overexpression of wild-type β-catenin/Arm is unable to overcome the effects of the degradation machinery, Wnt-stimulated β-catenin can resist the activity of the destruction complex. Although achieving stabilized pools of β-catenin represents the core goal of the Wnt pathway, high levels of β-catenin are not always coupled with elevated transcription. For example, in Xenopus, alanine substitution of one of the GSK3 target residues leads to elevated β-catenin levels, without causing an increase in Tcf-mediated transcription. Thus, further posttranslational modifications of β-catenin are necessary to potentiate its signaling activity (Lee, 2009b).

Furthermore, phosphorylation can affect β-catenin stability by affecting protein-protein interactions that regulate protein turnover and activity. Phosphorylation of β-catenin by Cdk5 inhibits APC binding to β-catenin, whereas phosphorylation by CK2 promotes β-catenin stability and transcriptional activity (Lee, 2009b).

Recent advances have begun to unravel the molecular complexity that controls β-catenin-mediated transcription within the nucleus. Upon pathway activation, Tcf recruits Arm to the enhancers of Wg-responsive genes where Arm forms multiple transcriptional complexes along its length. Formation of these transcriptional units is needed for the transmission of the Wg/Wnt signal. Recent studies have shown that phosphorylation (distinct from the N-terminal phosphorylation that triggers β-catenin destruction) may modulate its ability to recruit these co-factors. This study found that the Hipk-dependent stabilized form of Arm is transcriptionally active and induces the expression of Wg targets, suggesting modification by Hipk may promote protein interactions (Lee, 2009b).

APC and the cell-adhesion molecule E-cadherin compete with Tcf for overlapping binding sites on β-catenin. Competition between proteins may play an important role in the regulation of the Wnt signaling pathway. It is proposed that Hipks may promote the stability of Arm/β-catenin by excluding further interactions with other proteins, including those that antagonize Arm/β-catenin. Given that Hipks can also bind to Tcf/Lef1, it is predicted that these proteins may act synergistically to displace the inhibitory partners of β-catenin. In agreement with such a role, it is observed that Lef1 enhances the interaction between Hipk2 and β-catenin, and these interactions may insulate β-catenin from components of the degradation machinery (Lee, 2009b).

Although Hipk phosphorylates Arm, the functional significance of this modification has yet to be determined. Hipk might facilitate the interactions between Arm and its transcriptional co-factors, as Hipk2 phosphorylation has been shown to affect gene regulation by modifying the composition of various transcriptional complexes (reviewed by Calzado, 2007). Hipk may also enhance the formation of the β-catenin/Tcf transcriptional complex by inducing a conformational change and/or reducing the affinity of possible inhibitors for β-catenin through the phosphorylation of β-catenin. Recently, it has been reported that Hipk2 could antagonize β-catenin/Tcf-mediated transcription in a kinase-independent manner (Wei, 2007). Although these data appear in conflict with the current findings, it was observed that the effect of Hipk2 on transcription is very cell type- and target gene-dependent, suggesting Hipk2 function is affected by its cellular context, most probably owing to the availability of targets and co-factors (Lee, 2009b).

The dynamic localization of Hipk2 in the nucleus, nucleoplasm and in cytosolic speckles suggests that the protein may carry out distinct roles in each site (reviewed by Calzado, 2007; Rinaldo, 2007). Given the growing list of interacting proteins, it is tempting to speculate that specific Hipk function is determined in part through its particular localization. It is also possible that Hipk/Hipk2 may act as a scaffolding protein, bringing together multiple binding partners. Ongoing biochemical studies will further uncover the molecular significance of these interactions. Hipk proteins are emerging as important components of multiple signaling networks. The current study describes the roles of Hipk and Hipk2 as Wnt/Wg regulators and sheds light on the regulatory mechanisms governing this conserved pathway (Lee, 2009b).

Hipk is an essential protein that promotes Notch signal transduction in the Drosophila eye by inhibition of the global co-repressor Groucho

Homeodomain interacting protein kinase (Hipk) is a member of a novel family of serine/threonine kinases. Extensive biochemical studies of vertebrate homologs, particularly Hipk2, have identified a growing list of interactors, including proteins involved in transcriptional regulation, chromatin remodeling and essential signaling pathways such as Wnt and TGFβ. To gain insight into the in vivo functions of the single Drosophila Hipk loss of function alleles were characterized, that revealed an essential requirement for hipk. In the developing eye, hipk promotes the Notch pathway. Notch signaling acts at multiple points in eye development to promote growth, proliferation and patterning. Hipk stimulates the early function of Notch in promotion of global growth of the eye disc. It has been shown in the Drosophila eye that Hipk interferes with the repressive activity of the global co-repressor, Groucho (Gro). This paper proposes that Hipk antagonizes Gro to promote the transmission of the Notch signal, indicating that Hipk plays numerous roles in regulating gene expression through interference with the formation of Gro-containing co-repressor complexes (Lee, 2009a).

To study hipk function in vivo, deletions were generated at the hipk locus through imprecise excision of a transposable element. Two excisions (hipk² and hipk³) result in homozygous pupal lethality (with rare escaper adults) and trans-heterozygosity for any of these alleles and a deficiency removing hipk (Df(3L)ED4177) leads to lethality. A fourth allele, hipk⁴, was generated through targeted deletion of the DNA between two transposable elements flanking the locus and this allele causes lethality prior to the 3rd larval instar. Interallelic crosses reveal an allelic series in the order of weakest to strongest: hipk² < hipk¹ < hipk³ < hipk⁴. These findings demonstrate that the single hipk gene in Drosophila is essential. Indeed, the loss of both maternal and zygotic hipk results in embryonic lethality (Lee, 2009a).

hipk mutants consistently displayed small, rough eyes. Dissection of pharate adults from pupal cases revealed that 42% of hipk³ homozygotes displayed a preferential loss in the ventral region, leading to a small round eye. Additional eye phenotypes include the appearance of non-retinal tissue in 25% of hipk³ homozygotes (Lee, 2009a).

Staining of neuronal cells in 3rd instar eye imaginal discs with the neural anti-Elav antibody revealed that 25% of hipk³ homozygotes display a loss of photoreceptors. This loss was most prominent in the lateral poles of the eye disc as the Elav-positive cells did not extend to the dorsal and ventral margins of the eye disc as is seen in wildtype. The loss of photoreceptors likely correlates with the loss of eye structure in adults. Further reduction of Hipk activity by generating loss of function somatic clones with a stronger allele, hipk⁴, also led to a decrease of Elav staining. Under such conditions, neural differentiation is most sensitive to the loss of hipk near the MF. hipk⁴ clones proximal to the MF displayed diminished Elav staining. This effect is not restricted to the lateral poles, as was observed in hipk³ homozygotes. While photoreceptors in clones located posterior to the MF appeared to differentiate correctly, the spacing of these cells was reduced and irregular, suggesting hipk is also required for patterning of cells posterior to the MF. It was found that the loss of photoreceptors is likely not attributed to a defect in eye specification, since Ey expression is not diminished in hipk⁴ somatic clones (Lee, 2009a).

It was next determined if the loss of photoreceptors observed in hipk clones could be a secondary effect of altered cell cycle regulation or cell death during retinal specification. No apparent changes were observed in discs stained to visualize cell proliferation, using anti-phospho histone 3 antibody, or levels of apoptosis, as visualized by staining for the activated Drosophila ICE caspase (drICE). Hence it appears that loss of photoreceptors in hipk mutant cells may be linked to a modification in early eye development, rather than altered cell death (Lee, 2009a).

Consistent with loss of function analyses that implicate a role for hipk in eye patterning, it was found that ectopic expression of UAS-hipk also affected eye development. Using ey-Gal4 to drive wild type Hipk expression throughout larval eye development caused abnormal rough eyes, of which 33% also displayed cuticle-like structures. In these flies, a novel role was observed for hipk as a regulator of organ size. 39% of ey > hipk flies showed overgrown eyes that are likely caused during larval development, since overgrowths were also observed in imaginal discs. Thus Hipk plays a role in the patterning of the eye, although the underlying mechanism is still unknown (Lee, 2009a).

During eye development, hipk is expressed in a dynamic pattern throughout the eye disc. Antisense RNA in situ hybridization revealed that in the late second larval instar (L2) hipk is enriched in the medial domain of the visual primordium including the D/V boundary of the eye disc and this localization persists into early third instar. Beginning in mid 3rd instar larval stage, hipk expression is enriched in the anterior folds of the eye discs and becomes broadly expressed in the anterior region of the eye disc ahead of the MF. Later in late third instar, the localization varies between discs and likely reflects very dynamic changes in expression. In these disc, hipk is further refined to a narrow stripe covering much of the width of the disc. Using a combination of fluorescent in situ hybridization (FISH) and antibody staining, co-localization was observed of hipk and the retinal determination factor Dachshund (Dac) at the anterior-most edge of the Dac expression domain. This edge of Dac expression delimits the anterior boundary of the cells entering the neural program. This dynamic pattern of expression shows that hipk is expressed at the D/V organizing center early and later in undifferentiated cells anterior to the MF (Lee, 2009a).

N signaling controls many aspects of eye development such as proliferation and the establishment of the eye field. Loss of N signaling causes a small eye phenotype and gain-of-function mutants leads to an overproliferation of the eye. In addition, the dorsal and ventral eye regions are asymmetrically regulated, as the loss of the Ser regulator Lobe results in preferential loss of the ventral eye domain. The loss of eye tissue in hipk homozygous mutants and the overgrowth defects in ey > hipk resemble those observed with modulated activity of N and suggest a potential role for Hipk as a mediator of N-regulated growth processes (Lee, 2009a).

Genetic interaction studies were undertaken to investigate the interaction between the N pathway and hipk. Heterozygosity for the N ligand Dl enhances the small eye phenotype of hipk³ mutants. 30% of these small eyes are half the normal size and more dramatically, 20% were a quarter of the normal eye size. In contrast, in hipk³ homozygotes, only 4% of eyes were reduced to half the size and 2% were a quarter of the normal size, respectively. These phenotypes were much more severe than those observed with the hipk³ homozygous mutation alone and suggested a potential synergy between Dl and hipk. This interaction was also observed with the hipk2 allele. Similarly, the overproliferation defect observed in ey > Dl was enhanced by the co-expression of Hipk (Lee, 2009a).

Most strikingly, expression of dominant negative N with ey-Gal4 led to a dramatic loss of the eye which was suppressed by co-expression of Hipk. Such a rescue of reduced N signaling strongly suggests Hipk acts to promote N signaling downstream of the receptor. Further support for this model is seen upon examining imaginal disc phenotypes. Overexpression of the constitutively active NICD with ey-Gal4 leads to severely abnormal eye discs with dramatic overgrowths and reduced number of photoreceptors as a result of increased lateral inhibition. Decreasing hipk in these discs restored the population of photoreceptors. These findings suggest hipk likely regulates a subset of N-mediated processes (Lee, 2009a).

These analyses suggested that hipk cooperates with the N pathway. To assess whether Hipk is required to promote the transduction of this cascade, N activity was measured in hipk mutant cells by examining the expression of the products of the E(spl) complex, direct targets of Su(H). Using an antibody that recognizes 4 of 7 products of the E(spl) complex, a decrease was observed in E(spl) expression in mutant cells, most evident in cells located near the furrow. Therefore, hipk is required for the efficient transduction of the N signal and hipk mutant cells have reduced N signaling activity (Lee, 2009a).

Intriguingly, clones located in the posterior of the eye disc display slightly elevated expression of E(spl), suggesting additional mechanisms through which hipk patterns the eye. These findings, and the complexity of the hipk phenotype, demonstrate that hipk plays multiple roles during eye development in addition to its role as a positive regulator of the N signal (Lee, 2009a).

Hairless (H) is an antagonist of N that functions as an adaptor to bridge Gro and Su(H) to form a repressor complex. This mechanism is utilized to inhibit N signaling in multiple developmental processes. It was shown that Hipk phosphorylation could antagonize Gro function by promoting the disassembly of the repressor complex (Choi, 2005), so it was investigated whether this may be the route through which Hipk promotes N activity. It was hypothesized that Hipk may serve as a general antagonist of Gro and consequently promote Su(H)-mediated transcription by inhibiting the interactions between the repressor complex and Su(H). If such a mechanism exists, expression of a phospho-mimetic form of Gro, in which Hipk phosphorylation sites are mutated to glutamic acid residues, should exert effects reminiscent of those observed by expressing wildtype Hipk (Lee, 2009a).

To test this model, biochemical studies were performed to characterize the interaction between Hipk and Gro. Kinase assays were performed using purified Gro (full length and derivatives) from bacterial lysates in the presence of GST-Hipk. Hipk specifically phosphorylated the SP domain of Gro. Further analyses using synthetic Gro decapeptides identified two Hipk target residues, namely amino acids S297 and T300. S297 was also identified as a Hipk site by Choi (2005). These sites were mutated to alanine (GroAA) to test Hipk's specificity in a kinase assay. While full length Gro was phosphorylated by Hipk, the GroAA variant was resistant to phosphorylation, confirming S297 and T300 as Hipk target sites. These residues are also conserved in human Hipk2 (Lee, 2009a).

To generate a phospho-mimetic variant, these target residues were mutated to glutamic acid (GroEE). If Hipk can indeed repress Gro activity, then this form of Gro should be constitutively inhibited, while the GroAA variant should display constitutive activity. To test the properties of these Gro variants in vivo, transgenic fly strains expressing groAA and groEE, under control of the UAS promoter were generated. Expression of GroAA with ey-Gal4 produced a similar loss of eye phenotype to that seen in ey > groWT flies. Such phenotypic similarities suggested that GroAA is functionally equivalent to wild type Gro. ey > groEE flies displayed a much less severe phenotype upon misexpression than groWT, suggesting the activity of GroEE is compromised (Lee, 2009a).

Misexpression of Hipk can suppress the loss of eye phenotype caused by ey > groWT. Whether this rescue occurs by inhibiting Gro's repressive activity was examined. In contrast to the suppression of groWT, phenotypes induced by both groAA and groEE were less sensitive to elevated levels of Hipk. Co-expression of groAA or groEE with hipk showed a phenotype most similar to the Gro derivatives alone, indicating that these forms are not sensitive to the regulation by Hipk compared with the sensitivity seen with GroWT (Lee, 2009a).

To investigate whether Hipk can promote N activity via its regulation of Gro, a series of genetic interaction assays were carried out involving groWT, groEE and groAA in conjunction with the N antagonist H. Both inhibition of N or ectopic groWT led to loss of eye structures. Co-expression of Hipk can rescue the effect of dominant negative N. The model predicts that if the rescue of N signaling by Hipk is mediated through direct inhibition of Gro through phosphorylation, then a similar rescue should be observed with the phospho-mimetic form GroEE. However, it is expected that the Hipk-resistant form GroAA will phenocopy the effects of GroWT. Decreasing Notch activity via expression of the antagonist H caused a complete loss of eye, similar to that caused by expression of NDN. Expression of hipk at this temperature only induced a mild rough eye. Co-expression of hipk and H partially restored the development of retinal tissue as observed by the presence of a small eye. Furthermore, morphological defects in the head, including ocellar defects in the dorsal head, were also rescued, suggesting Hipk may also regulate other N-dependent processes (Lee, 2009a).

Next it was determined whether the mutated Gro forms could suppress the ey > H phenotype by mimicking the regulation seen with co-expression of Hipk. Consistent with the prediction, misexpressing groEE with ey-Gal4 was capable of restoring eye structures in the ey > H background, phenocopying the rescue by hipk. Conversely, co-expression of groAA led to a dramatic enhancement of the ey > H phenotype. In these flies, the eye fails to develop and head defects are magnified as indicated by the presence of only the dorsal vertex of the head in 41%, or more severely, the entire head is lost in 29% of the flies. As expected, similar interactions were also observed with groWT. Taken together, these results support a model in which Hipk phosphorylates and inhibits Gro to facilitate the N pathway (Lee, 2009a).

To further confirm that these effects on transduction of the Notch signal were indeed a consequence of decreasing Gro's repressive activity, it was asked if the addition of Hipk could modify the interactions between the Gro derivatives and H. It was predicted that introducing Hipk would modify the effects of GroWT on H, but not those induced by the Gro derivatives, since these mutations would render Gro less sensitive to elevated levels of Hipk. The results suggest that these mutations bypass the regulation of Gro by Hipk, since it was observed that co-expression of hipk, H and groWT led to a slight rescue of the very abnormal head structures seen with ey > H, GroWT, and suppressed the enhancement of the ey > H phenotype incurred by introducing gro. This indicates that Hipk can inhibit Gro activity, thereby quenching its antagonistic effect on the N signal. However, co-expression of H and hipk in the presence of groEE or groAA did not detectably modify the phenotype seen with the combination of H and the Gro derivatives alone. These observations support the model that Hipk contributes to the propagation of the N signal by inhibiting Gro's repressive activity through phosphorylation of S297 and T300 (Lee, 2009a).

These data support a model in which Hipk can promote N signaling during eye development through its repression of Gro. A previous report demonstrated that Hipk could promote the in vitro transcriptional activation activity of Eyeless by inhibiting Gro. In vivo data showed that Hipk could modify Gro activity and the resulting eye phenotypes were attributed to changes in Eyeless activity (Choi, 2005). Promotion of Ey activity would reflect a role solely in eye specification. The phenotypic consequences of modifying Hipk activity and hipk's dynamic expression profile both clearly suggest additional requirements for Hipk other than eye specification. To further clarify the mechanism underlying the genetic observations, it was examined whether the same phenotypic rescue of ey > H seen with Hipk could be seen with the misexpression of Ey. If the only function of Hipk were to promote Ey activity, then a similar rescue would be expected with elevated levels of Ey. However, it was found that ectopic ey failed to rescue the ey > H phenotype, and moreover, greatly enhanced it. Furthermore, it was also found that concomitant misexpression of hipk mildly modified ey > ey phenotype, rather than a potent synergistic modification. These genetic interactions suggest that the ability of Hipk to rescue diminished N signaling activity is independent of the ascribed role in promoting Ey activity (Choi, 2005). These findings are consistent with the genetic interaction studies and analyses of N target genes that indicate that Hipk acts to promote N signaling (Lee, 2009a).

N is a recurring player in eye development, a feat that is accomplished through its unique interplay with members of the Pax6 family of transcriptional regulators. For example, N-controlled eye growth is specifically mediated via Eyg. Reducing N signaling activity induces an eye-loss phenotype, which is caused by a deregulation of organ growth through Eyg, rather than a reliance on the eye specification players Ey or Toy. Overexpression of Eyg but not Ey nor Toy reliably restored eye development in N deficient flies (Lee, 2009a).

Several lines of evidence strongly suggested that Hipk promotes N-mediated eye growth. First, ey > hipk phenocopies the overgrowths seen with elevated levels of the N signal. Second, as was seen with Eyg, simultaneous misexpression of Hipk rescues the loss of eye phenotype in a dominant negative N background. Furthermore, hipk is expressed in a region encompassing the D/V organizing growth center where N acts to specifically control the global growth of the eye. The genetic examination were extended to confirm the model by further characterizing the interaction between Hipk and H. Specifically, it was addressed whether the ey > H phenotype was correlated with a defect in organ growth, rather than eye specification. Consistent with such a model, overexpression of the growth regulator eyg, but not ey, restored the eye in ey > H adults. These suppressive effects are identical to those observed with both hipk and groEE transgenes (Lee, 2009a).

Similar to what is seen with components of the N pathway, Hipk induces pleiotropic effects throughout eye development. Attempts were made to confirm that the genetic rescues were not attributed to a secondary effect of Hipk-mediated processes unrelated to growth. To address this, the consequences were examined of modifying Hipk levels on the phenotype induced upon misexpression of Fringe (Fng). The small eye phenotype seen in ey > Fng flies is attributed solely to a defect in eye growth. Thus any observed modification in this sensitized genetic background would validate a requirement for Hipk in eye growth. As predicted by the model, overexpression of hipk or groEE partially rescues the ey > fng phenotype. More strikingly, the eye fails to form when hipk activity is reduced, a phenotype similar to what is seen with eyg or Dl mutants in an ey > Fng background. Taken together, these genetic interactions demonstrate that Hipk promotes N-mediated eye growth (Lee, 2009a). The complementary expression domains of the N ligands Dl and Ser in the dorsal and ventral compartments, respectively, ensures that the N pathway is activated at the D/V boundary of the developing 2nd instar eye disc. N establishes the organizing center to mediate global growth by regulating eyg expression along the length of the D/V boundary. The expression of Ser and Dl appear normal in hipk⁴ clones suggesting that the D/V center is established normally in hipk mutant cells. However, it was observed that Eyg expression is autonomously reduced in hipk mutant clones. Such an effect on eyg expression is also observed in clones mutant for either Su(H) or Dl and Ser. Conversely, ey > hipk third instar eye discs display an expanded expression domain of Eyg. These observations indicate that Hipk is required for activation of normal Eyg expression, and loss of hipk induces a growth defect (Lee, 2009a).

In conclusion, the lethality of hipk mutant alleles demonstrates that Hipk is an essential kinase in Drosophila that plays a critical role during eye development. Eye patterning and growth defects are observed in both hipk homozygous mutants and somatic clones. hipk mutant clones display reduced N signaling activity, as measured by the diminished expression of the N targets, E(spl) and Eyg. Therefore, these studies implicate Hipk in the positive regulation of the N signaling cascade during eye development. Although these data demonstrate that Hipk regulates N-mediated eye growth, the neural patterning defects are less severe than previously published N mutant phenotypes. The possibility cannot be excluded that these neuronal defects are due to a secondary consequence of Hipk's requirement in earlier phases of eye development or a role for Hipk in modulating additional eye patterning pathways other than the N pathway (Lee, 2009a).

In vitro and in vivo data support a model in which Hipk phosphorylates Gro, and consequently relieves its repressive activity on the Su(H) transcriptional complex. Overexpression of the N antagonist H results in loss of eye. Hipk-mediated phosphorylation of Gro at S297 and T300 is necessary to rescue the phenotype caused by reduced N signaling. Indeed, genetic misexpression analyses clearly demonstrate that this phosphorylation event is necessary to relieve Gro's inhibitory effect on N, thereby permitting activation of downstream N targets. Since Gro is a global co-repressor, this interaction may represent a global mechanism through which Hipk can regulate gene expression during development (Lee, 2009a).

This study has identified Hipk as a key player in modulating growth in the eye. Given that a similar role for Hipk in promoting growth was observed in additional tissues, this likely represents a general role for Hipk in organ and tissue growth. Although Hipk can induce outgrowths in the wing, it does so via a Notch-independent mechanism. Future studies will reveal to what extent Hipk can integrate multiple signaling inputs or regulate transcriptional complexes (Lee, 2009a).

A positive feedback loop between Myc and aerobic glycolysis sustains tumor growth in a Drosophila tumor model

Cancer cells usually exhibit aberrant cell signaling and metabolic reprogramming. However, mechanisms of crosstalk between these processes remain elusive. This study shows that in an in vivo tumor model expressing oncogenic Drosophila Homeodomain-interacting protein kinase (Hipk), tumor cells display elevated aerobic glycolysis. Mechanistically, elevated Hipk drives transcriptional upregulation of Drosophila Myc (dMyc; MYC in vertebrates) likely through convergence of multiple perturbed signaling cascades. dMyc induces robust expression of pfk2 (encoding 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PFKFB in vertebrates) among other glycolytic genes. Pfk2 catalyzes the synthesis of fructose-2,6-bisphosphate, which acts as a potent allosteric activator of Phosphofructokinase (Pfk) and thus stimulates glycolysis. Pfk2 and Pfk in turn are required to sustain dMyc protein accumulation post-transcriptionally, establishing a positive feedback loop. Disruption of the loop abrogates tumorous growth. Together, this study demonstrates a reciprocal stimulation of Myc and aerobic glycolysis and identifies the Pfk2-Pfk governed committed step of glycolysis as a metabolic vulnerability during tumorigenesis (Wong, 2019).

In the 1920s, Otto Warburg first discovered that cancer cells vigorously take up glucose and preferentially produce lactate even in the presence of oxygen, a phenomenon now widely termed the Warburg effect or aerobic glycolysis. Despite his pioneering work, the Warburg effect was largely disregarded for the subsequent decades. After the discoveries of oncogenes and tumor suppressor genes, cancers are generally considered as genetic diseases rather than metabolic ones. Not until the 1980s did the revisiting of the Warburg effect in connection with oncogenes spark substantial research in cancer metabolism, laying the foundation for 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) positron emission tomography (PET) in clinical cancer diagnosis, recognition of metabolic reprogramming as a hallmark of cancer and development of anti-cancer agents targeting aerobic glycolysis (Wong, 2019).

How the Warburg effect arises and contributes to tumor progression have always been the center in the field of cancer metabolism. Warburg hypothesized that mitochondrial impairment is the cause of aerobic glycolysis and cancer, which has sparked much controversy among scientists. Contrary to his idea, the current, widely-accepted view is that oncogenic drivers such as RAS, MYC, hypoxia-inducible factors (HIFs) and steroid receptor coactivators (SRCs) promote cancer cell proliferation and directly stimulate aerobic glycolysis through regulating transcriptional expression or catalytic activities of metabolic enzymes. Several explanations for the Warburg effect have been put forward, including rapid adenosine triphosphate (ATP) production and de novo biosynthesis of macromolecules. Recently, Warburg effect functions are being re-evaluated as a growing body of evidence shows that metabolic rewiring in cancers impacts cell signaling and epigenetics (Wong, 2019).

Drosophila has proven to be a powerful genetic model organism for studying tumorigenesis in vivo largely due to high conservation of genes and signaling cascades between human and flies and reduced genetic redundancy. In a recent study, it was shown that elevation of Drosophila Hipk causes an in vivo tumor model characterized by tissue overgrowth, loss of epithelial integrity and invasion-like behaviors (Blaquiere, 2018). The tumorigenic roles of Drosophila Hipk seem to be conserved in mammals as the four members of the HIPK family (HIPK1-4) are also implicated in certain cancers. For example, HIPK1 is highly expressed in breast cancer cell lines, colorectal cancer samples and oncogenically-transformed mouse embryo fibroblasts. Also, HIPK2 is elevated in certain cancers including cervical cancers, pilocytic astrocytomas, colorectal cancer cells and in other proliferative diseases, such as thyroid follicular hyperplasia. To gain a better understanding of cancer metabolism, this study set out to use the fly Hipk tumor model to investigate whether and how cellular metabolism is altered in tumor cells. Hipk-induced tumorous growth was found to be accompanied by elevated aerobic glycolysis. Furthermore, novel feedback mechanisms were identified leading to prolonged dMyc expression and hence tumorigenesis. This study reveals potential metabolic vulnerabilities that could be exploited to suppress tumor growth (Wong, 2019).

Multiple approaches and tools have been developed to measure glycolysis including measurement of the extracellular acidification rate (ECAR), fluorescent probes, biosensors, fluorescent/colorimetric assays and mass spectrometry. In the Hipk tumor model, the tumor cells are surrounded by wild-type cells. This study primarily used fluorescence imaging such that it was possible to examine changes in metabolite levels, gene and protein expression between tumor cells and the adjacent wild-type cells. This work delineates the causes and significance of metabolic changes in tumor cells (Wong, 2019).

Drosophila tumor models frequently acquire metabolic changes, especially the Warburg effect. For instance, epidermal growth factor receptor (EGFR)-driven tumors, tumors with activated platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptor (Pvr) and tumors with polarity loss (such as scrib or dlg mutant tumors) feature robust upregulation of Ldh among other glycolytic genes. Depletion of Ldh was shown to reduce growth of EGFR-driven tumors but not tumors with polarity loss. In another study RasV12scrib^-/- tumors, elevated glucose uptake was evident, but its significance was not evaluated. Similar to the previously described models, Hipk tumor cells exhibited elevated glucose metabolism. Metabolic reprogramming was driven by dMyc upregulation. Although the transcript levels of another glycolytic inducer sima remained unchanged, it was not possible to eliminate the possibility that Sima protein levels are altered in the tumor cells. Thus, whether Sima is involved in the tumor growth warrants further studies. Genetical inhibition of Pfk or Pfk2 was sufficient to block Hipk-induced tumorigenesis whereas depletion of Pgk, Pyk or Ldh at most slightly reduced tumor growth. Therefore, the data suggest that targeted but not generic inhibition of glycolysis is required to abrogate tumor growth (Wong, 2019).

A feedback loop between dMyc and aerobic glycolysis as a metabolic vulnerability in tumor cells Although MYC functions in cancer metabolism are widely recognized, MYC is generally considered 'undruggable' largely due to its nuclear localization, lack of an enzymatic 'active site', and indispensable roles during normal development. Hence, limited therapeutic strategies have been developed, and identification of the 'druggable' regulatory proteins of MYC becomes critical (Wong, 2019).

This study reveals two modes of dysregulation of endogenous dMyc specifically during Hipk-induced tumorigenesis. The first mode is transcriptional stimulation of dMyc likely as a consequence of convergence of multiple signaling outputs. In a previous study, it was found that genetically targeting individual signaling cascades failed to restrain Hipk tumor growth (Blaquiere, 2018). This is likely due to the redundancy of signaling cascades in inducing dMyc upregulation. It is also interesting to note that dMyc upregulation and the associated tumor growth are most striking in the wing hinge. The pouch region, however, seems more refractory to such alterations. The lowest intracellular glucose levels and robust Ldh upregulation were observed in the hinge area. dMyc-dependent glucose uptake, on the other hand, was enhanced in both hinge and pouch regions even though dMyc protein buildup is undetectable in the wing pouch, suggesting that glucose uptake is particularly sensitive to changes in dMyc levels. The data therefore imply that the Warburg effect and the tumor growth phenotype may be linked to the dMyc levels being induced. The region-specific susceptibility to tumorigenic stimuli has been previously described and the hinge region was coined a 'tumor hotspot' because of its unique epithelial cell architectures and high endogenous JAK/STAT signaling. It is possible that such features in the hinge region also contribute to the sensitivity of dMyc upregulation by elevated Hipk and other signaling pathways. Further studies are required to verify this proposition (Wong, 2019).

The second mode of dMyc regulation is the metabolic control of dMyc protein levels by aerobic glycolysis. Specifically, this study found that the rate-limiting enzymes Pfk2 and Pfk are required to perpetuate dMyc buildup in tumor cells in a post-transcriptional manner. Similar to the effects on tumor growth, while pfk2/pfk knockdown prevented dMyc accumulation, knockdown of other glycolytic enzymes had little effect on sustaining dMyc accumulation. Such a disparity could possibly be explained by the potency of the glycolytic enzymes in controlling glycolytic flux. While Pfk2 and Pfk govern the rate-limiting, committed step of glycolysis, RNAi targeting other glycolytic genes may fail to render the corresponding steps rate-limiting, especially in tumors with elevated aerobic glycolysis. In other words, knockdown of other glycolytic enzymes may not reach the threshold that would restrict glycolytic flux as potently as pfk2/pfk knockdown. Given that Pfk2 is mis-expressed in tumor cells but not in normal cells and that dMyc accumulation is most sensitive to the Pfk2-Pfk mediated committed step of glycolysis specifically in tumor cells, targeting Pfk2 may be a favorable, selective metabolic strategy in the treatment of cancers, especially those displaying ectopic MYC expression (Wong, 2019).

The functions of mammalian PFK and PFKFB in controlling glycolytic flux are well-defined. The allosteric regulation of PFK by F2,6-BP seems to be conserved among mammals and insects as F2,6-BP is able to activate insect Pfk, likely due to the conserved amino acid residues for F2,6-BP binding. This suggests that Pfk2 can induce Pfk activation and hence glycolytic flux through biosynthesizing F2,6-BP. A previous report shows that flies lacking pfk2 exhibited levels of circulating glucose and reduced sugar tolerance, providing a functional link between Pfk2 and glucose metabolism. This study has provided evidence that both pfk2 and pfk knockdown larvae exhibited significant decreases in pyruvate contents, confirming a conserved role of Pfk2 in regulating glycolytic flux (Wong, 2019).

Recently, the roles of PFK and PFKFB in the regulation of transcription factors or cofactors have drawn considerable attention. For example, vertebrate PFK was shown to physically interact with TEAD factors to stimulate YAP/TAZ activity, thus promoting proliferation and malignancy in cancer cells. PFKFB4 (an isoform of PFK2) phosphorylates and activates oncogenic SRC-3 to promote breast cancers through stimulating purine synthesis. These studies point to the fact that PFK and PFKFB functions are not restricted to metabolic regulation. Thus, it is tempting to speculate that Pfk2/Pfk may sustain ectopic dMyc through a direct interaction with or even phosphorylation of dMyc. That being said, as knockdown of Pfk2 or Pfk had no effects on dMyc protein levels under normal conditions, it is likely that a tumorigenic environment with active aerobic glycolysis is necessary for the metabolic control of dMyc in particular through the committed step governed by Pfk2/Pfk. In summary, this study shows glycolytic enzymes Pfk and Pfk2, likely through their core biochemical role in stimulating glycolytic flux, glycolysis-independent actions or both, act as key players in coupling metabolic demands with growth signals to achieve cancer progression (Wong, 2019).

Drosophila Smt3 negatively regulates JNK signaling through sequestering Hipk in the nucleus

Post-translational modification by the small ubiquitin-related modifier (SUMO) is important for a variety of cellular and developmental processes. However, the precise mechanism(s) that connects sumoylation to specific developmental signaling pathways remains relatively less clear. This study shows that Smt3 (SUMO) knockdown in Drosophila wing discs causes phenotypes resembling JNK gain of function, including ectopic apoptosis and apoptosis-induced compensatory growth. Smt3 depletion leads to an increased expression of JNK target genes Mmp1 and puckered. Although knockdown of the homeodomain-interacting protein kinase (Hipk) suppresses Smt3 depletion-induced activation of JNK, Hipk overexpression synergistically enhances this type of JNK activation. This study further demonstrates that Hipk is sumolylated in vivo, and its nuclear localization is dependent on the sumoylation pathway. These results thus establish a mechanistic connection between the sumoylation pathway and the JNK pathway through the action of Hipk. It is proposed that the sumoylation-controlled balance between cytoplasmic and nuclear Hipk plays a crucial role in regulating JNK signaling (Huang, 2011).

Sumoylation is a post-translational modification that regulates multiple biological activities by modifying a variety of different substrates. This study shows that tissue-specific perturbation of the sumoylation pathway activates the JNK signaling pathway. In particular, knockdown of the Drosophila SUMO gene smt3 recapitulates several key gain-of-function features of the JNK pathway, including apoptosis and wg ectopic expression. These results suggest that sumoylation plays a crucial role in regulating JNK signaling. Further experiments demonstrate that Hipk is responsible for Smt3 depletion-induced JNK activation. These experiments show that Hipk itself is sumoylated and that its nuclear localization is dependent on the sumoylation pathway. Based on these findings, a model is proposed in which Hipk is normally kept in the nucleus, but a compromised sumoylation pathway (such as that produced by depletion of Smt3) allows some Hipk molecules to translocate to the cytoplasm and activate the JNK signaling pathway (Huang, 2011).

Sumoylation regulates the biological activities of its substrates through several distinct mechanisms. These mechanisms include altering subcellular localization of its substrate proteins and/or molecular shuttling between the nucleus and the cytoplasm, mediating protein-protein interactions, locking its substrates in a particular conformational state (i.e. active or inactive) or altering protein stability and clearance. This study highlights the importance of sumoylation-dependent subcellular localization of Hipk in regulating its biological activities. It is proposed that sumoylation normally restricts Hipk to the nucleus and facilitates the execution of its nuclear functions, such as interaction with and phosphorylation of transcriptional co-repressors. However, unsumoylated or desumoylated Hipk becomes accessible to the cytoplasm for executing its cytoplasmic function(s). As shown in this study, one such cytoplasmic function of Hipk is to modulate the JNK signaling pathway (Huang, 2011).

Hipk family members play roles in different biological processes, such as cell cycle progression, p53-dependent apoptosis, and transcriptional regulation. The mammalian cells contain four Hipk proteins that perform overlapping, but distinct functions. For example, Hipk1 and Hipk2 have functionally redundant roles in mediating cell proliferation and apoptosis during development. Hipk1 interacts with transcription factor c-Myb, while Hipk2 phosphorylates transcriptional co-repressor Groucho, suggesting their distinct roles in transcription regulation. Therefore, it would be interesting to elucidate whether Drosophila Hipk executes the functions of all the mammalian counterparts, although Drosophila Hipk shares most homology with Hipk2. This all-in-one mode of Hipk function requires different strategies to regulate its functions. Previous studies have shown that Drosophila Hipk promotes various signaling pathways such as the Wnt pathway through stabilizing Armadillo, and the Notch pathway through inhibiting the global co-repressor Groucho. This work reports that Drosophila Hipk potentiates JNK signaling through a sumoylation-dependent regulation of its subcellular localization. This work underscore the roles of Drosophila Hipk both inside and outside of the nucleus in fine-tuning signaling pathways. It remains to be determined precisely how Hipk regulates the JNK pathway and whether it involves a direct mechanism such as phosphorylating relevant components of this pathway (Huang, 2011).

The subcellular localization of Hipk represents an important mechanism in defining its functional specificity. In particular, Hipk controls the degradation of transcriptional co-repressor CtBP inside the nucleus, while the cytoplasmic Hipk interacts with the nonhistone chromosomal factor Hmga1 (high-mobility group A1) to inhibit cell growth. Hipk has also been shown to, within the speckled subnuclear structures, interact with p53 to promote its phosphorylation. The current results presented show that Hipk is normally sequestered in the nucleus but gains access to the cytoplasm, upon sumoylation perturbation, to activate the JNK signaling pathway. The idea that the subcellular localization of Hipk is crucial for its functional specificity also explains why overexpressing Hipk alone did not result in a robust activation of JNK. It is suggested that, without sumoylation perturbation, the majority of transgene-expressed Hipk is, like the endogenously expressed Hipk, sumoylated and kept in the nucleus, making it inaccessible to activating JNK (Huang, 2011).

The JNK signaling pathway is composed of stepwise actions of kinases. The canonical JNK pathway receives signals from death stimuli, such as tumor necrosis factor (TNF) and oxidative stresses. In addition to the JNK pathway, other factors such as Hipk proteins are also stimulated by a variety of stresses. For example, the human HIPK1 responds to the stimulation of TNFα to relocate itself from the nucleus to the cytoplasm. In addition, the mammalian Hipk2 phosphorylates p53 in response to UV irradiation and phosphorylates cyclic AMP response element-binding protein (CREB) to cope with genotoxic stress. It is proposed that stress signals such as TNF may activate not only the canonical JNK pathway but also the Hipk-dependent JNK activation mechanism(s). The idea that Hipk acts downstream of TNF is consistent with genetic evidence that RNAi ablation of Hipk partially rescues the Drosophila TNF (Egr)-induced phenotype in the eye. A major finding of the current study is that it establishes a cross-regulation between the sumoylation and the JNK pathways through the action of Hipk. It is currently unknown whether TNF or even JNK itself may regulate the sumoylation pathway, but it remains an interesting possibility that will require further investigation. It is noted that the relationship between sumoylation and JNK pathways is likely to be more complex than Hipk-mediated action described in this work. It has been shown that sumoylation is required for Axin-mediated JNK activation. Thus, it is possible that sumoylation may have different, even opposite, effects on the JNK pathway through distinct sumoylation targets. The robust increase of the JNK activity detected in Smt3-depleted cells in this study demonstrates an overall negative role of the sumoylation pathway in JNK signaling (Huang, 2011).

Homeodomain-interacting protein kinase regulates Yorkie activity to promote tissue growth

The Hippo (Hpo) tumor suppressor pathway regulates tissue size by inhibiting cell proliferation and promoting apoptosis. The core components of the pathway, Hpo, Salvador, Warts (Wts), and Mats, form a kinase cascade to inhibit the activity of Yorkie (Yki), the transcriptional effector of the pathway. Homeodomain-interacting protein kinases (Hipks) are a family of conserved serine/threonine kinases that function as regulators of various transcription factors to regulate developmental processes including proliferation, differentiation, and apoptosis. Hipk can induce tissue overgrowth in Drosophila. This study demonstrates that Hipk is required to promote Yki activity. Hipk affects neither Yki stability nor its subcellular localization. Moreover, hipk knockdown suppresses the overgrowth and target gene expression caused by hyperactive Yki. Hipk phosphorylates Yki and in vivo analyses show that Hipk's regulation of Yki is kinase-dependent. This is the first kinase identified to positively regulate Yki (Chen, 2012; see graphical abstract).

These findings indicate that Hipk promotes Yki transcriptional activity to regulate tissue growth during Drosophila development. Because Hipk functions in multiple signaling pathways, the possibility cannot be excluded that Hipk regulates Hpo signaling and Yki via multiple pathways; however, it is thought that this study has ruled out a regulation through known targets. Furthermore, it was shown that Hipk phosphorylates Yki, implying a direct regulatory role of Hipk on Yki. Although Hipk promotes Yki activity by phosphorylation, the functional significance of this modification has yet to be further investigated. These analyses have applied the same genetic criteria and obtained similar results as were used to demonstrate that Sd was an essential factor for Yki-mediated growth. Given that Hipk exerts its effects on nuclear Yki and is enriched in nuclear speckles , it may play an important role to facilitate the interactions between Yki and its transcriptional cofactors. Further studies should reveal whether this growth regulation by Hipk family members is evolutionarily conserved. Trapasso (2009) demonstrate that hipk2 mutant mice have a reduced body size and hipk2^-/- mouse embryo fibroblasts show reduced proliferation rates, suggesting that Hipks may also promote Yap activity to regulate tissue growth in vertebrates. Using mammalian cell culture, it has been observed that overexpressing WT Hipk2, but not kinase-dead Hipk2, is able to induce elevated Yap level. This finding suggests that vertebrate Hipks may regulate Hpo signaling through Yap stability and possibly also by regulating Yap nuclear activity (Chen, 2012).

Homeodomain-interacting protein kinase regulates Hippo pathway-dependent tissue growth

The Salvador-Warts-Hippo (SWH) pathway is an evolutionarily conserved regulator of tissue growth that is deregulated in human cancer. Upstream SWH pathway components convey signals from neighboring cells via a core kinase cassette to the transcription coactivator Yorkie (Yki). Yki controls tissue growth by modulating activity of transcription factors including Scalloped (Sd). To date, five SWH pathway kinases have been identified, but large-scale phosphoproteome studies suggest that unidentified SWH pathway kinases exist. To identify such kinases, this study performed an RNA interference screen and isolated homeodomain-interacting protein kinase (Hipk). Unlike previously identified SWH pathway kinases, Hipk is unique in its ability to promote, rather than repress, Yki activity and does so in parallel to the Yki-repressive kinase, Warts (Wts). Hipk is required for basal Yki activity and is likely to regulate Yki function by promoting its accumulation in the nucleus. Like many SWH pathway proteins, Hipk's function is evolutionarily conserved as its closest human homolog, HIPK2, promotes activity of the Yki ortholog YAP in a kinase-dependent fashion. Further, HIPK2 promotes YAP abundance, suggesting that the mechanism by which HIPK2 regulates YAP has diverged in mammals (Poon, 2012).

Hipk proteins dually regulate Wnt/Wingless signal transduction

The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic β-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes β-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, this study describes that Hipk proteins have a role independent of their effect on β-catenin/Armadillo stability to enhance Wnt/Wingless signaling (Verheyen, 2012).

In summary, Hipk proteins play multiple important roles during developmental signaling events, and have the capacity to simultaneously impact the outcomes of both Wg and Hh signal transduction. The finding that Hipk proteins act by blocking Slimb/β-TrCP-mediated ubiquitination of substrates suggests that additional targets may also be affected through the action of these kinases. Additionally a nuclear role for Hipk was observed that is independent of its role in stabilizing the Wg pathway effector Arm. The data suggest that Hipk can enhance the transcriptional activation by the Arm/TCF complex. This role thought to be independent of its role in blocking substrate ubiquitination (Verheyen, 2012).

Multiple roles have been described for Hipk family members in regulation of Wnt/Wg pathway activity. Such findings are reminiscent of the work performed to decipher the roles of several other kinases in the Wnt pathway. Both GSK3 and CK1 have distinct roles at different steps in the transduction of Wnt signaling and act to either promote or inhibit pathway activity and as a result gene expression. The finding that Hipk-stabilized Arm is phosphorylated by GSK3 and CK1 reveals a specific point of action in the targeting and destruction of Arm/β- catenin. Future work should reveal whether the stabilized N-terminally phosphorylated Arm is associated with the destruction complex, in effect blocked from being transferred to the proteasome as a result of Hipk acting on Slimb. The regulation of β-catenin stability and activity has important implications for normal growth and patterning, tissue homeostasis and the development of cancer (Verheyen, 2012).

Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito

The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. This study shows that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and several phosphorylation sites of Nito were mapped. Furthermore, it was possible to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase (Dewald, 2014).

A collective form of cell death requires Homeodomain interacting protein kinase

Post-eclosion elimination of the Drosophila wing epithelium was studied in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step (see Collective Cell Death and Canonical Pathways). Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype was leveraged in mosaic animals to discover relevant genes. This study establish that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).

Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. First, unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. Second, extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. Third, widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).

One study proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ—before cells are removed from the wing (Kimura, 2004 and this study). First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the current screen. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor (Kimura, 2004). Fourth, using time-lapse microscopy, condensing or pycnotic nuclei were clearly detected, followed by the rapid removal of all cell debris in time frames (minutes) not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).

This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death–defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).

By leveraging this distinct phenotype, novel cell death genes, were captured including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner (Kim, 1998), this gene was found to be an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted orthologue of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway, but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).

Phosphorylation by the DHIPK2 protein kinase modulates the corepressor activity of Groucho

Groucho function is essential for Drosophila development, acting as a corepressor for specific transcription factors that are downstream targets of various signaling pathways. Evidence is provided that Groucho is phosphorylated by the DHIPK2 protein kinase. Phosphorylation modulates Groucho corepressor activity by attenuating its protein-protein interaction with a DNA-bound transcription factor. During eye development, DHIPK2 modifies Groucho activity, and eye phenotypes generated by overexpression of Groucho differ depending on its phosphorylation state. Moreover, analysis of nuclear extracts fractionated by column chromatography further shows that phospho-Groucho associates poorly with the corepressor complex, whereas the unphosphorylated form binds tightly. It is proposed that Groucho phosphorylation by DHIPK2 and its subsequent dissociation from the corepressor complex play a key role in relieving the transcriptional repression of target genes regulated by Groucho, thereby controlling cell fate determination during development (Choi, 2005. Full text of article).

Search PubMed for articles about Drosophila Hipk

Blaquiere, J. A., Wong, K. K. L., Kinsey, S. D., Wu, J. and Verheyen, E. M. (2018). Homeodomain-interacting protein kinase promotes tumorigenesis and metastatic cell behavior. Dis Model Mech 11(1). PubMed ID: 29208636

Calzado, M. A., Renner, F., Roscic, A. and Schmitz, M. L. (2007). HIPK2: a versatile switchboard regulating the transcription machinery and cell death. Cell Cycle 6: 139-143. PubMed ID: 17245128

Chen, J. and Verheyen, E. M. (2012). Homeodomain-interacting protein kinase regulates Yorkie activity to promote tissue growth. Curr. Biol. 22(17): 1582-6. PubMed ID: 22840522

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date revised: 19 June 2024

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