InteractiveFly: GeneBrief

kramer: Biological Overview | References |

Gene name - kramer

Synonyms -

Cytological map position - 87F12-87F13

Function - signaling

Keywords - regulates Wnt signaling by antagonizing Kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination

Symbol - kmr

FlyBase ID: FBgn0085412

Genetic map position - chr3R:13,715,207-13,764,667

Classification - Phosphoinositol 3-phosphate binding proteins 1, 2, and 3 pleckstrin homology (PH) domain

Cellular location: cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Kramer orthologs: Biolitmine

The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/β-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, this study examined the cellular determinants of ISC proliferation, harnessing Kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation and kramer regulates Wnt signaling in this context by antagonizing Kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes Kramer as a physiological regulator of Wnt/β-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling (Sun, 2023).

The adult Drosophila melanogaster intestine is a powerful model to study stem cell proliferation.1-5 The fly gut has many important physiological functions, most notably nutrient absorption, acting as a physical barrier and providing immunity to enteric pathogens and chemical insults.1,2 A conserved hallmark of the gut is the dynamic nature of its architecture.3-5 The Drosophila midgut is the largest and central portion of the intestines, and it is analogous to the mammalian small intestine in function and, to some extent, cellular architecture and composition (Sun, 2023).

The signature feature of the midgut is its epithelium, a single cell layer acting as a barrier to separate the lumen from internal tissues. In Drosophila, the midgut epithelium comprises four principal cell types. Intestinal stem cells (ISCs) can self-renew and also give rise to progenitor cells termed enteroblasts (EBs), which in turn can fully differentiate into enterocytes (ECs) that comprise the bulk of the epithelium. The specification of the fourth cell type, enteroendocrine cells (EEs), has not been fully elucidated, despite the importance of these cells in mediating important paracrine signaling events. EBs have been proposed to differentiate into EEs; however, recent studies demonstrated that ISCs, when Prospero-positive, divide into a distinct progenitor type termed pre-EEs, which subsequently differentiate into EEs. Disruption of ISC function can lead to either excessive proliferation or precocious differentiation, often resulting in disease. Therefore, a detailed understanding of the pathways and mechanisms regulating ISC proliferation is an important long-term goal with therapeutic implications (Sun, 2023 and references therein).

Numerous studies have shown that Wnt/β-catenin signaling, a morphogen signaling pathway that is highly conserved in animals, promotes ISC proliferation and differentiation under both physiological conditions and upon challenges such as enteric infection or chemical insults, both of which can damage the gut epithelium. More broadly, Wnt/β-catenin signaling, also known as canonical Wnt signaling, controls diverse cellular processes during animal development and homeostasis, including stem cell maintenance, cell fate specification, neural patterning, spindle orientation, cell migration, cell polarity, and gap junction communication. Dysregulation of canonical Wnt signaling caused by mutations of core components of this pathway is frequently linked to birth defects and many types of cancer. During tissue development and homeostasis, canonical Wnt signaling is thought to be the main pathway for regulating ISC proliferation and self-renewal, which drives massive renewal processes of intestinal epithelial cells (Sun, 2023).

Despite the fundamental importance of Wnt signaling in regulating ISC proliferation and subsequent tissue renewal in the gut epithelium, understanding of how Wnt signaling in different cell types within the gut contributes to these effects on ISCs is still rudimentary. Furthermore, recent studies have elucidated roles for β-catenin-independent, or non-canonical Wnt signaling pathways, which share some upstream components in the Wnt-receiving cell but do not activate β-catenin-dependent gene expression, in regulating ISC proliferation in the Drosophila midgut. Thus, knowledge of which cell types exhibit canonical and non-canonical Wnt signaling that collectively contribute to ISC proliferation and thus tissue maintenance in the midgut are major fundamental and unanswered questions (Sun, 2023).

A key shared player in all Wnt signaling pathways is Dishevelled (Dsh/DVL), which is recruited to the plasma membrane upon activation of Wnt receptors and co-receptors from the Frizzled and LRP families. Such recruitment catalyzes the disassembly of a multiprotein complex that facilitates proteasomal degradation of β-catenin, enabling its accumulation and subsequent translocation to the nucleus to activate TCF/LEF-dependent gene expression in the canonical pathway. Dsh recruitment to the plasma membrane also activates planar cell polarity and other non-canonical Wnt pathways, including the Wnt/Ca2+ pathway, by activation of Frizzled receptors. Thus, regulation of Dsh levels, which occurs via the ubiquitin-proteasome system and involves the action of several distinct E3 ubiquitin ligases, is a key control point in all Wnt signaling pathways (Sun, 2023).

Notably, a mammalian multi-subunit phosphoinositide-binding protein, pleckstrin homology domain-containing family A number 4 (PLEKHA4), promotes both Wnt/β-catenin and non-canonical Wnt signaling in human cell lines by antagonizing DVL polyubiquitination by the Cullin-3 (CUL3)-Kelch-like protein 12 (KLHL12) E3 ubiquitin ligase. This study found as well that Wnt/β-catenin signaling and subsequent cell proliferation in mouse models of melanoma was dependent upon PLEKHA4 expression (Shah, 2021). In Drosophila, knockout of the closest fly ortholog of PLEKHA4, kramer (kmr), impairs planar cell polarity in the adult wing, larval wing imaginal disc, and pupal wing disc epithelium. The absence of any discernable defects in canonical Wnt signaling in kmr knockout flies led the authors to question whether kmr indeed controlled Wnt/β-catenin signaling in this organism. It is proposed that the extent to which PLEKHA4/kmr loss affected canonical or non-canonical Wnt pathways might depend on cellular and tissue contexts, where expression of other factors downstream of DVL/Dsh might govern how tuning of DVL/Dsh levels would differentially affect outcomes from these pathways (Sun, 2023).

To test this prediction, this study investigated the role of kmr in controlling ISC proliferation in the Drosophila midgut, a physiological process dependent upon canonical Wnt signaling and recently linked to non-canonical Wnt signaling pathways as well. The experimental model used in this study involved challenge of adult flies with E. carotovora carotovora 15 (Ecc15), a gram-negative bacterium that produces non-lethal infection, to damage the midgut epithelium and induce repair pathways dependent upon ISC proliferation. Global knockout and cell type-specific knockdown of kmr was performed and effects on tissue pathophysiology were compared to those induced by knockdown of other established components of Wnt signaling pathways. As such, this study accomplished several goals. First, roles for kmr were established in controlling canonical Wnt signaling in Drosophila. Second, kmr was used as a tool to elucidate roles for canonical and non-canonical Wnt signaling within different cell types in the Drosophila midgut. These studies reveal not only that kmr-dependent canonical Wnt signaling controls ISC proliferation in the midgut but that such signaling occurs in several cell types, including Prospero-positive cells, suggesting that EEs, which these studies support derive from pre-EE progenitors, may play an unexpectedly important role in these processes (Sun, 2023).

Proliferation and differentiation of intestinal stem cells in the adult Drosophila midgut is essential to maintain the balance of tissue homeostasis and prevent excessive proliferation in this tissue. Wnt/β-catenin signaling plays central roles in tissue maintenance during development, including in the midgut. However, how canonical Wnt signaling is activated within intestinal stem cell progenitors and by fully differentiated progeny cells is still not well understood. This study discovered a new player, kramer (kmr), that regulates canonical Wnt signaling in the Drosophila midgut using a challenge with the non-lethal pathogen Erwinia carotovora carotovora (Ecc15) to induce massive ISC proliferation required to rebuild the damaged gut epithelium (Sun, 2023).

Using this system, kmr was established as a positive regulator of ISC proliferation and Wnt/β-catenin signaling. Inducible, cell type-specific kmr knockdown was used as a tool to interrogate the requirements for Wnt signaling within each cell type in the gut for controlling ISC proliferation. In addition to known effects of Wnt signaling within intestinal stem cells (ISCs), their immediate downstream progenitors termed enteroblasts (EBs), and fully differentiated enterocytes (ECs) in governing this process, this study unexpectedly points to roles for Wnt signaling within enteroendocrine cells (EEs) as a mechanism controlling stem cell proliferation in the Drosophila midgut (Sun, 2023).

It was first shown that interruption of kmr function decreases the expression of a canonical Wnt signaling target gene, fz3, in the posterior midgut, consistent with studies demonstrating fz3RFP expression in both ISCs and ECs showing that knockout of Wnt signaling components led to loss of fz3 expression in midgut region R5. Though ECs are the primary cell type in which Wnt signaling is activated, studies using kmr knockdown in multiple cell types suggest that Wnt signaling is also active in ISCs/EBs and EEs in posterior end of the midgut. These studies also revealed that loss of kmr in multiple cell types results in fewer EE cells and decreased ISC proliferation in the posterior midgut, indicating a role for kmr in EE differentiation (Sun, 2023).

Further, kmr knockdown in all cell types caused a decrease in proliferating, phospho-H3 positive (pH3+) cells, though staining with Armadillo/Prospero antibodies, which enables identification of ISCs/EBs and EEs, suggests that kmr knockdown in EE cells specifically downregulates stem cell proliferation. This distinction is important because pH3+ cells include all dividing progenitor cells, including ISCs/EBs and pre-EEs, and the data showed that kmr knockdown in all cell types significantly decreased dividing stem cells. Notably, kmr knockdown in ISCs/EBs and in ECs did not result in a defect in number of Arm+/Pro- cells (i.e., ISCs/EBs); However, kmr knockdown in Pro+ cells led to a reduction in number of ISCs/EBs, suggesting that kmr expression in EE cells regulates the proliferation of neighboring ISCs in a non-autonomous manner. These data also provide support to a model wherein EEs derive not from EBs but instead from a distinct set of progenitors termed pre-EEs (Sun, 2023).

This study also sheds light on the mechanisms by which kmr expression in distinct midgut cell types might regulate the physiological response to Ecc15 infection. Prior work on the mammalian ortholog of kmr, PLEKHA4, revealed that it promotes Wnt signaling pathways via physical interaction with KLHL12, a CUL3 E3 ubiquitin ligase substrate-specific adaptor and negative regulator of DVL. By binding to KLHL12 and sequestering it in plasma membrane-associated clusters, PLEKHA4 prevents DVL polyubiquitination by CUL3-KLHL12, ultimately causing elevated DVL levels and enhanced Wnt signaling in Wnt-receiving cells (Sun, 2023).

Because of the pleiotropic roles for DVL in both canonical Wnt/β-catenin and non-canonical β-catenin-independent pathways, this study found that PLEKHA4 knockdown affected both of these pathways. Previous studies on kmr in Drosophila, focusing on hair patterning, identified defects in planar cell polarity (PCP), a Frizzled- and Dsh-dependent pathway in flies. However, the present study identifies for the first time a role for kmr in promoting canonical Wnt/β-catenin signaling in vivo. By analyzing knockdown of the two fly KLHL12 orthologs, kelch and diablo, this study found that kmr acts in opposition to kelch, much as PLEKHA4 does with KLHL12 in mammalian cells, supporting the established mechanism of action. Finally, Wnt/β-catenin and PCP signaling were independently blocked and ISC proliferation following Ecc15 challenge was assessed; only blockade of Wnt/β-catenin signaling was found to exactly phenocopy loss of kmr (Sun, 2023).

In conclusion, this study reveals that the Dsh regulator kmr is a new, important regulator of Wnt/β-catenin signaling and ISC proliferation in vivo. As well, kmr was harnessed as a tool to systematically investigate the role of Wnt signaling in each of the major cell types in the Drosophila midgut in controlling ISC proliferation and differentiation during epithelial repair following challenge with an enteric pathogen. These studies suggest that Wnt signaling within enteroendocrine cells can control this process, adding a new layer of regulation to this important physiological process. Future studies will be necessary to identify downstream mechanisms by which EEs may non-autonomously regulate ISC proliferation in the midgut, as well as the existence of similar pathways in mammalian systems (Sun, 2023).

Functions of Kramer orthologs in other species

PLEKHA4 Promotes Wnt/beta-Catenin Signaling-Mediated G(1)-S Transition and Proliferation in Melanoma

Despite recent promising advances in targeted therapies and immunotherapies, patients with melanoma incur substantial mortality. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. This study identified a regulator of Wnt/beta-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro decreased Dishevelled levels, attenuated Wnt/beta-catenin signaling, and blocked progression through the G(1)-S cell-cycle transition. In mouse xenograft and allograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and exhibited an additive effect with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid- and protein-binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. This work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/beta-catenin signaling in the control of melanoma proliferation. This study establishes that melanoma cell proliferation requires the protein PLEKHA4 to promote pathologic Wnt signaling for proliferation, highlighting PLEKHA4 inhibition as a new avenue for the development of targeted therapies (Shah, 2021).

Functions of Kramer orthologs in other species

Origin and Evolution of the Multifaceted Adherens Junction Component Plekha7

Plekha7 is a key adherens junction component involved in numerous functions in mammalian cells. Plekha7 is the most studied member of the PLEKHA protein family, which includes eight members with diverse functions. However, the evolutionary history of Plekha7 remains unexplored. This study outlines the phylogeny and identify the origins of this gene and its paralogs. Plekha7, together with Plekha4, Plekha5, and Plekha6, belong to a subfamily that we name PLEKHA4/5/6/7. This subfamily is distinct from the other Plekha proteins, which form two additional separate subfamilies, namely PLEKHA1/2 and PLEKHA3/8. Sequence, phylogenetic, exon-intron organization, and syntenic analyses reveal that the PLEKHA4/5/6/7 subfamily is represented by a single gene in invertebrates, which remained single in the last common ancestor of all chordates and underwent gene duplications distinctly in jawless and jawed vertebrates. In the latter species, a first round of gene duplications gave rise to the Plekha4/7 and Plekha5/6 pairs and a second round to the four extant members of the subfamily. These observations are consistent with the 1R/2R hypothesis of vertebrate genome evolution. Plekha7 and Plekha5 also exist in two copies in ray-finned fishes, due to the Teleostei-specific whole genome duplication. Similarities between the vertebrate Plekha4/5/6/7 members and non-chordate sequences are restricted to their N-terminal PH domains, whereas similarities across the remaining protein molecule are only sporadically found among few invertebrate species and are limited to the coiled-coil and extreme C-terminal ends. The vertebrate Plekha4/5/6/7 proteins contain extensive intrinsically disordered domains, which are topologically and structurally conserved in all chordates, but not in non-chordate invertebrates. In summary, this study sheds light on the origins and evolution of Plekha7 and the PLEKHA4/5/6/7 subfamily and unveils new critical information suitable for future functional studies of this still understudied group of proteins (Kourtidis, 2022).

Overexpression of Pleckstrin Homology Domain-Containing Family A Member 4 Is Correlated with Poor Prognostic Outcomes and Immune Infiltration in Lower-Grade Glioma

The global incidence of brain tumors, the most common of which is lower grade glioma (LGG), remains high. Pleckstrin homology domain-containing family A member 4 (PLEKHA4) has been reported to be related to tumor invasion and growth. However, its role and correlation with immunity in LGG remain elusive. This study evaluated the expression pattern, prognostic value, biological functions, and immune effects of PLEKHA4 in LGG. We also analyzed the association between PLEKHA4 levels in different tumors, patient prognosis, and its role in tumor immunity. Depending on the type of research data, statistical methods were used such as Student's t-tests, Mann-Whitney U tests, one-way ANOVA tests, Kruskal-Wallis tests, Pearson's or Spearman's correlation analysis, Chi-square and Fisher's exact tests. The results revealed that PLEKHA4 levels were markedly elevated in most tumors (such as LGG). High PLEKHA4 levels are associated with poor overall survival (OS), progression-free interval (PFI) rates, and disease-specific survival (DSS) in LGG patients. Cox regression analysis and nomograms showed that PLEKHA4 levels are independent prognostic factors for LGG patients. According to functional enrichment analysis, PLEKHA4 levels in LGG are associated with immune infiltration and immunotherapy. In conclusion, PLEKHA4 is a potential prognostic marker and immunotherapy target for LGG (Huang, 2022).


Search PubMed for articles about Drosophila Kramer

Huang, B., Pan, W., Wang, W., Wang, Y., Liu, P. and Geng, W. (2022). Overexpression of Pleckstrin Homology Domain-Containing Family A Member 4 Is Correlated with Poor Prognostic Outcomes and Immune Infiltration in Lower-Grade Glioma. Dis Markers 2022: 1292648. PubMed ID: 36408463

Kourtidis, A., Dighera, B., Risner, A., Hackemack, R. and Nikolaidis, N. (2022). Origin and Evolution of the Multifaceted Adherens Junction Component Plekha7. Front Cell Dev Biol 10: 856975. PubMed ID: 35399503

Shah, A. S., Cao, X., White, A. C. and Baskin, J. M. (2021). PLEKHA4 Promotes Wnt/beta-Catenin Signaling-Mediated G(1)-S Transition and Proliferation in Melanoma. Cancer Res 81(8): 2029-2043. PubMed ID: 33574086

Sun, H., Shah, A. S., Bonfini, A., Buchon, N. S. and Baskin, J. M. (2023). Wnt/β-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation. bioRxiv. PubMed ID: 36865263

Biological Overview

date revised: 12 August 2023

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