Biology subjects

Veraksa, A.

Publications and source records attributed to Veraksa, A..

5 recordsLinked to original sources

bioRxiv2024

ERK inhibits Cic repressor function via multisite phosphorylation

The receptor tyrosine kinase (RTK)/Extracellular Signal-Regulated Kinase (ERK) signaling pathway controls cell proliferation, differentiation, and survival. How ERK activation is relayed to its phosphorylation targets is not well understood. The transcriptional repressor Capicua (Cic) has emerged as a key target for ERK-mediated downregulation in Drosophila and mammals, and mutations in human CIC result in cancer and neurological diseases. Phosphorylation by ERK is critical for Cic downregulation, but the identities of phosphosites in Drosophila Cic are unknown. Here, we identify sites of phosphorylation in Cic that are directly targeted by ERK and validate their developmental functions in vivo using mutant Cic variants. Cic phosphosites are distributed throughout the length of the protein, and a group of centrally located sites appears to have a primary role in Cic downregulation. Cic mutated in 20 high-confidence sites behaves as a "super-repressor" in vivo that is largely insensitive to ERK-mediated downregulation, despite fully retaining the ability to bind to ERK. No single site is sufficient to turn off Cic activity; instead, we find that ERK must phosphorylate multiple sites in Cic simultaneously to achieve full downregulation. This multisite phosphorylation likely targets phosphodegrons that are recognized by ubiquitin ligases such as Ago/FBXW7 and contributes to Cic degradation. This study advances our understanding of the molecular mechanisms of signal interpretation downstream of the RTK/ERK signaling network.

developmental biology↗

bioRxiv2024

Regulation of brain development by the Minibrain/Rala signaling network

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized early role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in brain development. Significance statementThe kinase Minibrain(Mnb)/DYRK1A regulates the development of the brain and other tissues across many organisms. Here we show the critical importance of Mnb within the developing neuroepithelium. Advancing our understanding of Mnb function, we identified novel protein interactors of Mnb, Reps and Rlip, which function together with Mnb to regulate growth in Drosophila melanogaster. We also identify and characterize a role for the small GTPase Rala in Mnb-regulated growth and nervous system development. This work reveals an early role of Mnb in brain development and identifies a new Mnb/Reps/Rlip/Rala signaling axis.

developmental biology↗

bioRxiv2023

The Drosophila Tumour Suppressor Lgl and Vap33 activate the Hippo pathway by a dual mechanism, involving RtGEF/Git/Arf79F and inhibition of the V-ATPase.

The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly, Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using an in vivo affinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

cell biology↗

bioRxiv2022

Molecular basis of DNA recognition by the HMG-box-C1 module of Capicua

The HMG-box protein Capicua (CIC) is an evolutionarily conserved transcriptional repressor with key functions in development and disease-associated processes. CIC binds DNA using an exclusive mechanism that requires both its HMG-box and a separate domain called C1, but how these domains cooperate to recognize specific DNA sequences is not known. Here we report the crystal structure of the human CIC HMG-box and C1 domains in complex with an 18-base-pair DNA oligomer containing a consensus octameric CIC binding site. We find that both protein domains adopt independent tri-helical structures that pack against opposite sides of the DNA helix. The C1 domain in particular folds into a helix-turn-helix (HTH) structure that resembles the FF phosphoprotein binding domain. It inserts into the major groove of the DNA and plays a direct role in enhancing both the affinity and sequence specificity of CIC DNA binding. Our results reveal a unique bipartite protein module, ensuring highly specific DNA recognition by CIC, and show how this mechanism is disrupted by cancer mutations affecting either the HMG-box or C1 domains.

biochemistry↗

bioRxiv2021

Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye

The canonical function of the Hippo signaling pathway is regulation of organ growth. How this pathway controls cell fate determination is less well understood. Here, we uncover a function of the Hippo pathway in developmental cell fate decisions in the Drosophila eye-antennal disc exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian Transcriptional Intermediary Factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell fate decisions by recruiting transcriptional and post-transcriptional co-regulators, and by activating epidermal differentiation genes and repressing Notch target genes. Our work expands the range of functions and regulatory mechanisms under Hippo pathway control.

developmental biology↗