During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

• Klíčová slova
• C. elegans, DPY-23/AP2M1, SEL-5/AAK1, Wnt signalling, cell biology, developmental biology, excretory cell, retromer,
• MeSH
• Caenorhabditis elegans * genetika   metabolismus   MeSH
• frizzled receptory metabolismus   genetika   MeSH
• pohyb buněk * MeSH
• proteiny Caenorhabditis elegans * metabolismus   genetika   MeSH
• proteiny Wnt metabolismus   genetika   MeSH
• signální dráha Wnt * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CWN-1 protein, C elegans MeSH Prohlížeč
• CWN-2 protein, C elegans MeSH Prohlížeč
• Egl-20 protein, C elegans MeSH Prohlížeč
• frizzled receptory MeSH
• proteiny Caenorhabditis elegans * MeSH
• proteiny Wnt MeSH

Selection and internalization of cargo via clathrin-mediated endocytosis requires adaptor protein complexes. One complex, AP-2, acts during cargo selection at the plasma membrane. African trypanosomes lack all components of the AP-2 complex, except for a recently identified orthologue of the AP-2-associated protein kinase 1, AAK1. In characterized eukaryotes, AAK1 phosphorylates the μ2 subunit of the AP-2 complex to enhance cargo recognition and uptake into clathrin-coated vesicles. Here, we show that kinetoplastids encode not one, but two AAK1 orthologues: one (AAK1L2) is absent from salivarian trypanosomes, while the other (AAK1L1) lacks important kinase-specific residues in a range of trypanosomes. These AAK1L1 and AAK1L2 novelties reinforce suggestions of functional divergence in endocytic uptake within salivarian trypanosomes. Despite this, we show that AAK1L1 null mutant Trypanosoma brucei, while viable, display slowed proliferation, morphological abnormalities including swelling of the flagellar pocket, and altered cargo uptake. In summary, our data suggest an unconventional role for a putative pseudokinase during endocytosis and/or vesicular trafficking in T. brucei, independent of AP-2.

• Klíčová slova
• AAK1, AP-2 complex, African trypanosomes, endocytosis,
• MeSH
• buněčná membrána MeSH
• endocytóza fyziologie   MeSH
• klathrin metabolismus   MeSH
• paraziti * metabolismus   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• klathrin MeSH

In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL.

• Klíčová slova
• GH1 cells, TRH receptor, small GTPase-mediated signaling, taltirelin, thyrotropin-releasing hormone, β-arrestin2,
• MeSH
• beta arrestin 1 metabolismus   MeSH
• fosforylace MeSH
• hormon uvolňující thyreotropin * metabolismus   farmakologie   MeSH
• receptory thyroliberinu * metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta arrestin 1 MeSH
• hormon uvolňující thyreotropin * MeSH
• receptory thyroliberinu * MeSH

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

• Klíčová slova
• AAK1, AP2 endocytic adaptor, NECAP, NMR, Numb-associated kinases (NAK), SNX9, TIRF, clathrin-mediated endocytosis, crystallography, regulation by phosphorylation,
• MeSH
• adaptorový proteinový komplex - alfa-podjednotky genetika   MeSH
• adaptorový proteinový komplex 2 genetika   metabolismus   MeSH
• endocytóza genetika   MeSH
• fosforylace genetika   MeSH
• klathrin genetika   metabolismus   MeSH
• klathrinové vezikuly genetika   metabolismus   MeSH
• lidé MeSH
• potažené jamky v buněčné membráně genetika   metabolismus   MeSH
• třídící nexiny genetika   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorový proteinový komplex - alfa-podjednotky MeSH
• adaptorový proteinový komplex 2 MeSH
• klathrin MeSH
• NECAP1 protein, human MeSH Prohlížeč
• SNX9 protein, human MeSH Prohlížeč
• třídící nexiny MeSH