p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Tempes, A.; Brzozowska, A.; Wegierski, T.; Olek, K.; Jastrzebski, K.; Liszewska, E.; Misztal, K.; Machnicka, K.; Macias, M.; Szybinska, A.; Sitkiewicz, E.; Gozdz, A.; Wrobel, A.; Miaczynska, M.; Pokrzywa, W.; Jaworski, J.; Malik, A.

doi:10.1101/2025.02.13.638021

bioRxiv · 10.1101/2025.02.13.638021

p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Tempes, A.·Brzozowska, A.·Wegierski, T.·Olek, K.·Jastrzebski, K.·Liszewska, E.·Misztal, K.·Machnicka, K.·Macias, M.·Szybinska, A.·Sitkiewicz, E.·Gozdz, A.·Wrobel, A.·Miaczynska, M.·Pokrzywa, W.·Jaworski, J.·Malik, A.

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Abstract

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME. Summary statementClathrin-mediated endocytosis is essential for receptor internalization and cell signaling. This study identifies a novel regulatory mechanism in which p70S6K-mediated phosphorylation of the AP2 subunit 2 at Ser45 modulates AP2 conformational dynamics and facilitates receptor endocytosis.

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Tempes, A., Brzozowska, A., Wegierski, T., Olek, K., Jastrzebski, K., Liszewska, E., Misztal, K., Machnicka, K., Macias, M., Szybinska, A., Sitkiewicz, E., Gozdz, A., Wrobel, A., Miaczynska, M., Pokrzywa, W., Jaworski, J., Malik, A.. 2025-02-16. p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.. https://doi.org/10.1101/2025.02.13.638021

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