Roles of Protein Kinase D2 during actin-driven vesicle scission at the Trans-Golgi-network (TGN).
Final Report Abstract
Cellular secretion is vital for tissue homeostasis and intercellular communication. Dysregulation of cargo trafficking can contribute to different diseases, such as cancer or diabetes. The Protein kinase D enzymes (PKDs) were first described as vital mediators of vesicle fission at the Trans-Golginetwork (TGN) by modulating the lipid environment to a fission competent state. Major additional functions also include control of several actin regulatory processes in the cell periphery via phosphorylation of actin-regulatory proteins. Interestingly, the actin cytoskeleton was also suggested to facilitate different steps during vesicle biogenesis and fission from donor membranes, e.g., during clathrin-mediated endocytosis, where branching actin polymerization by the Actinrelated protein 2/3 complex (ARP2/3 complex) supports vesicle scission. Similar mechanisms are proposed to be in place at the TGN, yet the composition and regulatory interactions for the respective complexes still need to be elucidated in detail. To initiate actin nucleation the Arp2/3 complex is activated by Wiskott-Aldrich Syndrome proteins, such as the nucleation promoting factors (NPF) N-WASP. Another actin-regulatory protein, Cortactin has been also implicated in the regulation of Arp2/3 complex activity as well as in the stabilization of F-actin branch points. Interestingly Cortactin is a PKD kinase substrate, and this phosphorylation was shown to modulate synergistic actin polymerization in conjunction with WASP family NPFs in vitro. Since our previous studies have demonstrated vital functions of the PKD2 isoform in regulating constitutive secretion and vesicle biogenesis at the TGN, we were prompted to elucidate in this grant proposal, whether PKD2 an Cortactin would coordinate and time actin polymerization as well as vesicle pinching by the GTPase Dynamin2 during constitutive secretion at TGN. To this end, we have demonstrated that all the above-described actin regulatory factors reside at the TGN. Once activated, PKD2 acts as a negative regulator of actin polymerization at the TGN, until vesicles are ready for scission. In this state, we determined that phosphorylation of Cortactin by PKD2 at S298 impairs the interaction between the inhibitory WASP-interacting protein (WIP) and its binding partner Cortactin, thus enabling WIP to sequester N-WASP in an autoinhibited conformation. Consequently, this resulted in an inhibition of synergistic branching actin polymerization, as N-WASP was unable to initiate activation of the Arp2/3 complex. When vesicles are ready to initiate filial fission steps, CDC42 is activated to facilitate N-WASP activation by direct binding. We also show that CDC42 acts upstream of PKD2 and causes inactivation of the kinase, thereby shifting the balance to non-S298-phosphorylated-Cortactin, which now preferentially binds WIP and relieves N-WASP auto-inhibition. In conjunction with activated N-WASP, Cortactin then initiates synergistic actin nucleation by the Arp2/3 complex, which is thought to generate forces that drives vesicles away from donor membranes. Consequently, we were able to show that Cortactin, when its not phosphorylated by PKD, facilitates an increase in constitutive secretion from the TGN that is dependent on N-WASP. Thus, with this grant proposal, we have unraveled the molecular regulation of branching actin polymerization at the TGN during constitutive secretion and further describe similar processes, which involve branching actin polymerization at the plasma membrane downstream of the PKD1 isoform and Cortactin that mediate the effective secretion of small extracellular vesicles (sEVs, exosomes).
Publications
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(2019) Concerted regulation of actin polymerization during constitutive secretion by cortactin and PKD2. J Cell Sci 132
Weeber, F., Becher, A., Seibold, T., Seufferlein, T., and Eiseler, T.
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(2020) Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice. Gastroenterology 159, 1019-1035.e1022
Armacki, M., Polaschek, S., Waldenmaier, M., Morawe, M., Ruhland, C., Schmid, R., Lechel, A., Tharehalli, U., Steup, C., Bektas, Y., Li, H., Kraus, J. M., Kestler, H. A., Kruger, S., Ormanns, S., Walther, P., Eiseler, T., and Seufferlein, T
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(2021) Pancreatic Cancer Small Extracellular Vesicles (Exosomes): A Tale of Short- and Long-Distance Communication. Cancers (Basel) 13
Waldenmaier, M., Seibold, T., Seufferlein, T., and Eiseler, T.
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(2021) SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas. Nat Metab 3, 149-165
Müller, J. A., Groß, R., Conzelmann, C., Krüger, J., Merle, U., Steinhart, J., Weil, T., Koepke, L., Bozzo, C. P., Read, C., Fois, G., Eiseler, T., Gehrmann, J., van Vuuren, J., Wessbecher, I. M., Frick, M., Costa, I. G., Breunig, M., Grüner, B., Peters, L., Schuster, M., Liebau, S., Seufferlein, T., Stenger, S., Stenzinger, A., MacDonald, P. E., Kirchhoff, F., Sparrer, K. M. J., Walther, P., Lickert, H., Barth, T. F. E., Wagner, M., Münch, J., Heller, S., and Kleger, A.
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(2021) Small Extracellular Vesicles and Metastasis—Blame the Messenger. Cancers 13, 4380
Seibold, T., Waldenmaier, M., Seufferlein, T., and Eiseler, T.
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(2021) Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma. Adv Sci (Weinh), e2102381
Seibold, T., Schonfelder, J., Weeber, F., Lechel, A., Armacki, M., Waldenmaier, M., Wille, C., Palmer, A., Halbgebauer, R., Karasu, E., Huber-Lang, M., Kalbitz, M., Radermacher, P., Paschke, S., Seufferlein, T., and Eiseler, T.
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(2022) Comparative Panel Sequencing of DNA Variants in cf-, ev- and tumorDNA for Pancreatic Ductal Adenocarcinoma Patients. Cancers (Basel) 14
Waldenmaier, M., Schulte, L., Schonfelder, J., Furstberger, A., Kraus, J. M., Daiss, N., Seibold, T., Morawe, M., Ettrich, T. J., Kestler, H. A., Kahlert, C., Seufferlein, T., and Eiseler, T.
