Study of the establishment of epithelial polarity: search for new proteins that interact with apkc

Póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.A key issue in developmental biology is the relationship between cell polarity and signal transduction pathways. Most eukaryotic cells are polarized with an asymmetric distribution of molecules and organelles resulting in different functional regions required for cell physiology. The control of this polarity in space and time is essential to coordinate changes in cell morphology with proliferation and morphogenetic movements required for the development of the organism. This control is carried out by signalling pathways, which in many cases are regulated by the subcellular localization of their components. In fact, there is a close relationship between polarity and the control of cell proliferation, since many receptors of intercellular communication pathways that regulate proliferation are located and activated in specific domains of the plasma membrane. Therefore, the understanding of the signalling pathways‐cell polarity relationship is crucial for the knowledge of how signals are integrated to induce morphogenesis but also how are modified in aberrant processes as those occurring in cancer. The atypical protein kinase C (aPKC) is a crucial protein in the cell polarity establishment or maintenance and also can participate in many other processes in the cell. aPKC has an enzymatic activity and can regulate different signaling pathways in the cell. In all these processes aPKC interact, depending on the process, with different regulators and modifies different substrates. In addition, aPKC is an oncogene. To understand how cell polarity is established, maintained and modified and also how this polarity can regulate signalling processes we have focused on to find out new proteins that interact with aPKC.Peer Reviewe

Estudio del establecimiento de la polaridad epitelial : búsqueda de nuevas proteínas que interaccionan con aPKC

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología del DesarrolloClave Programa: DBICódigo Línea: 9Las células eucarióticas presentan una distribución asimétrica de moléculas y orgánulos que genera diferentes regiones funcionales necesarias para la fisiología celular. La maquinaría de tráfico vesicular tiene un papel esencial en la formación de esta polaridad celular, estando ambos procesos regulados mutuamente para llevar a cabo los cambios en la morfología celular necesarios durante el desarrollo de un organismo. Dentro de la maquinaría de la polaridad celular, destaca la Proteína Quinasa C atípica (aPKC), una de las pocas quinasas implicada en el establecimiento y mantenimiento de la polaridad celular. aPKC participa en procesos como migración, división celular, supervivencia o proliferación; estando implicada además en diversos tipos de cáncer. La actividad de aPKC está regulada por interacción con otras proteínas que modifican su localización y/o regulan su actividad quinasa. Además, aPKC fosforila a multitud de substratos modificando su compartimiento. Estos interactores de aPKC son diferentes dependiendo del proceso celular, de modo que encontrar nuevas proteínas que interaccionen con aPKC resulta fundamental para entender mejor cómo funciona esta quinasa. En este trabajo he realizado una búsqueda bioquímica de nuevas proteínas que interaccionen con aPKC. Para ello he sobreexpresado en embriones de Drosophila la proteína aPKC fusionada a una serie de epítopos que me han permitido purificar complejos proteicos en condiciones nativas en los que aPKC participa. Usando esta metodología he aislado la proteína Nuclear fallout (Nuf) como un nuevo interactor de aPKC. Nuf es homólogo a Rab11-FIP3 de vertebrados y participa en el transporte de los endosomas de reciclaje (RE) a través de su interacción con Rab11 y las proteínas motoras Dineína y Kinesina. En esta tesis he demostrado que aPKC se une a Nuf directamente. Esta unión se produce entre la mitad N-terminal de Nuf y el dominio quinasa de aPKC, solo cuando aPKC está activa (autofosforilada). He identificado a Nuf como un nuevo substrato de aPKC. Dicha fosforilación se produce en la serina 155 y elimina la unión aPKC-Nuf, ya que la versión fosfomimética de Nuf (Nuf S155D) es incapaz de unirse a aPKC. En el disco imaginal de ala, cuyas células están altamente polarizadas en un eje apico-basal, he descubierto que la falta de función de aPKC produce una acumulación de Nuf y Rab11 en el córtex subapical, sugiriendo que aPKC afecta el tráfico de RE. Esto se debe al efecto de la fosforilación de aPKC en la localización subcelular de Nuf. La versión no fosforilable de Nuf (Nuf S155A) se acumula en la región subapical co-localizando con aPKC en la membrana plasmática, mientras que la versión fosfomimética Nuf S155D aparece en la región subapical pero evita el contacto con la membrana. También he observado que la sobreexpresión de Nuf S155A aumenta los niveles de aPKC en la membrana subapical, mientras que la falta de función de nuf produce una disminución aPKC en la membrana, lo que sugiere un transporte de aPKC vía Nuf. Esto lo he corroborado al eliminar la función de Sec5, Sec6 o Rab11, componentes de la ruta de reciclaje. En estas condiciones, aPKC se acumula intracelularmente y desaparece de las uniones celulares. Los resultados de este trabajo muestran la existencia de un reciclaje de aPKC en un tejido maduro, en el que aPKC es transportada hacia la membrana subapical vía Rab11-Nuf. Este reciclaje estaría regulado en una retroalimentación negativa por la propia aPKC, que fosforila a Nuf evitando su unión a la membrana.Universidad Pablo de Olavide. Escuela de Doctorad

Study ot the establishment of epithelial polarity: search for new proteins that interact with aPKC

Resumen del póster presentado a la 1ª Jornada de los másteres de biotecnología de la UPO, celebrada en Sevilla el 3 de mayo de 2012.Most eukaryotic cells are polarized with an asymmetric distribution of molecules and organelles resulting in different functional regions required for cell physiology. It is essential the regulation of this polarity in space and time to coordinate changes in cell morphology with proliferation and morphogenetic movements required for the organism development1. This regulation is accomplished by signalling pathways, which in many cases are regulated by the subcellular localization of their components. Consequently, the understanding of the relation between signalling pathways and cell polarity is crucial for the knowledge of how signals are integrated to induce morphogenesis but also how are modified in aberrant processes as those occurring in cancer. The atypical protein kinase C (aPKC) is a critical protein in cell polarity establishment and maintenance, and also participate in many other processes like migration o asymmetric cell division. aPKC has an enzymatic activity and can regulate different signaling pathways in the cell. In all these processes aPKC interacts with different regulators and modifies different substrates. In addition, aPKC is an oncogene. To understand how cell polarity is established, maintained and modified and also how this polarity can regulate signalling processes we have focused on to find out new proteins that interact with aPKC.Peer reviewe

Study ot the establishment of epithelial polarity: search for new proteins that interact with aPKC

Most eukaryotic cells are polarized with an asymmetric distribution of molecules and organelles resulting in different functional regions required for cell physiology. It is essential the regulation of this polarity in space and time to coordinate changes in cell morphology with proliferation and morphogenetic movements required for the organism development1. This regulation is accomplished by signalling pathways, which in many cases are regulated by the subcellular localization of their components. Consequently, the understanding of the relation between signalling pathways and cell polarity is crucial for the knowledge of how signals are integrated to induce morphogenesis but also how are modified in aberrant processes as those occurring in cancer. The atypical protein kinase C (aPKC) is a critical protein in cell polarity establishment and maintenance, and also participate in many other processes like migration o asymmetric cell division. aPKC has an enzymatic activity and can regulate different signaling pathways in the cell. In all these processes aPKC interacts with different regulators and modifies different substrates. In addition, aPKC is an oncogene. To understand how cell polarity is established, maintained and modified and also how this polarity can regulate signalling processes we have focused on to find out new proteins that interact with aPKC.D. St Johnston and B. Sanson, Current opinion in cell biology 23 (5), 540 (2011).540 (2011). T. Vaccari and D. Bilder, Molecular oncology 3 (4), 354 (2009)

Nuclear fallout provides a new link between aPKC and polarized cell trafficking

[Background]: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood. [Results]: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling. [Conclusions]: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.S.S. and, JM. E-V. are supported through grants of the Ministerio de Educación y Ciencia to J. C-G Hombría. This work was supported by grants of the MICINN/FEDER to J. C-G Hombría and S.S (BFU2010-15020, BFU2010-15851 and BFU2013-45866), grant of the Junta de Andalucía (P11-CVI7256) and by NIH Grants R01CA172025 to J.M., and R01CA192642 to M.T.D.-M. and 5P30CA030199 to M.T.D-M. and J.M.Peer Reviewe