Cyclin-Dependent Kinase 1 And Aurora Kinase Choreograph Mitotic Storage And Redistribution Of A Growth Factor Receptor

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis

Mitotic rounding drives Ciona robusta pre-cardiac cell asymmetric division and cardiac cell fate specification through influencing polarized FGFR redistribution

Asymmetric division plays a key role in cell fate specification and tissue homeostasis. Recent studies have demonstrated that polarized trafficking of membrane proteins during cell division contributes to asymmetric division. Mitotic rounding is the biophysical process in which cells balloon up in mitotic entry to adopt a spherical shape. Mitotic rounding has been shown to influence cell division and development in a variety of ways, but rarely in the context of mitotic protein trafficking. During early Ciona robusta development, the pre-cardiac founder cells undergo asymmetric division to give rise to a ventral cardiac progenitor and a dorsal tail muscle progenitor. Previous work has shown that cardiac fate induction is a result of polarized redistribution of fibroblast growth factor receptors (FGFRs) to the ventral adherent membrane, and that FGFR trafficking occurs in a mitotic stage-dependent fashion. In our present work, we investigate the impact mitotic rounding has on FGFR redistribution. According to our model, mitotic rounding leads to shape changes that deadhere the pre-cardiac founder cells from the ventral epidermis. Localized focal adhesions polarizes FGFR redistribution to the ventral side, ultimately specifying cardiac cell fate in the ventral daughter post mitosis. To test this model, we blocked mitotic rounding using EIPA, an inhibitor of the Na+/H+ antiporter. This ion channel is responsible for generating the osmotic pressure that helps drive the ballooning of the cell. We found that EIPA treatment led to shorter, flatter cells during mitotic entry, showing that mitotic rounding was blocked. Through analyzing the distribution of FGFR, we found that loss of mitotic rounding led to the depolarization of FGFR in early mitosis. We also used induction assays to show that the loss of mitotic rounding resulted in a loss of asymmetric heart progenitor fate specification. Our results support a previously undocumented role for mitotic rounding in mitotic receptor trafficking and cell fate specification

Identification And Characterization Of A B-Raf Kinase α-Helix Critical For The Activity Of MEK Kinase In MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf–MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas

Identification And Characterization Of A B-Raf Kinase Alpha Helix Critical For The Activity Of MEK Kinase In MAPK Signaling

In the mitogen-activated protein kinase (MAPK) pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we used information from the available B-Raf-MEK crystal structure to generate an active form of B-RafV600E that can be expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We introduced mutations along this alpha helix to pinpoint regions that are important for the B-Raf-MEK interaction and tested their effects on binding and phosphorylation. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry. We also assessed phosphorylation levels of MEK, as well as its downstream target ERK, in vitro and in cells. These studies showed that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. This result suggests that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas. Our cell-based data with a point mutation in B-Raf further suggests that combination therapies with ATP-competitive inhibitors would be useful to further reduce B-Raf activity and prevent the development of resistanc