BRFAA - Biomedical Research Foundation Academy Of Athens
Biomedical Research Foundation Academy Of AthensAcademy Of Athens

Research

Protein-phosphorylation induced protein-protein interactions during the intracellular signalling of Growth Factor receptors (RTKs) and of G-protein-coupled receptors (GPCRs) are linear in early development, leading irreversibly to program induction for cell divisions. As development progresses multiple cross-talk loops occur, turning these pathways into machineries that sense the environment in order to elicit complex functions. 
The major hypothesis we follow is that such switches are served by protein populations and protein dosage changes, imposed by the introduction of developmentally-regulated proteins, which then lead to differential regulation of transcriptional/translational programs and post-translational targeting of proteins. 
Focusing on the major PKC/NF1/Ras/Raf/MEK/ERK pathway in neural cells, we have identified PKC-ε and the RasGAP neurofibromin, a protein kinase and its specific substrate (our previous work), as the dipole to study for such affecting switches on the biological outcomes of receptors.

Τhe PKC/Neurofibromin/Ras/Raf/MEK/ERK pathway in development and disease

Our extensive research on CB1 signalling in neurons has revealed novel insights for the CB1-proximal signaling events, as well as the importance of neurofibromin function as a RasGAP in this pathway.  More specifically, we have identified kinases PKCε and Src as physically associating, immediate effectors of CB1 for ERK activation, both of which generate, after intermolecular phosphorylations, a loop of signal amplification via transactivation of the Fibroblast Growth Factor receptor (FGFR).  In addition, we established that lipid rafts (LR) are the membrane platform for CB1 signaling to ERK for an array of processes, from neuronal differentiation and microglia inactivation to complex behaviors such as memory formation and appetite. Lipid rafts are regulatory for intracellular signalling, as most signalling proteins preferentially segregate at the lipid raft-cytosol interphase.

Using the CB1 agonism paradigm, we addressed the urgent question of how neurofibromin attains membrane association, in response to receptor-mediated incoming signals, in order to act as a RasGAP. We demonstrated that PKCε activation precedes Ras activation, while an activated PKCε, also instantly recruited to lipid rafts, leads to a transient and specific enrichment of SOS1 in the ordered fractions. At the same time frame, lateral flowing out from the lipid rafts of the activated H-Ras is readily seen in Western blots of the fractions, yet soon H-Ras reappears in the ordered fractions, due to the action of the RasGAP neurofibromin that also acutely associates with lipid rafts in a PKCε-regulated manner. This “treadmilling” mobility documents that neurofibromin filters out the plethora of SOS1-activated H-Ras molecules, thus controlling downstream signalling output in cells.

Most importantly, our efforts on CB1 signalling have helped establish the significance of biomolecular condensates, that is concentrations of macromolecules not surrounded by a membrane, in the biological outcomes of membrane receptor signalling.


Neurofibromin is the product of the tumor suppressor gene NF1 that causes the disease Neurofibromatosis (NF-1) when mutated. NF-1 presents with variability, ranging from autism to cancer predisposition, especially for glioblastoma. Despite the > 3000 identified mutations, genotype-phenotype associations remain elusive. This quest is complicated by development- and cell type-dependent alternative splicings of the NF1, which produce different neurofibromin isoforms.

Towards this, we have followed the strategy to systemically unravel the functions of all neurifibromin domains in the context of each possible isoform.  For example we have discovered that specific phosphorylations on the N-terminus (CSR Domain) confer as high an allosteric regulation on the adjacent RasGAP domain (GRD), as to switch the EGF outcome from differentiation to proliferation; this fact has been recently recognized by geneticists as the sole explanation for a major genotype-phenotype correlation in NF-1 of single amino acid mutations in the CSRD and high possibility for malignacy.

Our most important recent finding concerns the function of neurofibromin as a microtubule associated protein (MAP).  Having shown that a PKC-ε-phosphorylation of Ser2808, adjacent to a nuclear localization signal (NLS) in the C-terminus domain (CTD), drives in a Ran-dependent manner neurofibromin into the nucleus and onto the mitotic spindle microtubules (MTs), we found that siRNA-mediated depletion of all transcripts and isoforms of this tumor suppressor protein leads to aberrant chromosome congression, revealing the importance of neurofibromin as a mitotic MAP.  Next, in studying the individual properties of neurofibromin isoforms that differ in the sequence of the 41 amino acids encoded by exon 51 that bears the NLS, namely of ΔNLS- and NLS-neurofibromins, we established that they differentially  regulate a.cytosolic MT intersection occurrence, cytosolic or spindle β-tubulin polymers, and cell migration patterns at interphase,  and b. actively participate, possibly by exerting opposing effects, in the formation of mitotic asters and spindles, and efficient, error-free chromosome congression and faithful genome transmission.