PubMed · 2159170
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T Isobe, T Ichimura, T Okuyama. 1990. [Nervous system-specific proteins].. https://pubmed.ncbi.nlm.nih.gov/2159170/
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RAF inhibitor "paradoxical activation" (PA) is a phenomenon where RAF kinase inhibitors increase RAF kinase signaling. Through mathematical modeling and experimental data analysis, we recently demonstrated that the combination of conformational autoinhibition (CA) with the disruption of CA by RAF inhibitors plays an important role in PA. 14-3-3 proteins are known to modulate RAF CA and RAF dimerization. We here extend our mathematical model to include both roles of 14-3-3 proteins, and we derive rigorous analytical expressions of RAF signal regulation as modulated by 14-3-3 proteins. We then use the model to investigate how 14-3-3 proteins may modulate PA. We mathematically show 14-3-3 protein stabilization of the autoinhibited form of RAF should potentiate PA, while 14-3-3 protein stabilization of the active RAF dimer should reduce PA. Our analysis suggests that the net-effect will often be a potentiation of PA, and that 14-3-3 proteins may be capable of inducing PA for RAF inhibitors that normally show little to no PA. We test model-based insights experimentally with two different approaches: forced increases in 14-3-3 expression (which we find amplifies PA) and evolved resistance assays (which suggest increased 14-3-3 expression may contribute to resistance to RAF inhibitors). Overall, this work supports a role for 14-3-3 in modulating RAF-inhibitor mediated paradoxical activation.
The roles of protein kinase C (PKC) and Exo1 in exocytosis from digitonin-permeabilized adrenal chromaffin cells were explored by using exogenous purified proteins in a run-down/reconstitution system. The stimulatory action of Exo1 on exocytosis from run-down cells was found to be completely dependent on the continuous presence of exogenous MgATP, suggesting that it acts on the slow phase of exocytosis [Holz, Bittner, Peppers, Senter & Eberhard (1989), J. Biol. Chem. 264, 5412-5419]. Partially purified rat brain PKC was found to be able to stimulate Ca(2+)-dependent exocytosis from run-down cells in a dose-dependent manner. This effect was indeed due to PKC and not a contaminant in the PKC fraction, since the PKC activator phorbol 12-myristate 13-acetate (PMA), under conditions in which control secretion was not affected, potentiated the effect of the exogenous PKC in stimulating secretion. Furthermore, although either PKC or Exo1 alone could stimulate exocytosis from run-down cells, the effect of combining the fractions was synergistic, as had previously been observed using PMA treatment combined with Exo1 incubation [Morgan & Burgoyne (1992) Nature (London) 355, 833-836]. The observed synergy between PKC and Exo1 was not due to PKC-mediated phosphorylation of Exo1, and Exo1 was found not to affect PKC activity in enzyme assays. We conclude that PKC and Exo1 act synergistically in the slow phase of Ca(2+)-dependent exocytosis from adrenal chromaffin cells. Furthermore, PKC does not directly affect Exo1, but rather enhances the activity of Exo1 by a putative phosphorylation of another, unidentified, component of the exocytotic machinery which facilitates the action of Exo1 in exocytosis.
The 14-3-3 proteins are a family of acidic proteins found mainly in the brain and are suggested to have a role in monoamine synthesis based on their ability to activate tyrosine and tryptophan hydroxylases in the presence of type II Ca2+/calmodulin-dependent protein kinase. Recently, however, it has been demonstrated that a member of the 14-3-3 family, termed Exo1, stimulates Ca(2+)-dependent exocytosis in permeabilized adrenal chromaffin cells, suggesting that this protein family may influence the protein kinase C-mediated control of Ca(2+)-dependent exocytosis. Here we show that the 14-3-3 proteins activate protein kinase C at about 2-fold more than the known level of the activated protein kinase, i.e. the activity of protein kinase C in the presence of Ca2+ and phospholipids. This raises the possibility that the cellular activity of protein kinase C is regulated by diverse members of the 14-3-3 family and that the reported ability of Exo1 to reactivate Ca(2+)-dependent exocytosis is based on its stimulatory effect on protein kinase C activity. The 14-3-3 family, therefore, appears to be a multifunctional regulator of cell signalling processes mediated by two types of Ca(2+)-dependent protein kinase, protein kinase C and type II calmodulin-dependent protein kinase.