Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling▿

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation

Effect of caspase cleavage-site phosphorylation on proteolysis.

Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1' positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation

KSR2 is a Calcineurin Substrate that Promotes ERK Cascade Activation in Response to Calcium Signals

Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling

Evidence supporting exercise interventions for persons in early-stage Alzheimer's disease

The purpose of this article is to grade research evidence supporting exercise-based interventions for persons with early-stage dementias and to report the recommendations of a consensus panel. The search produced 11 data based articles testing the effects of exercise interventions on a variety of outcomes. The body of evidence to support exercise interventions in the prevention and treatment of Alzheimer’s disease is growing and has potential as a treatment modality following translational studies in recreation therapy and other fields

Evidence supporting exercise interventions for persons in early-stage Alzheimer's disease

The purpose of this article is to grade research evidence supporting exercise-based interventions for persons with early-stage dementias and to report the recommendations of a consensus panel. The search produced 11 databased articles testing the effects of exercise interventions in a variety of outcomes. The body of evidence to support exercise interventions in the prevention and treatment of Alzheimer's disease is growing and has potential as a treatment modality following translational studies in recreation therapy and other fields

Sentinel lymph node biopsy at the time of mastectomy does not increase the risk of lymphedema: implications for prophylactic surgery

Women diagnosed with or at high risk for breast cancer increasingly choose prophylactic mastectomy. It is unknown if adding sentinel lymph node biopsy (SLNB) to prophylactic mastectomy increases the risk of lymphedema. We sought to determine the risk of lymphedema after mastectomy with and without nodal evaluation. 117 patients who underwent bilateral mastectomy were prospectively screened for lymphedema. Perometer arm measurements were used to calculate weight-adjusted arm volume change at each follow-up. Of 234 mastectomies performed, 15.8 % (37/234) had no axillary surgery, 63.7 % (149/234) had SLNB, and 20.5 % (48/234) had axillary lymph node dissection (ALND). 88.0 % (103/117) of patients completed the LEFT-BC questionnaire evaluating symptoms associated with lymphedema. Multivariate analysis was used to assess clinical characteristics associated with increased weight-adjusted arm volume and patient-reported lymphedema symptoms. SLNB at the time of mastectomy did not result in an increased mean weight-adjusted arm volume compared to mastectomy without axillary surgery (p = 0.76). Mastectomy with ALND was associated with a significantly greater mean weight-adjusted arm volume change compared to mastectomy with SLNB (p < 0.0001) and without axillary surgery (p = 0.0028). Patients who underwent mastectomy with ALND more commonly reported symptoms associated with lymphedema compared to those with SLNB or no axillary surgery (p < 0.0001). Patients who underwent mastectomy with SLNB or no axillary surgery reported similar lymphedema symptoms. Addition of SLNB to mastectomy is not associated with a significant increase in measured or self-reported lymphedema rates. Therefore, SLNB may be performed at the time of prophylactic mastectomy without an increased risk of lymphedema

Oxfendazole mediates macrofilaricidal efficacy against the filarial nematode Litomosoides sigmodontis in vivo and inhibits Onchocerca spec. motility in vitro.

A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing