SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics.

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

SHOC2-MRAS-PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis.

Dephosphorylation of the inhibitory "S259" site on RAF kinases (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. The MRAS GTPase, a close relative of RAS oncoproteins, interacts with SHOC2 and protein phosphatase 1 (PP1) to form a heterotrimeric holoenzyme that dephosphorylates this S259 RAF site. MRAS and SHOC2 function as PP1 regulatory subunits providing the complex with striking specificity against RAF. MRAS also functions as a targeting subunit as membrane localization is required for efficient RAF dephosphorylation and ERK pathway regulation in cells. SHOC2's predicted structure shows remarkable similarities to the A subunit of PP2A, suggesting a case of convergent structural evolution with the PP2A heterotrimer. We have identified multiple regions in SHOC2 involved in complex formation as well as residues in MRAS switch I and the interswitch region that help account for MRAS's unique effector specificity for SHOC2-PP1. MRAS, SHOC2, and PPP1CB are mutated in Noonan syndrome, and we show that syndromic mutations invariably promote complex formation with each other, but not necessarily with other interactors. Thus, Noonan syndrome in individuals with SHOC2, MRAS, or PPPC1B mutations is likely driven at the biochemical level by enhanced ternary complex formation and highlights the crucial role of this phosphatase holoenzyme in RAF S259 dephosphorylation, ERK pathway dynamics, and normal human development.

An investigation of the mutagenic activity of salamide - a major impurity of hydrochlorothiazide.

Hydrochlorothiazide is a widely used antihypertensive agent and one of its major impurities, salamide (4-amino-6-chlorobenzene-1,3-disulphonamide), has a chemical structure containing a primary amino group, a functional group that has previously been reported to be associated with carcinogenic activity. It is known that hydrochlorothiazide purity is a challenging problem for the pharmaceutical industry. As there were no prior mutagenicity data for the impurity salamide, the aim was to investigate its mutagenicity in this study. Salamide was tested for mutagenic potential in Salmonella typhimurium TA98, TA100, TA 1535, TA 1537, and E. coli WP2 uvrA + E. coli WP2 [pKM101] strains at six different concentrations, the highest concentration being the 5000 µg/plate. In both the presence and absence of the metabolic activation system, no mutagenic activity was observed. Results indicated that salamide should be classified as an ordinary impurity and controlled according to Q3A(R2) and Q3B(R2) guidelines.

Pulmonary hypertension in hemodialysis patients without arteriovenous fistula: the effect of dialyzer composition.

Pulmonary hypertension (PHT) increases mortality rate in hemodialysis (HD) patients. Numerous clinical, hemodynamic, and metabolic abnormalities have been suggested to be associated with the development of PHT in HD patients. We aimed to investigate the acute effects of two different dialyzer membranes on pulmonary arterial pressure (PAP) throughout a HD session in maintenance HD patients. Seventy-four HD patients dialyzed through permanent tunneled jugular central venous catheter were enrolled. A first-use cellulose acetate and high-flux polysulfone dialysis membrane were tested using a crossover design. For each membrane, pre- and post-dialysis pulmonary artery pressures were measured echocardiographically. Elevated pulmonary artery pressure was observed in 68.8% of patients (n = 51), whereas mild PHT was observed in 28.3% of patients (n = 21) and moderate PHT in 40.5% (n = 30). Decrease in pulmonary artery pressure following HD procedure performed using high-flux polysulfone membrane was significantly higher than the decrease observed following HD procedure performed using cellulose acetate membrane (p < 0.05). Significant decrease in pulmonary artery pressures was observed only after HD procedures performed using high-flux polysulfone membrane (p < 0.05). Ultrafiltered volume was only significantly correlated with the decrease in pulmonary artery pressure observed after HD procedure performed through high-flux polysulfone membrane (ß = 0.411, p < 0.05). PHT seems to be prevalent among HD patients even in the absence of AV fistula and abnormal cardiac functions. Membrane composition seems to be important, which may overwhelm the improving effects of ultrafiltration.

Nephrotic-range proteinuria with small glomerular fibrillary deposits in a patient on diphenylhydantoin treatment.

BACKGROUND: Although the exact role of the diphenylhydantoin treatment on the immune system is still unclear, prolonged administration of diphenylhydantoin has been suggested to be associated with immune-based disorders and syndromes. CASE REPORT: A case of fibrillary glomerulonephritis associated with DPH treatment is reported. This is the first reported case of fibrillary glomerulonephritis associated with this commonly used common anticonvulsive drug. CONCLUSIONS: A more routine surveillance for proteinuria in patients receiving DPH is recommended.

SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers.

Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.

Perioperative type I aortic dissection during conventional coronary artery bypass surgery: risk factors and management.

OBJECTIVES: Perioperative iatrogenic type I aortic dissection (PIAD) is a rare but potentially fatal complication of conventional coronary artery bypass surgery (CCABG). Prompt recognition and repair of PIAD may significantly improve outcomes. METHODS: We reviewed the hospital records of patients with PIAD occurring as a complication of CCABG at Siyami Ersek Thoracic and Cardiovascular Surgery Center from January 2001 through June 2007. During this period, 10,130 CCABG were performed and 21 patients (0.20%) with PIAD were identified. We compared variables for these 21 patients with 603 patients without PIAD (control group). RESULTS: PIAD occurred intraoperatively in 19 patients (90%) and during the early postoperative period (first 6 hours) in 2 patients (10%) who underwent CCABG. Dissections were noticed after removal of the aortic crossclamp in 11 patients, during aortic cannulation in 3 patients, and after removal of the partial-occlusion clamp in 5 patients. Patients with and without PIAD differed significantly in regard to sex (P = .05), history of hypertension (P = .001), and history of severe concomitant peripheral arterial disease (PAD) (P = .001). The diameter of the aorta was significantly wider in patients with PIAD. (3.83 +/- 0.9 vs 2.93 +/- 0.46 cm, P = .019). The occurrence of high cardiopulmonary bypass (CPB) pressure (>==120 mmHg) was significantly higher in the PIAD patients than the non-PIAD patients (28.6% vs 3.3%, P = .0001). Seven PIAD patients (33.3%) died preoperatively and 3 (14.2%) died postoperatively. CONCLUSION: PIAD is frequently fatal. Risk factors for PIAD during or after CCABG include female sex, history of PAD and hypertension, increased aortic diameter, and high CPB pressure.