The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch.

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

Identification of urinary protein pattern in type 1 diabetic adolescents with early diabetic nephropathy by a novel combined proteome analysis.

Diabetic nephropathy is the main cause of morbidity and mortality in patients with Type 1 diabetes mellitus. Microalbuminuria has been established as a risk factor for the development and the progression of diabetic renal disease. A strong demand exists for better technologies to provide accurate diabetic nephropathy risk estimates before renal functional or structural disturbances already become established. Here, we present the application of a novel proteomics technology identifying urinary polypeptides and proteins. In this pilot study, we investigated 44 Type 1 diabetic patients with more than 5 years of diabetes duration compared with an age-matched control group. Random spot urine samples were examined utilizing high-resolution capillary electrophoresis (CE), coupled to mass spectrometry (MS). More than 1000 different polypeptides, characterized by their separation time and mass, were found between 800 Da and 66.5 kDa. Mathematical analysis revealed specific clusters of 54 polypeptides only found in Type 1 diabetic patients and an additional 88 polypeptides present or absent in patients with beginning nephropathy defined by the albumin-to-creatinine ratio (ACR; >35 mg/mmol). We observed polypeptide patterns characteristic for healthy controls and diabetic patients and subdivision of patients according to the excretion of polypeptides typical for diabetic nephropathy. Our study revealed that the urinary proteome contains a much greater variety of polypeptides than previously recognized and demonstrated the successful application of a novel high-throughput technology towards the human urinary proteome. Future prospective studies with the application of this technique may enable the earlier and more accurate detection of individuals at high risk to develop diabetic nephropathy.

Impact of diabetic nephropathy and angiotensin II receptor blockade on urinary polypeptide patterns.

BACKGROUND: New insights into the pathogenesis and treatment of diabetic renal disease may emerge from recent advances in proteomics using high-throughput mass spectrometry (MS) of urine. METHODS: Using a combination of online capillary electrophoresis (CE) and MS we evaluated urinary polypeptide patterns in four groups of type 2 diabetic patients matched for age, gender, and diabetes duration, including 20 normoalbuminuric patients with and 20 without diabetic retinopathy, 20 microalbuminuric patients with diabetic retinopathy, and 18 macroalbuminuric patients with diabetic retinopathy. Furthermore, changes in urinary polypeptide patterns during treatment with the angiotensin II receptor blocker (ARB) candesartan were evaluated in the macroalbuminuric patients in a randomized double-blinded, cross-over trial where each patient received treatment with placebo, candesartan 8, 16, and 32 mg daily each for 2 months. RESULTS: Overall, 4551 different polypeptides were found in the samples. Urinary polypeptide patterns were comparable in normo- (with and without diabetic retinopathy) and microalbuminuric patients, whereas distinct differences were found in macroalbuminuric patients. Differences in urinary polypeptide patterns between normo- and macroalbuminuric patients permitted the establishment of a "diabetic renal damage" pattern consisting of 113 polypeptides. Eleven of these polypeptides had been sequenced and identified. Candesartan treatment in macroalbuminuric patients significantly changed 15 of the 113 polypeptides in the diabetic renal damage pattern toward levels in normoalbuminuric patients. Change in the diabetic renal damage pattern was not candesartan dose-dependent but individual changes correlated with changes in urinary albumin excretion at each dose level. CONCLUSION: CE-MS serves as a fast and sensitive tool for identification of biomarkers and urinary polypeptide patterns specific for macroalbuminuric type 2 diabetic patients and may be used to explore and monitor renoprotective effects of ARB.

Proteomic analysis for the assessment of diabetic renal damage in humans.

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Hence early identification of patients prone to develop this complication is important. Diabetic renal damage should be reflected by a change in urinary polypeptide excretion at a very early stage. To analyse these changes, we used an online combination of CE/MS (capillary electrophoresis coupled with MS), allowing fast and accurate evaluation of up to 2000 polypeptides in urine. Employing this technology, we have examined urine samples from 39 healthy individuals and from 112 patients with Type II diabetes mellitus and different degrees of albumin excretion rate. We established a 'normal' polypeptide pattern in the urine of healthy subjects. In patients with Type II diabetes and normal albumin excretion rate, the polypeptide pattern in urine differed significantly from normal, indicating a specific 'diabetic' pattern of polypeptide excretion. In patients with higher grade albuminuria, we were able to detect a polypeptide pattern indicative of 'diabetic renal damage'. We also found this pattern in 35% of those patients who had low-grade albuminuria and in 4% of patients with normal albumin excretion. Moreover, we could identify several of the indicative polypeptides using MS/MS sequencing. We conclude that proteomic analysis with CE/MS permits fast and accurate identification and differentiation of polypeptide patterns in urine. Longitudinal studies should explore the potential of this powerful diagnostic tool for early detection of diabetic renal damage.

Capillary electrophoresis coupled to mass spectrometry to establish polypeptide patterns in dialysis fluids.

Combination of capillary electrophoresis with mass spectrometry (CE-MS) allows generation of polypeptide patterns of body fluids. In a single CE-MS (45 min) run more than 600 polypeptides were analyzed in hemodialysis fluids obtained with different membranes (high-flux/low-flux). Larger polypeptides (M(r) > 10 000) were almost exclusively present in high-flux dialysates only, while in low-flux dialysates additional small polypeptides were detected. Comparison to the normal urine pattern yielded a surprisingly low consensus: a number of polypeptides present in urine were missing. We established a fast and sensitive technique, easily applicable to the monitoring of different modalities of dialyzers.

Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers.

The on-line coupling of capillary electrophoresis (CE) with electrospray-time-of-flight mass spectrometry (MS) has been used to obtain patterns of peptides and proteins present in the urine of healthy human individuals. This led to the establishment of a "normal urine polypeptide pattern", consisting of 247 polypeptides, each of which was found in more than 50% of healthy individuals. Applying CE-MS to the analysis of urine of patients with kidney disease revealed differences in polypeptide pattern. Twenty-seven polypeptides were exclusively found in samples of patients. Another 13, present in controls, were missing. These data indicate that CE-MS can be applied as powerful tool in clinical diagnostics.

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway.

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.