Cardiovascular risk and kidney function profiling using conventional and novel biomarkers in young adults: the African-PREDICT study.

BACKGROUND: Low- and middle-income countries experience an increasing burden of chronic kidney disease. Cardiovascular risk factors, including advancing age, may contribute to this phenomenon. We (i) profiled cardiovascular risk factors and different biomarkers of subclinical kidney function and (ii) investigated the relationship between these variables. METHODS: We cross-sectionally analysed 956 apparently healthy adults between 20 and 30 years of age. Cardiovascular risk factors such as high adiposity, blood pressure, glucose levels, adverse lipid profiles and lifestyle factors were measured. Various biomarkers were used to assess subclinical kidney function, including estimated glomerular filtration rate (eGFR), urinary albumin, uromodulin and the CKD273 urinary proteomics classifier. These biomarkers were used to divide the total population into quartiles to compare extremes (25th percentiles) on the normal kidney function continuum. The lower 25th percentiles of eGFR and uromodulin and the upper 25th percentiles of urinary albumin and the CKD273 classifier represented the more unfavourable kidney function groups. RESULTS: In the lower 25th percentiles of eGFR and uromodulin and the upper 25th percentile of the CKD273 classifier, more adverse cardiovascular profiles were observed. In multi-variable adjusted regression analyses performed in the total group, eGFR associated negatively with HDL-C (ß= -0.44; p < 0.001) and GGT (ß= -0.24; p < 0.001), while the CKD273 classifier associated positively with age and these same risk factors (age: ß = 0.10; p = 0.021, HDL-C: ß = 0.23; p < 0.001, GGT: ß = 0.14; p = 0.002). CONCLUSION: Age, lifestyle and health measures impact kidney health even in the third decade.

Understanding glomerular diseases through proteomics.

INTRODUCTION: Chronic kidney disease is avery common and complex chronic disease. Uncovering the pathological patterns of CKD on the molecular level of bio-fluids and tissue appears to be both vital and promising for a more favorable outcome. We reviewed recently discovered proteomics biomarkers for CKD to provide new insight into disease pathology. AREAS COVERED: We review the application of proteome analysis in the context of CKD with various etiologies within the last 5 years. Proteins and peptides associated with CKD as derived from multiple sources (urine, blood and tissue) are reported along with their various biological pathways. EXPERT OPINION: A systematic and theoretical comprehension of the CKD pathology is essential for its successful management. The underlying complexity of the disease further requires specific conditions for reliable and interpretable results. In this context, clinical proteomics has resulted in first encouraging findings in CKD. A more complete understanding of the biological pathways related to the disease, based on the scope of a holistic proteomic approach, could improve substantially the management of CKD, especially when in conjunction with the current trend of personalized medicine.

Characteristics of high- and low-risk individuals in the PRIORITY study: urinary proteomics and mineralocorticoid receptor antagonism for prevention of diabetic nephropathy in Type 2 diabetes.

AIM: To compare clinical baseline data in individuals with Type 2 diabetes and normoalbuminuria, who are at high or low risk of diabetic kidney disease based on the urinary proteomics classifier CKD273. METHODS: We conducted a prospective, randomized, double-blind, placebo-controlled international multicentre clinical trial and observational study in participants with Type 2 diabetes and normoalbuminuria, stratified into high- or low-risk groups based on CKD273 score. Clinical baseline data for the whole cohort and stratified by risk groups are reported. The associations between CKD273 and traditional risk factors for diabetic kidney disease were evaluated using univariate and logistic regression analysis. RESULTS: A total of 1777 participants from 15 centres were included, with 12.3% of these having a high-risk proteomic pattern. Participants in the high-risk group (n=218), were more likely to be men, were older, had longer diabetes duration, a lower estimated GFR and a higher urinary albumin:creatinine ratio than those in the low-risk group (n=1559, P<0.02). Numerical differences were small and univariate regression analyses showed weak associations (R2 < 0.04) of CKD273 with each baseline variable. In a logistic regression model including clinical variables known to be associated with diabetic kidney disease, estimated GFR, gender, log urinary albumin:creatinine ratio and use of renin-angiotensin system-blocking agents remained significant determinants of the CKD273 high-risk group: area under the curve 0.72 (95% CI 0.68-0.75; P<0.01). CONCLUSIONS: In this population of individuals with Type 2 diabetes and normoalbuminuria, traditional diabetic kidney disease risk factors differed slightly between participants at high risk and those at low risk of diabetic kidney disease, based on CKD273. These data suggest that CKD273 may provide additional prognostic information over and above the variables routinely available in the clinic. Testing the added value will be subject to our ongoing study. (European Union Clinical Trials Register: EudraCT 2012-000452-34 and Clinicaltrials.gov: NCT02040441).

Urine peptidomic biomarkers for diagnosis of patients with systematic lupus erythematosus.

Background Systematic lupus erythematosus (SLE) is characterized with various complications which can cause serious organ damage in the human body. Despite the significant improvements in disease management of SLE patients, the non-invasive diagnosis is entirely missing. In this study, we used urinary peptidomic biomarkers for early diagnosis of disease onset to improve patient risk stratification, vital for effective drug treatment. Methods Urine samples from patients with SLE, lupus nephritis (LN) and healthy controls (HCs) were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS) for state-of-the-art biomarker discovery. Results A biomarker panel made up of 65 urinary peptides was developed that accurately discriminated SLE without renal involvement from HC patients. The performance of the SLE-specific panel was validated in a multicentric independent cohort consisting of patients without SLE but with different renal disease and LN. This resulted in an area under the receiver operating characteristic (ROC) curve (AUC) of 0.80 ( p < 0.0001, 95% confidence interval (CI) 0.65-0.90) corresponding to a sensitivity and a specificity of 83% and 73%, respectively. Based on the end terminal amino acid sequences of the biomarker peptides, an in silico methodology was used to identify the proteases that were up or down-regulated. This identified matrix metalloproteinases (MMPs) as being mainly responsible for the peptides fragmentation. Conclusions A laboratory-based urine test was successfully established for early diagnosis of SLE patients. Our approach determined the activity of several proteases and provided novel molecular information that could potentially influence treatment efficacy.

[Proteome analysis of undiluted vitreous humor in patients with branch retinal vein occlusion]. / Proteomanalyse unverdünnter Glaskörperflüssigkeit bei Patienten mit einem Venenastverschluss.

BACKGROUND: The pathophysiological mechanisms of macular edema secondary to branch retinal vein occlusion (BRVO) remain unclear. OBJECTIVES: To analyze the protein profile of human vitreous of patients with BRVO and to identify specific dysregulated proteins. MATERIALS AND METHODS: Undiluted vitreous humor samples from patients with treatment naïve BRVO and 15 controls with idiopathic floaters were analyzed in this clinical-experimental study using capillary electrophoresis coupled to a mass spectrometer (CE-MS) and tandem mass spectrometry (MS/MS). Quantitative analysis of the dysregulated proteins was performed with enzyme-linked immunosorbent assay (ELISA). Protein-protein interactions were depicted with the STRING database. RESULTS: A total of 84 proteins were found in the human vitreous samples of 15 patients with BRVO and 15 controls. In all, 14 proteins were significant when comparing the signal intensities of BRVO and control samples. Six significant dysregulated proteins with p < 0.001 were further verified with ELISA. Clusterin, complement factor C3, prostaglandin-H2 D­isomerase and vitronectin were significantly upregulated in the BRVO group and opticin was downregulated. The protein interactions analysis showed associations with inflammatory cascades, matrix changes, mechanisms of cell survival und death. CONCLUSIONS: The results of the study reveal that the proteomic composition of vitreous humor differed significantly between the patients with BRVO and the controls. Whether the identified proteins may serve as potential biomarkers for pathophysiology, diagnostics or therapy should be examine in further studies.

Proteomic peptide profiling for preemptive diagnosis of acute graft-versus-host disease after allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation is one curative treatment for hematological malignancies, but is compromised by life-threatening complications, such as severe acute graft-versus-host disease (aGvHD). Prediction of severe aGvHD as early as possible is crucial to allow timely initiation of treatment. Here we report on a multicentre validation of an aGvHD-specific urinary proteomic classifier (aGvHD_MS17) in 423 patients. Samples (n=1106) were collected prospectively between day +7 and day +130 and analyzed using capillary electrophoresis coupled on-line to mass spectrometry. Integration of aGvHD_MS17 analysis with demographic and clinical variables using a logistic regression model led to correct classification of patients developing severe aGvHD 14 days before any clinical signs with 82.4% sensitivity and 77.3% specificity. Multivariate regression analysis showed that aGvHD_MS17 positivity was the only strong predictor for aGvHD grade III or IV (P<0.0001). The classifier consists of 17 peptides derived from albumin, ß2-microglobulin, CD99, fibronectin and various collagen α-chains, indicating inflammation, activation of T cells and changes in the extracellular matrix as early signs of GvHD-induced organ damage. This study is currently the largest demonstration of accurate and investigator-independent prediction of patients at risk for severe aGvHD, thus allowing preemptive therapy based on proteomic profiling.

A urinary peptide biomarker set predicts worsening of albuminuria in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Microalbuminuria is considered the first clinical sign of kidney dysfunction and is associated with a poor renal and cardiovascular prognosis in type 2 diabetes. Detection of patients who are prone to develop micro- or macroalbuminuria may represent an effective strategy to start or optimise therapeutic intervention. Here we assessed the value of a urinary proteomic-based risk score (classifier) in predicting the development and progression of microalbuminuria. METHODS: We conducted a prospective case-control study. Cases (n = 44) and controls (n = 44) were selected from the PREVEND (Prevention of Renal and Vascular End-stage Disease) study and from the Steno Diabetes Center (Gentofte, Denmark). Cases were defined by transition from normo- to microalbuminuria or from micro- to macroalbuminuria over a follow-up of 3 years. Controls with no transitions in albuminuria were pair-matched for age, sex and albuminuria status. A model for the progression of albuminuria was built using a proteomic classifier based on 273 urinary peptides. RESULTS: The proteomic classifier was independently associated with transition to micro- or macroalbuminuria (OR 1.35 [95% CI 1.02, 1.79], p = 0.035). The classifier predicted the development and progression of albuminuria on top of albuminuria and estimated GFR (eGFR, area under the receiver operating characteristic [ROC] curve increase of 0.03, p = 0.002; integrated discrimination index [IDI]: 0.105, p = 0.002). Fragments of collagen and α-2-HS-glycoprotein showed significantly different expression between cases and controls. CONCLUSIONS/INTERPRETATION: Although limited by the relatively small sample size, these results suggest that analysis of a urinary biomarker set enables early renal risk assessment in patients with diabetes. Further work is required to confirm the role of urinary proteomics in the prevention of renal failure in diabetes.

Proteomic biomarkers for the early detection of acute kidney injury.

Acute kidney injury (AKI) comprises several syndromes that are associated with a sudden decrease in renal function. AKI is a common condition especially among critically ill patients. It is typically multifactorial and of great prognostic significance. The incidence of AKI has increased while the associated mortality rate has remained unchanged over the last years. Recent definitions of AKI, namely the Risk, Injury, Failure, Loss of renal function and End-stage kidney disease (RIFLE) classifycation or the Acute Kidney Injury Network (AKIN) criteria, incorporate serum creatinine and urine output as the principal markers to define and detect AKI. However, elevated serum creatinine or oliguria were demonstrated to detect AKI at late stages of renal injury when preventive strategies may be less effective. Therefore, there has recently been a great scientific interest in obtainng valuable markers for early AKI detection. In the last 5 years numerous new markers such as neutrophil-gelatinase associated lipo-calin, interleukin-18, cystatin C and kidney injury molecule 1 in the urine and/or serum have been studied and proposed as early detection markers of AKI. Persistently, these markers performed well in initial pilot trials. However, these promising results could often not be confirmed in later, larger multicentre trials and limitation of these biomarkers in the early diagnosis of renal injury were discovered. Furthermore, as AKI is multi-factorial and heterogeneous in origin, it seems likely that not one single marker but a panel of biomarkers will be required to detect all subtypes of AKI early during their evolution. This has initiated proteomic studies to develop panels of biomarkers which may facilitate early detection of AKI. The present review will focus on the most important clinical studies evaluating the ability of single AKI biomarkers and on those in clinical proteomics that attempted to establish panels of biomarkers in urine for early and accurate AKI diagnosis and prognosis.

A comparison between MALDI-MS and CE-MS data for biomarker assessment in chronic kidney diseases.

Non-invasive detection of diseases, based on urinary proteomics, is becoming an increasingly important area of research, especially in the area of chronic kidney disease (CKD). Different platforms have been used in independent studies, mostly capillary-electrophoresis coupled ESI-MS (CE-MS), liquid chromatography coupled mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have compared the performance of CE-MS with MALDI-MS in detecting CKD, based on a cohort of 137 urine samples (62 cases and 75 controls). Data cross-talk between the two platforms was established for the comparison of detected biomarkers. The results demonstrate superior performance of the CE-MS approach in terms of peptide resolution and obtained disease prediction accuracy rates. However, the data also demonstrate the ability of the MALDI-MS approach to separate CKD patients from controls, at slightly reduced accuracy, but expected reduced cost and time. As a consequence, a practical approach can be foreseen where MALDI-MS is employed as an inexpensive, fast, and robust screening tool to detect probable CKD. In a second step, high resolution CE-MS could be used in those patients only that scored negative for CKD in the MALDI-MS analysis, reducing costs and time of such a program.

A pilot study on the effect of short-term consumption of a polyphenol rich drink on biomarkers of coronary artery disease defined by urinary proteomics.

Polyphenol rich diets have been associated with a reduced risk of cardiovascular disease. We examined the effect of a polyphenol rich (P-R) drink on biomarkers assessed by urinary proteomics. Thirty nine middle aged and overweight subjects were randomized to P-R drink (n = 20) or placebo (n = 19) in addition to their normal diet. After two weeks urine samples were obtained for assessment of the urinary proteome using capillary electrophoresis coupled to a mass spectrometer. A total of 93 polypeptides were found to be candidates for differential distribution with a nominal p-value <0.05, though these differences did not reach significance when multiple testing was accounted for. Sequences were determined in 19 of these demonstrating that they originate from alpha-1 antitrypsin, collagens, fibrinogen alpha and IgG kappa. Levels of 27 polypeptides were greater than 4-fold different between the two groups. Of these, 7 were previously found to be part of a coronary artery disease (CAD) specific urinary biomarker pattern. Their direction of expression was closer to the healthy state in the P-R drink group and closer to CAD state in the placebo group. Our data suggest that the P-R drink may have beneficial effects on urinary biomarkers of CAD. The data encourage the planning of future prospective studies, aimed at investigating significant effects of polyphenol rich dietary products.

Urine proteomics in clinical applications: technologies, principal considerations and clinical implementation.

The technology platforms for proteome analysis have advanced considerably over the last few years. Due to these improvements the number of studies on the analysis of the proteome/peptidome with the aim of defining biomarkers has escalated. In this review, we will summarise the technical aspects that relate to the proteomics field targeting the discovery of biomarkers for disease diagnosis. We will describe the course from biomarker discovery or 'potential' biomarkers to those that are clinically important. We also present several examples of successful proteomic studies that have defined 'biomarker patterns' for clinical applications, focussed on urine as a material source and capillary electrophoresis coupled mass spectrometry as a technology. Finally, current challenges and considerations for future studies will be discussed.

Proteome analysis in hematology using capillary electrophoresis coupled on-line to mass spectrometry.

Proteome analysis, the key technology for biomarker discovery, continues to gain importance in clinical diagnosis and follow-up. In this review we describe proteome analysis in the context of allogeneic, hematopoietic stem cell transplantation concentrating on capillary electrophoresis coupled on-line to mass spectrometry.

Proteomics in gerontology: current applications and future aspects--a mini-review.

BACKGROUND: Aging is closely related to the onset of chronic diseases, such as coronary artery disease, diabetic nephropathy or different types of malignancies, reflecting the demand for novel biomarkers to manage theses diseases. OBJECTIVE: The analysis of the human proteome for biomarkers has made considerable advances in the last years. METHODS: We describe the main technological approaches taken, their advantages and disadvantages. RESULTS: We will review the different clinical sources of material and attempt to highlight the different challenges and approaches associated with these. Age-related changes in the proteome have been described and were found to be highly similar to changes associated with chronic diseases. We will give several examples on the successful application of proteomics in the diagnosis, prognosis and therapy of these chronic diseases. CONCLUSIONS: A boost in disease-related proteomic information is expected in the very near future, and will also result in its broad clinical application. However, this view appears to be dependent on the strict adherence to proper technological/analytical parameters, correct statistics, and large databases that allow comparison of datasets provided by different scientists. Clearly, the proteome is by far too complex to be tackled by one laboratory on its own.

Activation of protein kinase A (PKA) by 8-Cl-cAMP as a novel approach for antileukaemic therapy.

Activation of PKA by cAMP agonists, such as 8-Cl-cAMP activation, selectively causes rapid apoptosis in v-abl transformed fibroblasts by inhibiting the Raf-1 kinase. Here we investigated whether 8-Cl-cAMP is useful for the treatment of chronic myelogenous leukaemia (CML), which is hallmarked by the expression of the p210(bcr/abl) oncogene. Autologous bone marrow transplantation is a feasible alternative for patients with no suitable donor, but hampered by the risk of relapse due to the persistence of leukaemia cells in the transplant. To study the effects of 8-Cl-cAMP on primary leukaemic cells, bone marrow cells (BMCs) from eight CML patients (one at diagnosis, three in chronic and four in accelerated phase) were treated. Ex vivo treatment of BMCs obtained in chronic phase of CML with 100 microM 8-Cl-cAMP for 24-48 h led to the selective purging of Philadelphia Chromosome (Ph1 chromosome) without toxic side effects on BMCs from healthy donors as measured by colony-forming unit (CFU) assays. BMCs from patients in accelerated phase showed selective, but incomplete elimination of Ph1 chromosome positive colony forming cells. The mechanism of 8-Cl-cAMP was investigated in FDCP-mix cells transformed by p210(bcr/abl), a cell culture model for CML. The results showed that 8-Cl-cAMP reduced DNA synthesis and viability independent of Raf inhibition as Raf inhibitors had no effect. MEK inhibitors interfered with DNA synthesis, but not with viability. In summary, our results indicate that 8-Cl-cAMP could be useful to purge malignant cells from the bone marrow of patients with CML and certain other forms of leukaemias.

Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin.

G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta. Two-dimensional peptide mapping of PKCalpha-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulin-binding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.

Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry.

We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.

The PKC targeting protein RACK1 interacts with the Epstein-Barr virus activator protein BZLF1.

Phorbol esters reactivate Epstein-Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate-early gene BZLF1. BZLF1 is a member of the extended AP-1 family of transcription factors that binds to specific BZLF1-binding motifs within early EBV promoters and to consensus AP-1 sites. Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post-translational modifications. Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA [Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J. & Hammerschmidt, W., (1998) J. Virol. 72, 8105-8114]. The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay. RACK stands for receptor of activated C-kinase and is involved in targeting activated PKCs and other signaling proteins. In vivo, RACK1 binds directly to the transactivation domain of BZLF1. Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays. We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.

Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation.

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.

Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein.

We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.