Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.Signaling cascades that control fundamental cellular processes need to be tightly regulated. The MAPK 1 cascade is an evolutionarily conserved signaling module that is activated by a diverse set of signals and that stimulates numerous biological processes, including growth and differentiation. RKIP is a ubiquitously expressed and highly conserved protein with homologs in Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster that display remarkable degrees of interspecies sequence and structural similarity (reviewed in Ref. 6). Mammalian RKIP is distinct from other known proteins, and its function has remained largely enigmatic. A role for RKIP in signaling cascades was demonstrated when it was shown that RKIP binds to Raf-1 (7). However, RKIP also has a number of other reported functions, including inhibition of other kinases such as GRK2 (G protein-coupled receptor kinase-2) (8) and upstream kinase activators of IB kinase (9). Recently, RKIP has been identified as a metastasis suppressor gene, and this function correlates with MAPK activity (10).Elucidating the mechanism of RKIP action is important both for a complete understanding of Raf regulation and for generating potential therapeutic reagents. Previous studies have suggested that RKIP binds to and acts downstream of Raf-1 by competitive interference with MEK binding to Raf-1 (11). We previously identified a mechanism for overcoming inhibition of MAPK signaling by RKIP (12). Protein kinase C, activated by either phorbol esters or epidermal growth factor (EGF), phosphorylates RKIP at Ser 153 , and this phosphorylation causes the dissociation of RKIP from Raf-1 and the subsequent activation of ...
The case of a 26-year-old man with pneumonia due to Legionella pneumophila associated with acute renal failure is presented, and the English-language literature on legionnaires' disease is reviewed. For this review, acute renal failure was defined as rapid deterioration in renal function indicated by a rise in levels of blood urea nitrogen and creatinine with or without the presence of oliguria. Our patient experienced renal failure and underwent hemodialysis. His condition gradually improved after treatment of legionnaires' disease with erythromycin. Biopsy of the kidney showed acute tubulointerstitial nephritis. Immunofluorescence microscopy demonstrated the presence of L. pneumophila serogroup 1. The laboratory findings suggested rhabdomyolysis. To our knowledge, this is the first case report of a patient with legionnaires' disease who recovered from acute renal failure and in whom the presence of L. pneumophila was demonstrated, and we believe it is the first case in which morphology of the kidney demonstrated the presence of L. pneumophila in a patient with legionnaires' disease, rhabdomyolysis, and renal failure.
Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA reductase and thus the synthesis of cholesterol, are remarkably effective in the treatment of cardiovascular disease. In addition to their favorable effect on lipid profile, these drugs may also prevent the proliferation of vascular smooth muscle that is characteristic of atherosclerosis. We hypothesize that statins prevent the posttranslational prenylation, and thus inhibit the function, of critical small GTPases in vascular smooth muscle cells. We have therefore assayed the effect of lovastatin on both the growth of A10 vascular smooth muscle cells and the status of their Ras and RhoB proteins. We find that Յ1 M lovastatin potently inhibits the proliferation of A10 cultures, and higher concentrations (Ն3 M) induce apoptosis. We have also tested the effect of 3-allylfarnesol (3-alFOH), an inhibitor of farnesyl transferase (FTI). The data show that although Ն10 M 3-alFOH is required for a cytostatic effect, the action of 3 M 3-alFOH can be greatly potentiated by even nanomolar levels of lovastatin. We also find that lovastatin and 3-alFOH exhibit synergism to cause the up-regulation and relocalization of RhoB from the membrane to cytosolic compartments. This relocalization of RhoB, which is presumed to reflect an inhibition of its prenylation, correlates with the proapoptotic activities of combined 3-alFOH and lovastatin treatment. These data suggest that RhoB may be a valuable pharmacological target in cardiovascular disease, and that combinations of statins and certain FTIs may be of value in treatment of disorders that are characterized by excess cell proliferation.
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