The Raf family includes three members, of which B-Raf is frequently mutated in melanoma and other tumors. We show that Raf-1 and A-Raf require Hsp90 for stability, whereas B-Raf does not. In contrast, mutated, activated B-Raf binds to an Hsp90 -cdc37 complex, which is required for its stability and function. Exposure of melanoma cells and tumors to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in the degradation of mutant B-Raf, inhibition of mitogen-activated protein kinase activation and cell proliferation, induction of apoptosis, and antitumor activity. These data suggest that activated mutated B-Raf proteins are incompetent for folding in the absence of Hsp90, thus suggesting that the chaperone is required for the clonal evolution of melanomas and other tumors that depend on this mutation. Hsp90 inhibition represents a therapeutic strategy for the treatment of melanoma.17-allylamino-17-demethoxygeldanamycin ͉ cdc37 ͉ melanoma I n the last several years, it has become clear that the Ras͞Raf͞ mitogen-activated protein kinase (MAPK)͞extracellular signalregulated kinase kinase (MEK)͞MAPK signaling pathway is mutationally activated in most melanomas. One member of the Ras family, N-Ras, is mutated in Ϸ25% of melanomas (1), whereas mutations in the H-ras and K-ras genes are rare (2). The Raf gene family (Raf-1, A-Raf, and B-Raf) encodes closely related serine͞ threonine protein kinases that are important effectors of Ras activation. However, no mutations in the Raf gene were found until recently, when Davies et al. (3) showed that Raf-1 and A-Raf are rarely mutated but that mutations in the B-Raf gene are common in human cancer, especially in melanoma.Refs. 3 and 4 showed that B-Raf is mutated in Ϸ70% of human melanomas, 35-70% of papillary thyroid carcinomas, and less commonly in lung and colorectal carcinomas. Mutations are almost always in the B-Raf kinase domain and, in melanomas, the vast majority are V600E missense mutations (3). Marais and coworkers (5) have shown in heterologous systems that the V600E mutation leads to activation of B-Raf kinase.The frequency and activating nature of the B-Raf mutations suggest that they have an important role in the biology of melanoma and perhaps other tumors in which they have been detected. Moreover, small interfering RNA against mutated B-Raf but not Raf-1 inhibits the transformed phenotype of melanoma cells harboring B-Raf mutations (6, 7). N-Ras and B-Raf mutations seem to be mutually exclusive in melanoma, suggesting that they make similar contributions to transformation and that activation of this pathway is a key event in the development of this disease (3,8).Together, these data suggest that inhibition of B-Raf͞MEK͞ MAPK signaling could be a powerful means for treating melanomas and other tumors with B-Raf mutation. There is no validated therapy that potently inhibits mutated B-Raf function in patients. Selective inhibitors of MEK have been developed and have antitumor activity in xenograft models of melanoma (9). Putative Raf inhibitor is ...

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both A␤40 and A␤42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of ␤-cleaved amyloid precursor protein (APP) carboxylterminal fragments (␤CTF), the rate-limiting proteolytic intermediate in A␤ generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in ␤-cleaved soluble APP relative to ␣-cleaved soluble APP was also observed following Rab5 overexpression. ␤CTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, ␤CTF generation, and A␤ production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a ␤-amyloidogenic pathway.In the brain parenchyma, the deposition of the small peptide A␤, a proteolyic fragment of the amyloid precursor protein (APP), 1 is an invariant feature of Alzheimer's disease (AD) (1). Generation of A␤ requires two sequential proteolytic steps:␤-cleavage in the lumenal domain of APP followed by intramembranous ␥-cleavage (reviewed in Ref.2). ␤-Cleavage appears to be mediated by the BACE family of aspartyl-proteases (3, 4), whereas ␥-cleavage requires the presenilin protein complex (5, 6). The intracellular site(s) of A␤ generation has been the subject of intense investigation for a number of years, and both the secretory and endocytic pathways have been implicated (7-13). Confounding the clear delineation of A␤ generation sites is the broad intracellular distribution of the key proteins involved. APP is predominantly localized to the trans-Golgi network (TGN), but significant amounts of the protein are found at the cell surface and within endosomes and other transport vesicles (2). BACE has been placed within early endosomes and/or throughout the endosomal-lysosomal system (3, 14, 15), although a predominant TGN localization has also been reported (16,17). The presenilin proteins were initially thought to be located primarily within the endoplasmic reticulum and Golgi apparatus (18 -23), but more recent evidence suggests a distribution also within the plasma membrane and endosomes (18, 24 -27), and studies of nicastrin also strongly suggest that the mature presenilin complex is found in compartments distal to the endoplasmic reticulum (28).Our previous studies have shown that abnormalities of the neuronal endocytic pathwa...

Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer's disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPP␣ and sAPP␤ were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human A␤40 and A␤42 into the growth media nearly tripled in CD-MPR-and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells. Comparable increases were confirmed for endogenous mouse A␤40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating A␤ generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.Deposition in the brain parenchyma and cerebral vessel walls of ␤-amyloid and intracellular neurofibrillary tangles are diagnostic hallmarks of AD.1 ␤-Amyloid consists primarily of the short peptide A␤, which is derived from the proteolytic cleavage of the amyloid precursor protein (APP) at a site within its luminal domain (the ␤-cleavage site) and cleavage within the transmembrane domain (the ␥-cleavage site). Additionally, ␣-cleavage of APP may occur within the A␤ domain of APP, adjacent to the plasma membrane (see Ref.1 for a review of APP proteolytic processing). ␤-Amyloidogenesis in familial AD caused by mutation of APP or the presenilins involves, at least in part, the overproduction of A␤ (2); however, the mechanisms promoting ␤-amyloidogenesis in sporadic forms of AD, which account for Ͼ90% of AD cases, remain unclear. Experimental evidence has shown that early endosomes are an important site for APP processing, including the generation of A␤. Expression of trafficking mutants of APP lacking endocytosis signals reduces A␤ production compared with endocytosis-competent wild-type APP (3, 4). There is also evidence that at least some of the proteases responsible for A␤ generation reside within early endosomes. For example BACE, a recently identified transmembrane aspartic protease with ␤-secretase activity, resides in part within early endosomes (5-7). The lysosomal protease cathepsin D (Cat D) was recently confirmed ...

In murine L cells, treatment with calpeptin or calpain inhibitor III increased A␤42, but not A␤40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the ␤-cleaved CTF showed that generation of both ␣-and ␤-cleaved CTFs increase proportionately following inhibition of calpains. Pulsechase metabolic labeling confirmed that inhibiting calpains increases the production of ␣-and ␤-cleaved APP metabolites. Immunolabeling showed greater ␤CTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/ luminal epitope found on both APP and ␤CTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both ␣-and ␤-cleavage without changing the ratio of ␣CTFs/␤CTFs, and 3) the bulk of the ␤CTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing.The deposition of the small (ϳ40 -42 residues) A␤ peptide as insoluble ␤-amyloid plaque in the brain parenchyma is an invariant feature of Alzheimer's disease. A␤ is generated by proteolysis of the ϳ100-kDa amyloid precursor protein (APP), 1 a broadly expressed type 1 transmembrane protein that is found primarily in the trans-Golgi network (TGN) and at the cell surface (reviewed in Ref. 1). The ␤-cleavage of APP occurs within the luminal/extracellular domain of APP and generates two APP fragments: a large, soluble amino-terminal fragment (sAPP␤) that is secreted from the cell and a transmembrane, carboxyl-terminal fragment (CTF) that remains associated with the cell. This ␤CTF consists of 99 amino acids, contains the whole A␤ peptide, and has a molecular mass of ϳ10 kDa. An alternative pathway involves the cleavage of APP 16 residues downstream of this site at the ␣-cleavage site. Like ␤-cleavage, ␣-cleavage generates a sAPP fragment (sAPP␣) that is secreted from the cell and an ␣CTF (of 84 residues and ϳ8 kDa) that remains membrane-associated. ␣-Cleavage occurs within the A␤ peptide sequence and as such prevents the generation of A␤ from a given APP molecule. A␤ is generated from the ␤CTF by an intramembrane cleavage (␥-cleavage) that occurs primarily at 40 residues, and to a lesser extent 42 residues, downstream from the ␤-cleavage site, releasing A␤1-40 or A␤1-42.Much progress has been made recently in identifying the major proteases/protease complexes responsible for ␣-, ␤-, and ␥-cleavage (the ␣-, ␤-, and ␥-secretases). ␣-Cleavage appears to be due to...