Wanping Xu

Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression in vivo is associated with poor prognosis. We now show that the chaperone-binding ubiquitin ligase CHIP efficiently ubiquitinates and down-regulates ErbB2. CHIP expression shortens the half-life of both nascent and mature ErbB2 protein. In vitro ubiquitination assay shows that CHIP serves as a ubiquitin ligase for ErbB2, and both exogenously expressed and endogenous CHIP coprecipitate with the kinase. Furthermore, CHIP association with ErbB2 requires a chaperone intermediate and is increased by the chaperone-binding drug geldanamycin, a potent stimulator of ErbB2 ubiquitination and degradation. These data describe a previously unrecognized pathway, amenable to pharmacologic manipulation, that mediates ErbB2 stability.

CHIP activates HSF1 and confers protection against apoptosis and cellular stress.

Induction of molecular chaperones is the characteristic protective response to environmental stress, and is regulated by a transcriptional program that depends on heat shock factor 1 (HSF1), which is normally under negative regulatory control by molecular chaperones Hsp70 and Hsp90. In metazoan species, the chaperone system also provides protection against apoptosis. We demonstrate that the dual function co‐chaperone/ubiquitin ligase CHIP (C‐terminus of Hsp70‐interacting protein) regulates activation of the stress‐chaperone response through induced trimerization and transcriptional activation of HSF1, and is required for protection against stress‐induced apoptosis in murine fibroblasts. The consequences of this function are demonstrated by the phenotype of mice lacking CHIP, which develop normally but are temperature‐sensitive and develop apoptosis in multiple organs after environmental challenge. CHIP exerts a central and unique role in tuning the response to stress at multiple levels by regulation of protein quality control and transcriptional activation of stress response signaling.

Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer therapy

Overexpression of ErbB‐2/HER2 is associated with aggressive human malignancies, and therapeutic strategies targeting the oncoprotein are currently in different stages of clinical application. Tyrosine kinase inhibitors (TKIs) that block the nucleotide‐binding site of the kinase are especially effective against tumors. Here we report an unexpected activity of TKIs: along with inhibition of tyrosine phosphorylation, they enhance ubiquitylation and accelerate endocytosis and subsequent intracellular destruction of ErbB‐2 molecules. Especially potent is an irreversible TKI (CI‐1033) that alkylates a cysteine specific to ErbB receptors. The degradative pathway stimulated by TKIs appears to be chaperone mediated, and is common to the heat shock protein 90 (Hsp90) antagonist geldanamycin and a stress‐induced mechanism. In agreement with this conclusion, CI‐1033 and geldanamycin additively inhibit tumor cell growth. Based upon a model for drug‐induced degradation of ErbB‐2, we propose a general strategy for selective destruction of oncoproteins by targeting their interaction with molecular chaperones.

Targeting the Molecular Chaperone Heat Shock Protein 90 Provides a Multifaceted Effect on Diverse Cell Signaling Pathways of Cancer Cells

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of several conditionally activated and/or expressed signaling proteins as well as multiple mutated, chimeric, and/or overexpressed signaling proteins, which promote cancer cell growth and/or survival (see

Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex

The molecular chaperone Hsp90 modulates the function of specific cell signaling proteins. Although targeting Hsp90 with the antibiotic inhibitor geldanamycin (GA) may be a promising approach for cancer treatment, little is known about the determinants of Hsp90 interaction with its client proteins. Here we identify a loop within the N lobe of the kinase domain of ErbB2 that determines Hsp90 binding. The amino acid sequence of the loop determines the electrostatic and hydrophobic character of the proteins surface, which in turn govern interaction with Hsp90. A point mutation within the loop that alters ErbB2 surface properties disrupts Hsp90 association and confers GA resistance. Notably, the immature ErbB2 point mutant remains sensitive to GA, suggesting that mature and nascent client kinases may use distinct motifs to interact with the Hsp90 chaperone complex.

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis.

Significance TNF receptor-associated protein (TRAP1) is found predominantly in mitochondria. A possible direct impact of TRAP1 on mitochondrial metabolism remains unexplored. We used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1 deficiency promotes increased mitochondrial respiration, fatty acid oxidation, tricarboxylic acid cycle intermediates, ATP and reactive oxygen species, while concomitantly suppressing glucose metabolism. TRAP1 deficiency also results in strikingly enhanced cell motility and invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by geldanamycin and proteasome inhibition by velcade.

Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER-associated protein degradation, a sequential process requiring valosin-containing protein (VCP)–dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from geldanamycin and Velcade exposure overwhelms the ability of the VCP-centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the “small VCP-interacting protein” exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP-enhanced yellow fluorescent protein fluorescent construct, geldanamycin plus Velcade treatment redistributed VCP-enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination. (Mol Cancer Res 2006;4(9):667–81)

Asymmetric Hsp90 N Domain SUMOylation Recruits Aha1 and ATP-Competitive Inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90s function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.

Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity.

ABSTRACT The receptor tyrosine kinase ErbB2 plays a crucial role in tumorigenesis. We showed previously that the molecular chaperone Hsp90 protects ErbB2 from proteasome-mediated degradation by binding to a short loop structure in the N-lobe of the kinase domain. Here we show that loss of Hsp90 binding correlates with enhanced ErbB2 kinase activity and its transactivating potential, concomitant with constitutively increased phosphorylation of Tyr877, located in the activation loop of the kinase domain. We show further that Tyr877 phosphorylation is mediated by Src and that it is necessary for the enhanced kinase activity of ErbB2. Finally, computer modeling of the kinase domain suggests a phosphorylation-dependent reorientation of the activation loop, denoting the importance of Tyr877 phosphorylation for ErbB2 activity. These findings suggest that Hsp90 binding to ErbB2 participates in regulation of kinase activity as well as kinase stability.