Mehdi Mollapour

Targeting the dynamic HSP90 complex in cancer

The molecular chaperone heat shock protein 90 (HSP90) has been used by cancer cells to facilitate the function of numerous oncoproteins, and it can be argued that cancer cells are addicted to HSP90. However, although recent reports of the early clinical efficacy of HSP90 inhibitors are encouraging, the optimal use of HSP90-targeted therapeutics will depend on understanding the complexity of HSP90 regulation and the degree to which HSP90 participates in both neoplastic and normal cellular physiology.

Hog1 Mitogen-Activated Protein Kinase Phosphorylation Targets the Yeast Fps1 Aquaglyceroporin for Endocytosis, Thereby Rendering Cells Resistant to Acetic Acid

ABSTRACT Aquaporins and aquaglyceroporins form the membrane channels that mediate fluxes of water and small solute molecules into and out of cells. Eukaryotes often use mitogen-activated protein kinase (MAPK) cascades for the intracellular signaling of stress. This study reveals an aquaglyceroporin being destabilized by direct MAPK phosphorylation and also a stress resistance being acquired through this channel loss. Hog1 MAPK is transiently activated in yeast exposed to high, toxic levels of acetic acid. This Hog1 then phosphorylates the plasma membrane aquaglyceroporin, Fps1, a phosphorylation that results in Fps1 becoming ubiquitinated and endocytosed and then degraded in the vacuole. As Fps1 is the membrane channel that facilitates passive diffusional flux of undissociated acetic acid into the cell, this loss downregulates such influx in low-pH cultures, where acetic acid (pKa, 4.75) is substantially undissociated. Consistent with this downregulation of the acid entry generating resistance, sensitivity to acetic acid is seen with diverse mutational defects that abolish endocytic removal of Fps1 from the plasma membrane (loss of Hog1, loss of the soluble domains of Fps1, a T231A S537A double mutation of Fps1 that prevents its in vivo phosphorylation, or mutations generating a general loss of endocytosis of cell surface proteins [doa4Δ and end3Δ]). Remarkably, targetting of Fps1 for degradation may be the major requirement for an active Hog1 in acetic acid resistance, since Hog1 is largely dispensable for such resistance when the cells lack Fps1. Evidence is presented that in unstressed cells, Hog1 exists in physical association with the N-terminal cytosolic domain of Fps1.

Post-translational modifications of Hsp90 and their contributions to chaperone regulation.

Molecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of cytosolic and nuclear Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Swe1Wee1-Dependent Tyrosine Phosphorylation of Hsp90 Regulates Distinct Facets of Chaperone Function

Saccharomyces WEE1 (Swe1), the only true tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.

Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

Summary Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

Threonine 22 Phosphorylation Attenuates Hsp90 Interaction with Cochaperones and Affects Its Chaperone Activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both inxa0vitro and inxa0vivo. This α helix participates inxa0a hydrophobic interaction with the catalytic loop in Hsp90s middle domain, helping to stabilize the chaperones ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.

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.

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.

Hsp90 phosphorylation, Wee1, and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1∆ yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.

Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes, as it regulates diverse signal transduction nodes that integrate numerous environmental cues to maintain cellular homeostasis. Hsp90 also is secreted from normal and transformed cells and regulates cell motility. Here, we have identified a conserved hydrophobic motif in a β-strand at the boundary between the N domain and charged linker of Hsp90, whose mutation not only abrogated Hsp90 secretion but also inhibited its function. These Hsp90 mutants lacked chaperone activity in vitro and failed to support yeast viability. Notably, truncation of the charged linker reduced solvent accessibility of this β-strand and restored chaperone activity to these mutants. These data underscore the importance of β-strand 8 for Hsp90 function and demonstrate that the functional consequences of weakened hydrophobic contacts in this region are reversed by charged-linker truncation.