Studies of a phosphatidylinositol 3-kinase complex linked to vesicular trafficking in human cells

Phosphoinositide 3-kinases (PI 3-kinases) are signal transducing molecules that catalyse the addition of phosphate to the 3-OH position of the inositol ring of phosphoinositides. PI 3-kinase has emerged as an important mediator in the events leading to mitogenesis, cytoskeletal re-arrangement, receptor internalisation and glucose transport. In mammalian cells numerous PI 3-kinases have been discovered, including those regulated by Receptor Tyrosine Kinases (RTK) and heterotrimeric G proteins. These PI 3-kinases can generate Phosphatidylinositol (PtdIns)3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 as potential second messengers. In contrast, genetic and biochemical studies have shown that yeast contain only one PI 3-kinase, termed Vps34p. This enzyme uses PtdIns as its only substrate and is not activated by any known ligand. Vps34p is found associated with Vps15p, a 170kDa serine/threonine protein kinase. This heterodimeric complex is required for the efficient sorting and delivery of proteins to the yeast vacuole. A human homologue of the yeast Vps34p protein, PtdIns 3-kinase has been identified and shown to form a complex with a protein of 150kDa (termed p150), raising the possibility that a human Vpsl5p homologue exists. In this study the cDNA encoding the p150 protein was isolated and used to express a 150kDa protein in insect (Sf9) and mammalian (COS-7) cells, p150 was shown to display extensive amino acid homology with Vps15p and sequence comparisons between both proteins have been used to delineate potential functional domains. The data presented in this thesis also show that recombinant p150 associates with PtdIns 3-kinase both in vitro and in vivo. Using immunofluorescence techniques, p150 expressed in COS-7 cells was localised to perinuclear punctate compartments. To investigate which of the p150 domains are responsible for its subcellular localisation and association with PtdIns 3-kinase, deletion and amino acid substitution studies were undertaken. Additional biochemical studies investigating the 3'-phosphoinositides produced in cells expressing PtdIns 3-kinase demonstrated elevated levels of PtdIns3P and PtdIns(3,5)P2. The increase in PtdIns3P production by the p150/PtdIns 3-kinase complex in the presence of phosphatidylinositol transfer protein (PI-TP) also suggests a role for PI-TP in PtdIns substrate presentation to PtdIns 3-kinase. The results presented in this study demonstrate that p150 is the human Vps15p homologue, suggesting an evolutionary conservation between yeast and humans in the basic molecular mechanisms of vesicle transport. Based on the data presented, mechanisms by which the p150/PtdIns 3-kinase complex and its 3'-phosphoinositide products might be implicated in membrane trafficking events are proposed

Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo

c-Abl mediated tyrosine phosphorylation of Aha1 activates its co-chaperone function in cancer cells

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo

Essays on the role of narrative disclosures in financial reporting

This thesis contains two essays on the role of narrative disclosures in financial reporting. The first essay, “Tightening rating standards: The effect of narrative risk-related disclosures” (co-authored with Argyro Panaretou and Grzegorz Pawlina), examines how narrative disclosures affect rating stringency, a phenomenon where credit rating agencies assign ratings worse than what firm fundamentals justify. Results suggest that narrative disclosures about risk and uncertainty in Form 10-K reports moderate rating stringency. Moreover, this moderating effect is more pronounced when Form 10-K reports have textual attributes that can affect how users contextualize firm risk. The second essay, “Context matters: The role of fair value footnote narratives” (co-authored with Argyro Panaretou and Catherine Shakespeare), investigates how narrative disclosures in Form 10-K report footnotes that discuss the measurement of fair values affect investor uncertainty. The findings of this essay show that longer fair value footnote narratives reduce investor uncertainty for opaque fair values, and are particularly informative to sophisticated investors. Further test results suggest that standardized and non-specific fair value narratives increase investor uncertainty for Level 3 fair values, and that fair value narratives offer incremental information to investors relative to tabulated fair value footnote disclosures. Finally, the thesis includes a technical appendix, “A guide on extracting, processing, and operationalizing Form 10-K report narratives,” on the advantages and challenges in identifying, collecting, and integrating narrative disclosure data from Form 10-K reports into archival accounting studies

Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essen- tial component of the cellular homeostatic machinery in eukary- otes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co- chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilita- tor of Hsp90-mediated folding of kinase and non-kinase clients— including Tsc2—thereby preventing their ubiquitination and proteasomal degradation

Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors

The molecular chaperone Hsp90 protects deregu- lated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensi- tivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and dis- rupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensi- tizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors

Increase of enzyme activity through specific covalent modification with fragments

Modulation of enzyme activity is a powerful means of probing cellular function and can be exploited for diverse applications. Here, we explore a method of enzyme activation where covalent tethering of a small molecule to an enzyme can increase catalytic activity (k cat/K M) up to 35-fold. Using a bacterial glycoside hydrolase, BtGH84, we demonstrate how small molecule "fragments", identified as activators in free solution, can be covalently tethered to the protein using Michael-addition chemistry. We show how tethering generates a constitutively-activated enzyme-fragment conjugate, which displays both improved catalytic efficiency and increased susceptibility to certain inhibitor classes. Structure guided modifications of the tethered fragment demonstrate how specific interactions between the fragment and the enzyme influence the extent of activation. This work suggests that a similar approach may be used to modulate the activity of enzymes such as to improve catalytic efficiency or increase inhibitor susceptibility