Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP3Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP3R1, focusing on the ∼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914-1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP3R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP3R1-derived peptides revealed high affinity binding to amino acids 1898-1940 of IP3R1 (Kd ∼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP3R1 interaction was demonstrated by the ability of IP3R1 or Bok to recruit transmembrane domain-free Bok or IP3R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP3R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.

Structural and functional basis of protein phosphatase 5 substrate specificity.

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.

The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors.

Bok is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, although the role that Bok plays in this pathway is unclear. We have shown previously in cultured cell lines that Bok interacts strongly with inositol 1,4,5-trisphosphate receptors (IP3Rs), suggesting that it may contribute to the structural integrity or stability of IP3R tetramers. Here we report that Bok is similarly IP3R-assocated in mouse tissues, that essentially all cellular Bok is IP3R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP3Rs, that Bok is dramatically stabilized by binding to IP3Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP3Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP3R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP3Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP3R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP3Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.

The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage.

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895-1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

Endogenous Bok is stable at the endoplasmic reticulum membrane and does not mediate proteasome inhibitor-induced apoptosis.

Controversy surrounds the cellular role of the Bcl-2 family protein Bok. On one hand, it has been shown that all endogenous Bok is bound to inositol 1,4,5-trisphosphate receptors (IP3Rs), while other data suggest that Bok can act as a pro-apoptotic mitochondrial outer membrane permeabilization mediator, apparently kept at very low and non-apoptotic levels by efficient proteasome-mediated degradation. Here we show that 1) endogenous Bok is expressed at readily-detectable levels in key cultured cells (e.g., mouse embryonic fibroblasts and HCT116 cells) and is not constitutively degraded by the proteasome, 2) proteasome inhibitor-induced apoptosis is not mediated by Bok, 3) endogenous Bok expression level is critically dependent on the presence of IP3Rs, 4) endogenous Bok is rapidly degraded by the ubiquitin-proteasome pathway in the absence of IP3Rs at the endoplasmic reticulum membrane, and 5) charged residues in the transmembrane region of Bok affect its stability, ability to interact with Mcl-1, and pro-apoptotic activity when over-expressed. Overall, these data indicate that endogenous Bok levels are not governed by proteasomal activity (except when IP3Rs are deleted) and that while endogenous Bok plays little or no role in apoptotic signaling, exogenous Bok can mediate apoptosis in a manner dependent on its transmembrane domain.

Bok regulates mitochondrial fusion and morphology.

Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP3Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP3Rs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IP3R binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IP3Rs, Bok does not alter IP3R-mediated Ca2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.

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.