Background: Bok is a Bcl-2 protein family member, with largely unknown properties. Results: Bok binds constitutively to IP 3 receptors and protects them from proteolysis, but does not appear to alter their Ca

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 (IP 3 Rs), suggesting that it may contribute to the structural integrity or stability of IP 3 R tetramers. Here we report that Bok is similarly IP 3 R-assocated in mouse tissues, that essentially all cellular Bok is IP 3 R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP 3 Rs, that Bok is dramatically stabilized by binding to IP 3 Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP 3 Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP 3 R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP 3 Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP 3 R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP 3 Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.Bok is a member of the Bcl-2 protein family that controls the intrinsic apoptosis pathway (1-3). Bok contains four Bcl-2 homology domains (BH1-4) 2 and shares greatest sequence homology with the pro-apoptotic proteins Bak and Bax (1-4). However, unlike Bak and Bax, which have clearly defined roles in mediating mitochondrial outer membrane permeabilization (5, 6), the cellular role of Bok is unclear. Key observations that pertain to our current understanding of the function of Bok are (i) that the atypical C-terminal transmembrane (TM) domain of Bok localizes it to membranes of the endoplasmic reticulum (ER) and Golgi (7), (ii) that Bok over-expression leads to apoptosis (7-10) if Bak or Bax are present (7), indicating that Bok lies upstream of Bak and Bax, (iii) that Bok Ϫ/Ϫ mice are phenotypically normal (4, 11, 12), while Bax Ϫ/Ϫ Bak Ϫ/Ϫ mice exhibit multiple severe defects (1), indicating that Bok cannot substitute for Bak and Bax, and (iv) that ER stress-induced apoptosis (13) can be suppressed in Bok Ϫ/Ϫ mouse cells in vitro and in vivo (12), although this result has not been seen by all groups (7). Overall, these data suggest that Bok plays a very different role from Bax and Bak, and that it may participate in the pathway between ER stress and apoptosis.Another intriguing facet of Bok cell biology is that it binds very strongly to inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) (14), proteins that form tetrameric, IP 3 -, and Ca 2ϩ -gated Ca 2ϩ channels in ER membranes and play a key role in vertebrate cell signaling (15,16). Of the three mammalian IP 3 R types, Bok binding is strongest to IP 3 R1 and IP 3 R2 (14) and the...

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 1,4,5trisphosphate receptors (IP 3 Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP 3 Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPRassociated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP 3 Rs 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 IP 3 R 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 IP 3 Rs, Bok does not alter IP 3 R-mediated Ca 2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.

SummaryThe 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.