Oncogene (2000) 19, 4385 ± 4395 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-g and Hsp70/Hsc70 Howard Doong1, John Price1, Young Sook Kim1, Christopher Gasbarre1, Julie Probst1, Lance A Liotta1, Jay Blanchette1, Kathryn Rizzo1 and Elise Kohn*,1 1Molecular Signaling Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland, MD 20892, USA CAIR-1/BAG-3 forms an EGF-regulated ternary com- Introduction plex with Hsp70/Hsc70 and latent phospholipase C-g (PLC-g). The expression of CAIR-1, CAI stressed-1, Understanding the crosstalk between cellular signal was induced in A2058 human melanoma cells by pathways is critical to decipher the regulation of continuous exposure to CAI, an inhibitor of nonvol- cellular physiology. Intracellular calcium concentra- tage-gated calcium in¯ux. CAIR-1 sequence is identical, tions are tightly regulated and are altered during save 2 amino acids, to BAG-3 also cloned recently as transmembrane signaling events, such as those stimu- Bis, a member of the bcl-2-associated athanogene family. lated by growth factors, and also in response to cellular We show that CAIR-1/BAG-3 binds to Hsp70/Hsc70 in injury or stress (Berridge et al., 1998; Bukau and intact cells and this binding is increased by short term Horwich, 1998; Zwick et al., 1999). We have identi®ed exposure to CAI (P50.007). CAIR-1/BAG-3 is phos- an inhibitor of calcium in¯ux in nonexcitable cells, phorylated in vivo in the absence of stimulation. Basal CAI (Felder et al., 1991; Gusovsky et al., 1993; Kohn phosphorylation is inhibited by treatment with d-erythro- et al., 1994a). Exposure to CAI causes tumor and sphingosine (d-ES), a broad inhibitor of the protein endothelial cell cytostasis and inhibits cell attachment, kinase C family. CAIR-1/BAG-3 contains several PXXP migration, and angiogenesis and tumorigenesis in vitro SH3 binding domains leading to the hypothesis that it is and in vivo (Kohn et al., 1992, 1994a,b, 1995; Kohn a partner protein of phospholipase C-g. PLC-g is bound and Liotta, 1990). We hypothesized that continuous to CAIR-1/BAG-3 in unstimulated cells. It is increased exposure to CAI by altering stimulated and basal by CAI or d-ES (P=0.05) treatment, and abrogated by intracellular calcium concentrations would stress the EGF (r2=0.99); d-ES treatment blocks the EGF- tumor cells. This would then either stimulate a stress mediated dissociation. We show that CAIR-1/BAG-3 recovery response or alter regulation or expression of binds to PLC-g and Hsp70/Hsc70 through separate and proteins required for calcium activation pathways, such distinct domains. Hsp70/Hsc70 binds to the BAG as the phospholipases. Activated phospholipases C domain of BAGs-1 and -3. CAIR-1/BAG-3 from control produce inositol trisphosphate, a potent second and EGF-treated cell lysates bound selectively to the messenger inducing internal calcium release and SH3 domain of PLC-g, but not its N-SH2 or C-SH2 subsequent transmembrane calcium in¯ux (Berridge et domains. Con®rming the SH3 interaction, PLC-g was al., 1998). We developed a stress model in which tumor pulled down by CAIR-1/BAG-3 PXXP-GST fusions, but cell sublines were isolated after long term continuous GST-PXXP constructs confronted with lysates from exposure to increasing concentrations of CAI. We now EGF-treated cells did not bind PLC-g as was seen in report cloning of CAIR-1, CAI stressed-1, a protein intact cells. Hsp70/Hsc70 was brought down by the nearly identical to BAG-3 (Takayama et al., 1999) PLC-g SH3 construct equally from native and EGF- from CAI-stressed cells, and demonstrate novel regula- treated cells, but did not bind the PXXP construct under tion and function of CAIR-1/BAG-3. either condition. We propose that CAIR-1/BAG-3 may BAG-3 was cloned from a screen for homologs of act as a multifunctional signaling protein linking the the bcl-2 binding protein BAG-1 (bcl-2-associated Hsp70/Hsc70 pathway with those necessary for activa- athanogene) family (Takayama et al., 1995, 1999). tion of the EGF receptor tyrosine kinase signaling BAG-1 was identi®ed in a screen for bcl-2 binding pathways. Oncogene (2000) 19, 4385 ± 4395. proteins and has been shown to inhibit apoptosis and promote tumorigenesis (Stuart et al., 1998; Takayama Keywords: calcium; EGF; phospholipase C; Hsp70/ et al., 1995, 1997). Recent homology cloning Hsc70; signal transduction identi®ed fragments of at least four other BAG family members, all of which contain a conserved C- terminal domain, the BAG domain (Takayama et al., 1999). There is little other homology between BAG-1 and BAG-3, including lack of the BAG-1L nuclear localization signal or the BAG-1 ubiquitin region. The BAG domain was shown by pull down experiments and functional assays to bind to and promote substrate release from both HSP-70 and HSC-70 (Stuart et al., 1998; Takayama et al., 1997, *Correspondence: EC Kohn, 10 Center Drive, 10/2A33, Bethesda, 1999). Direct binding of BAG-1 to bcl-2 could not be MD 28092-1500, USA Received 21 January 2000; revised 21 June 2000; accepted 11 July demonstrated upon direct confrontation, however 2000 addition of ATP markedly enhanced BAG-1/bcl-2 An EGF-regulated complex of CAIR-1/BAG-3, PLC-g and Hsc70 H Doong et al 4386 binding, suggesting the requirement for an HSP-70 from a K; 237Q from an R) from BAG-3, a member intermediate (Takayama et al., 1997). HSP-70 and its of the bcl-2-associated athanogene family of bcl-2 constitutive form, HSC-70, are molecular chaperones binding proteins (Takayama et al., 1995, 1999). activated by varied cellular stresses including heat Genomic clones from chromosome 10q25 con®rm shock, oxygen-free radicals, transition heavy metals, the CAIR-1 sequence. Northern analysis revealed a in¯ammation, ischemia, anti-neoplastics, oncogenes, twofold net increase in expression of CAIR-1/BAG-3 and proto-oncogenes (Morimoto, 1998). They bind in cells stressed by constant exposure to 10 ± 30 mM ATP through an ATPase domain and have a CAI (10 ± 30R, Figure 1b) but not in cells exposed to separate substrate binding domain (Bukau and 10 mM CAI for 424 h (Figure 1c). Domain analysis Horwich, 1998; Pilon and Schekman, 1999). The site of CAIR-1/BAG-3 reveals the previously reported of Hsp70/Hsc70 binding to BAG-1 has been de®ned BAG region (Takayama et al., 1999) and WW as the ATPase domain (Bukau and Horwich, 1998; domain, as well as seven putative protein kinase C Takayama et al., 1997); a 1 : 1 molar binding results (PKC) and ®ve putative CKII phosphorylation sites, in reduction in the refolding function of HSP-70 multiple potential tyrosine phosphorylation sites, and (Stuart et al., 1998). While other functions for BAG- a series of proline-rich PXXP repeats of the SH3 1 such as binding to and activating Raf-1 have been binding type (Pawson and Scott, 1997). No genomic reported, no link between those functions and HSP- ampli®cation of CAIR-1/BAG-3 gene in the CAI- 70 have been demonstrated. CAIR-1/BAG-3 was stressed cells was demonstrated by Southern analysis recently cloned also as bis (Lee et al., 1999), in a or ¯uorescent in situ hybridization (data not shown). protein interaction cloning procedure using bcl-2 as Broad tissue expression of CAIR-1/BAG-3 was found bait. Lee and coworkers demonstrated a weak anti- in adult and embryonic tissues with lowest expression apoptotic activity of transfected bis and further seen in hematopoietic tissues (Figure 1d). Genomic showed synergy when suboptimal doses of both bis conservation was limited to the higher eukaryotes by and bcl-2 were transfected, simultaneously. They zoo blot (not shown, Bios, New Haven, CT, USA). localized the site of interaction of bis to the BH1 Immunoprecipitation from A2058 cells followed by domain of bcl-2. immunoblot with anti-peptide antibodies to CAIR-1/ Phospholipase C-g (PLC-g) regulates cytosolic free BAG-3 recognized a 74 kDa protein which was calcium concentration in response to transmembrane increased in quantity in 20 mM CAI-stressed cells signal transduction, and has been linked to malig- (Figure 1e). No similar induction of CAIR-1/BAG-3 nant transformation, invasive potential, and angio- protein was observed in wild type A2058 or MDA-435 genesis (Chang et al., 1997; Khoshyomn et al., 1999; human breast cancer cells upon exposure to 10 mM Smith et al., 1998; Turner et al., 1996, 1997; Yang CAI for up to 24 h (Figure 1f), consistent with the et al., 1998). Several investigators have demonstrated lack of increased gene expression with acute CAI transforming capacity of both full length PLC-g and exposure. the isolated SH2-SH2-SH3 moiety (Bar-Sagi et al., 1993; Chang et al., 1997; Schlessinger, 1994; Smith CAIR-1/BAG-3 is phosphorylated in vivo et al., 1998). Its link to calcium homeostasis, transmembrane signal transduction, and transforming CAIR-1/BAG-3 has not been demonstrated pre- potential made it a putative target protein in the viously to be a phosphoprotein. Domain analysis calcium in¯ux stress model. We demonstrate that of CAIR-1/BAG-3 indicated the presence of poten- CAIR-1, which is increased in expression in CAI- tial PKC and CKII phosphorylation sites, as well as treated cells, is a partner protein for PLC-g,and putative tyrosine phosphorylation sites. In vivo forms a ternary complex with HSP-70.