regulated ternary complex with phospholipase C
Correspondence to: E C Kohn, 10 Center Drive, 10/2A33, Bethesda, MD 28092 1500, USACAIR 1/BAG 3 forms an EGF regulated ternary complex with Hsp70/Hsc70 and latent phospholipase C (PLC ). The expression of CAIR 1, CAI stressed 1, was induced in A2058 human melanoma cells by continuous exposure to CAI, an inhibitor of nonvoltage gated calcium influx. CAIR 1 sequence is identical, save 2 amino acids, to BAG 3 also cloned recently as Bis, a member of the bcl 2 associated athanogene family. We show that CAIR 1/BAG 3 binds to Hsp70/Hsc70 in intact cells and this binding is increased by short term exposure to CAI (Pin vivo in the absence of stimulation. Basal phosphorylation is inhibited by treatment with d erythrosphingosine (d ES), a broad inhibitor of the protein kinase C family. CAIR 1/BAG 3 contains several PXXP SH3 binding domains leading to the hypothesis that it is a partner protein of phospholipase C . PLC is bound to CAIR 1/BAG 3 in unstimulated cells. It is increased by CAI or d ES (P=0.05) treatment, and abrogated by EGF (r2=0.99); d ES treatment blocks the EGF mediated dissociation. We show that CAIR 1/BAG 3 binds to PLC and Hsp70/Hsc70 through separate and distinct domains. Hsp70/Hsc70 binds to the BAG domain of BAGs 1 and 3. CAIR 1/BAG 3 from control and EGF treated cell lysates bound selectively to the SH3 domain of PLC , but not its N SH2 or C SH2 domains. Confirming the SH3 interaction, PLC was pulled down by CAIR 1/BAG 3 PXXP GST fusions, but GST PXXP constructs confronted with lysates from EGF treated cells did not bind PLC as was seen in intact cells. Hsp70/Hsc70 was brought down by the PLC SH3 construct equally from native and EGF treated cells, but did not bind the PXXP construct under either condition. We propose that CAIR 1/BAG 3 may act as a multifunctional signaling protein linking the Hsp70/Hsc70 pathway with those necessary for activation of the EGF receptor tyrosine kinase signaling pathways. Intracellular calcium concentrations are tightly regulated and are altered during transmembrane signaling events, such as those stimulated by growth factors, and also in response to cellular injury or stress (Berridge et al., 1998; Bukau and Horwich, 1998; Zwick et al., 1999). We have identified an inhibitor of calcium influx in nonexcitable cells, CAI (Felder et al., 1991; Gusovsky et al., 1993; Kohn et al., 1994a). Exposure to CAI causes tumor and endothelial cell cytostasis and inhibits cell attachment, migration, and angiogenesis and tumorigenesis in vitro and in vivo (Kohn et al., 1992, 1994a,b, 1995; Kohn and Liotta, 1990). We hypothesized that continuous exposure to CAI by altering stimulated and basal intracellular calcium concentrations would stress the tumor cells. This would then either stimulate a stress recovery response or alter regulation or expression of proteins required for calcium activation pathways, such as the phospholipases. Activated phospholipases C produce inositol trisphosphate, a potent second messenger inducing internal calcium release and subsequent transmembrane calcium influx (Berridge et al., 1998). We developed a stress model in which tumor cell sublines were isolated after long term continuous exposure to increasing concentrations of CAI. We now report cloning of CAIR 1, CAI stressed 1, a protein nearly identical to BAG 3 (Takayama e converse uk t al., 1999) from CAI stressed cells, and demonstrate novel regulation and function of CAIR 1/BAG 3.
BAG 3 was cloned from a screen for homologs of the bcl 2 binding protein BAG 1 (bcl 2 associated athanogene) family (Takayama et al., 1995, 1999). BAG 1 was identified in a screen for bcl 2 binding proteins and has been shown to inhibit apoptosis and promote tumorigenesis (Stuart et al., 1998; Takayama et al., 1995, 1997). Recent homology cloning identified 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, 1999). Direct binding of BAG 1 to bcl 2 could not be demonstrated upon direct confrontation, however addition of ATP markedly enhanced BAG 1/bcl 2 binding, suggesting the requirement for an HSP 70 intermediate (Takayama et al., 1997). HSP 70 and its constitutive form, HSC 70, are molecular chaperones activated by varied cellular stresses including heat shock, oxygen free radicals, transition heavy metals, inflammation, ischemia, anti neoplastics, oncogenes, and proto oncogenes (Morimoto, 1998). They bind ATP through an ATPase domain and have a separate substrate binding domain (Bukau and Horwich, 1998; Pilon and Schekman, 1999). The site of Hsp70/Hsc70 binding to BAG 1 has been defined as the ATPase domain (Bukau and Horwich, 1998; Takayama et al., 1997); a 1 : 1 molar binding results in reduction in the refolding function of HSP 70 (Stuart et al., 1998). While other functions for BAG 1 such as binding to and activating Raf 1 have been reported, no link between those functions and HSP 70 have been demonstrated. CAIR 1/BAG 3 was recently cloned also as bis (Lee et al., 1999), in a protein interaction cloning procedure using bcl 2 as bait. Lee and coworkers demonstrated a weak anti apoptotic activity of transfected bis and further showed synergy when suboptimal doses of both bis and bcl 2 were transfected, simultaneously. They localized the site of int converse uk eraction of bis to the BH1 domain of bcl 2.
Phospholipase C (PLC ) regulates cytosolic free calcium concentration in response to transmembrane signal transduction, and has been linked to malignant transformation, invasive potential, and angiogenesis (Chang et al., 1997; Khoshyomn et al., 1999; Smith et al., 1998; Turner et al., 1996, 1997; Yang et al., 1998). Several investigators have demonstrated transforming capacity of both full length PLC and the isolated SH2 SH2 SH3 moiety (Bar Sagi et al., 1993; Chang et al., 1997; Schlessinger, 1994; Smith et al., 1998). Its link to calcium homeostasis, transmembrane signal transduction, and transforming potential made it a putative target protein in the calcium influx stress model. We demonstrate that CAIR 1, which is increased in expression in CAI treated cells, is a partner protein for PLC , and forms a ternary complex with HSP 70. This provides a previously unknown link between transmembrane signaling and the HSP 70 system.
Identification and cloning of CAIR 1/BAG 3
A2058 human melanoma cells were exposed chronically to continuing and escalating quantities of the calcium entry blocker, CAI. A subline previously shown to be sensitive in vitro and in vivo to the anti invasive and anti tumorigenic activity of CAI was used as the control for subtractive hybridization. A chemical cross link solution hybridization protocol was used as described to minimize loss of low copy number transcripts (Hampson et al., 1992) and yielded 35 non overlapping clones. A clone containing the 3′ 1200 bp was upregulated by approximately threefold and was chosen for further analysis. CAIR 1, CAI stressed 1, was cloned as a 2.6 kb transcript encoding a 74 kDa protein (Figure 1a), found by GenBank BLAST analysis to differ by 2 amino acids (227Q from a K; 237Q from an R) from BAG 3, a member of the bcl 2 associated athanogene family of bcl 2 binding proteins (Takayama et al., 1995, 1999). Genomic clones from chromosome 10q25 confirm the CAIR 1 sequence. Northern analysis revealed a twofold net increase in expression of CAIR 1/BAG 3 in cells stressed by constant exposure to 10 M CAI (10 Figure 1b) but not in cells exposed to 10 M CAI for 24 h (Figure 1c). Domain analysis of CAIR 1/BAG 3 reveals the previously reported BAG region (Takayama et al., 1999) and WW domain, as well as seven putative protein kinase C (PKC) and five putative CKII phosphorylation sites, multiple potential tyrosine phosphorylation sites, and a series of proline rich PXXP repeats of the SH3 binding type (Pawson and Scott, 1997). No genomic amplification of CAIR 1/BAG 3 gene in the CAI stressed cells was demonstrated by Southern analysis or fluorescent in situ hybridization (data not shown). Broad tissue expression of CAIR 1/BAG 3 was found in adult and embryonic tissues with lowest expression seen in hematopoietic tissues (Figure 1d). Genomic conservation was limited to the higher eukaryotes by zoo blot (not shown, Bios, New Haven, CT, USA). Immunoprecipitation from A2058 cells followed by immunoblot with anti peptide antibodies to CAIR 1/BAG 3 recognized a 74 kDa protein which was increased in quantity in 20 M CAI stressed cells (Figure 1e). No similar induction of CAIR 1/BAG 3 protein was observed in wild type A2058 or MDA 435 human breast cancer cells upon exposure to 10 M CAI for up to 24 h (Figure 1f), consistent with the lack of increased gene expression with acute CAI exposure.
CAIR 1/BAG 3 is phosphorylated in vivo
CAIR 1/BAG 3 has not been demonstrated previously to be a phosphoprotein. Domain analysis of CAIR 1/BAG 3 indicated the presence of potential PKC and CKII phosphorylation sites, as well as putative tyrosine phosphorylation sites. In vivo phosphorylation demonstrated that CAIR 1/BAG 3 is phosphorylated in unstimulated wild type A2058 cells and unstimulated MDA 435 human breast cancer cells (Figure 2). No change in in vivo phosphorylation of CAIR 1/BAG 3 was found after cell treatment with PMA (100 ng/ml) for 30 min to stimulate endogenous PKC or 24 h to down regulate PMA sensitive classical and novel PKC isotypes (Figure 2a) (Hata et al., 1993; Nishizuka, 1988). Incubation with a broad spectrum inhibitor of all classes of PKC isotypes, d ES (10 g/ml for 2 h), markedly reduced CAIR 1/BAG 3 incorporation of 32P o phosphate without effecting total cellular CAIR 1/BAG 3 (Figure 2b). No tyrosine phosphorylation of CAIR 1 was demonstrated in unstimulated A2058 or MDA 435 human breast cancer cells. EGF treatment of MDA 435 cells, which did not effect the quantity of CAIR 1, resulted in tyrosine phosphorylation of CAIR 1/BAG 3 (Figure 2c). CAIR 1/BAG 3 remained restricted to the cytosol upon EGF exposure. A small but statistically significant effect of CAI (10 M, 2 h) was seen on CAIR 1/BAG 3 basal phosphorylation in MDA 435 cells (87 of control, P of control, Pin vivo incorporation of 32P o phosphate could be detected after 1 min exposure to EGF (100 ng/ml; Pn=3; Figure 2d).
In vivo binding of CAIR 1/BAG 3 to Hsp70/Hsc70 is increased by CAI exposure
BAG 1 and BAG 3 have been shown through ex vivo binding experiments to bind to HSP 70 (BAG 1 only) and HSC 70 (both) through the newly described BAG domain (Takayama et al., 1999). We demonstrate in vivo binding of CAIR 1/BAG 3 to Hsp70/Hsc70 and further show its regulation by CAI treatment. An antibody recognizing both molecular forms HSP 70 and HSC 70 was used for the CAIR 1/BAG 3 studies. CAIR 1/BAG 3 co immunoprecipitated Hsp70/Hsc70 under unstimulated conditions from MDA 435 cells (Figure 3). Treatment with 10 M CAI for 90 min, a time that does not alter expression of either CAIR 1/BAG 3 or Hsp70/Hsc70, resulted in a 207% increase in bound Hsp70/Hsc70 to CAIR 1/BAG 3 (Pn=5). Nor did d ES exposure significantly change Hsp70/Hsc70 complex formation (Figure 3b).
CAIR 1/BAG 3 is a binding protein for latent PLC
PLC was chosen as a putative CAIR 1/BAG 3 binding protein because of its potential involvement in the response to CAI stress, because its activation has been shown to be CAI sensitive (Gusovsky et al., 1993), and because it contains an SH3 domain to which the CAIR 1/BAG 3 PXXP regions could target. Unstimulated A2058 and MDA 435 cells were used initially to determine if CAIR 1 interacted with PLC independently of growth factor receptor induced interactions. Untreated cells were lysed under high stringency conditions (Buffer A adjusted to 300 mM NaCl with added 0.1% Triton X 100 and 0.1% SDS) and subjected to immunoprecipitation with anti CAIR 1/BAG 3 Ab 2 or Ab 8. Immunocomplexes were detected by immunoblot for PLC (Figure 4a). Similar results were seen after precipitation with anti PLC antibody and blotting with Ab2 or Ab8; approximately 3 of total cellular PLC is bound to CAIR 1/BAG 3 under basal conditions (data not shown). No association of PLC with CAIR 1/BAG 3 was found by coimmunoprecipitation in MDA 435 cells under standard or high stringency RIPA lysate conditions (Figure 4c), indicating a selectivity of CAIR 1/BAG 3 for PLC .
EGF stimulation results in rapid diss converse uk ociation of PLC from CAIR 1/BAG 3
We hypothesized that phosphorylation of CAIR 1/BAG 3 would regulate its binding to PLC . EGF treatment of MDA 435 cells induced a dose dependent reduction of the binding of PLC to CAIR 1/BAG 3 (Figure 5a). The EGF dose response for this dissociation is log linear (r2=0.99) with an EC50 of 55 ng/ml. EGF induces a rapid loss of PLC binding from CAIR 1/BAG 3, with almost all loss of binding within the first minute (Figure 5b). The potential tyrosine phosphorylation sites in the BAG domain may regulate CAIR 1 Hsp70/Hsc70 complex formation. However, no significant effect of EGF treatment on this complex was observed (n=4, Figure 5c). Since d ES alters the basal phosphorylation status of CAIR 1/BAG 3, we tested its ability to alter binding to PLC . Cell treatment with d ES caused an approximately 150% increase in PLC binding to CAIR 1/BAG 3 in unstimulated cells (PP binding to CAIR 1/BAG 3 (P=0.49 control vs dES/EGF, Figure 6a). Treatment of MDA 435 cells with d ES did not effect the quantity of EGF receptor protein or the tyrosine phosphorylation status of the EGF receptor under the conditions used for the co immunoprecipitation studies. CAI (10 M) incubation for 90 min also increased binding of PLC to CAIR 1/BAG 3 without altering quantity of either protein (Pn=4, Figure 6b).
CAIR 1/BAG 3 binds to PLC and Hsp70/Hsc70 through separate and distinct domains
CAIR 1/BAG 3 binds to the PLC SH3 domain: PLC can interact with partner proteins through multiple domains, including its two SH2 domains and an SH3 domain (Koch et al., 1991; Pawson and Scott, 1997). CAIR 1/BAG 3 was pulled down from unstimulated MDA 435 cell lysates by the GST PLC SH3 domain but not t converse uk he PLC N SH2 or C SH2 domains (Figure 7a). Treatment with EGF of the cells donating CAIR 1/BAG 3 did not alter CAIR 1/BAG 3 binding to the PLC SH3 domain, in contrast to what was observed with whole cell coimmunoprecipitation studies where EGF treatment reduced binding of CAIR 1/BAG 3 to PLC in whole cell lysates (Figure 7b). No binding to SH2 domain constructs was seen after EGF stimulation. Hsp70/Hsc70 has been reported to bind to BAG 1 and BAG 3 through the BAG domain (Takayama et al., 1995, 1997, 1999). No whole cell in vivo co precipitation of PLC and Hsp70/Hsc70 was observed (data not shown). Reprobing of the PLC SH3 domain binding blot for Hsp70/Hsc70 demonstrated that the PLC SH3 construct pulled down Hsp70/Hsc70 from unstimulated and EGF treated cell lysates equally (Figure 7c).
PLC binds to the CAIR 1/BAG 3 PXXP domain: Pull down experiments were done using the PXXP4 region of CAIR 1/BAG 3, a region containing four different PXXP repeats (Figure 1) of SH3 binding type (Koch et al., 1991; Pawson and Scott, 1997). This region does not include the BAG domain. This was done to determine if this region is the PLC binding site on CAIR 1/BAG 3 and thus the potential mechanism through which PLC associates with Hsp70/Hsc70. A second construct containing the three C terminal PXXP repeats was also generated and tested. Both constructs bound PLC from unstimulated MDA 435 cell lysates (Figure 8a) demonstrating that the PLC binding site on CAIR 1/BAG 3 is a functional PXXP SH3 binding site. In contrast to the PLC SH3 experiments and consistent with the whole cell immunoprecipitation experiments, no or minimal binding of PLC from EGF treated lysates to the CAIR 1/BAG 3 PXXP4 domain was found. Further, no Hsp70/Hsc70 could be detected in the GST PXXP4 construct experiments (Figure 8b). These data indicate a novel role for CAIR 1/BAG 3 as a binding partner of latent PLC , that PLC binding to the CAIR 1/BAG 3 SH3 binding domain is regulated by EGF treatment, and that PLC and Hsp70/Hsc70 bind CAIR 1/BAG 3 through different domains.
We demonstrate that CAIR 1/BAG 3 forms a ternary complex between latent PLC and Hsp70/Hsc70. This is the first demonstration of a regulated crosstalk between Hsp70/Hsc70 and the EGFR PLC signaling pathway. The lack of binding of Hsp70/Hsc70 to the CAIR 1/BAG 3 PXXP PLC binding domain suggests that PLC and Hsp70/Hsc70 interact with CAIR 1/BAG 3 at distinct, non overlapping locations on the CAIR 1/BAG 3 molecule. Partner protein binding of both CAIR 1/BAG 3 with PLC and CAIR 1/BAG 3 with Hsp70/Hsc70 is increased by CAI, the nonvoltage gated calcium influx inhibitor used to stress cells for cloning of CAIR 1/BAG 3; d ES, the PKC inhibitor, increased CAIR 1/BAG 3 binding to PLC but not to Hsp70/Hsc70. Release of PLC from CAIR 1/BAG 3 is stimulated by EGF treatment but abrogated by exposure to d ES. CAIR 1/BAG 3 binds in vivo and in vitro to PLC from lysates from unstimulated cells but not from EGF treated cells whereas wild type PLC SH3 pulls CAIR 1/BAG 3 from both unstimulated and EGF treated cells. This suggests that the PLC SH3 domain is a target site in the EGF receptor signaling pathway and argues against tyrosine phosphorylation of CAIR 1/BAG 3 as playing a role in the release of PLC after EGF treatment. These data are consistent with the concept that CAIR 1/BAG 3 is a stimulus to bring Hsp70/Hsc70 into proximity of PLC to allow or facilitate the change in the PLC SH3 domain and subsequent release from CAIR 1/BAG 3 (Figure 9).
CAIR 1/BAG 3 is a protein containing multiple protein interaction motifs and phosphorylation sites (Figure 1), in addition to a conserved BAG domain (Takayama et al., 1999). We have focused on the PXXP region, a putative SH3 binding domain, and have demonstrated that this region binds the latent form of PLC . There is a rapid reduction in PLC binding to CAIR 1/BAG 3 seen after cellular EGF treatment. Release of PLC from CAIR 1/BAG 3 is rapid, nearly complete by 1 min. The kinetics of PLC phosphorylation as previously reported (Wahl et al., 1988; Yang et al., 1994, 1998) and confirmed by us in this model system shows the peak of phosphorylation occurring between 1 and 2 min. Maintenance of PLC binding is seen when a wild type PLC SH3 construct is used but a marked reduction in partner protein complex is observed when the CAIR 1/BAG 3 PXXP region is presented with lysates from EGF treated cells. This implicates a change(s) in the conformation of the PLC SH3 domain is necessary for the process of CAIR 1/BAG 3 release of PLC upon EGF stimulation.
The BAG domain of CAIR 1/BAG 3 contains two tyrosine and three threonine residues that are not conserved from the BAG domain of BAG 1 (Takayama et al., 1999). We demonstrate endogenous binding of CAIR 1/BAG 3 to Hsp70/Hsc70 and show an increase in complex formation when cells are stressed for 90 min by exposure to 10 M CAI. Previous studies have shown a BAG 3 interaction with Hsp70/Hsc70 using BAG GST pull down experiments, from BAG 3 expressed in intact cells, and using a kDa recombinant BAG 3 fragment (Takayama et al., 1999). These studies demonstrated that the 24 kDa BAG 3 fragment had an attenuated binding to Hsc70 in a BIA Core assay and that Hsc70 dissociated more rapidly from the BAG 3 fragment BIA Core chip than did the purified BAG 1 or BAG 2 molecules. Our demonstration of endogenous binding of CAIR 1/BAG 3 to Hsp70/Hsc70 confirms that the interaction of CAIR 1/BAG 3 occurs in intact cells. The increase seen after