title: CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-gamma and Hsp70/Hsc70.
abstract: CAIR-1/BAG-3 forms an EGF-regulated ternary complex with Hsp70/Hsc70 and latent phospholipase C-gamma (PLC-gamma). 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 (P<0.007). CAIR-1/BAG-3 is phosphorylated in 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-gamma. PLC-gamma 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-gamma 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-gamma, but not its N-SH2 or C-SH2 domains. Confirming the SH3 interaction, PLC-gamma 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-gamma as was seen in intact cells. Hsp70/Hsc70 was brought down by the PLC-gamma 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.
authors.affiliation: Molecular Signaling Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland, MD 20892, USA.
authors.forename: H
authors.initials: H
authors.lastname: Doong
authors.forename: J
authors.initials: J
authors.lastname: Price
authors.forename: Y S
authors.initials: YS
authors.lastname: Kim
authors.forename: C
authors.initials: C
authors.lastname: Gasbarre
authors.forename: J
authors.initials: J
authors.lastname: Probst
authors.forename: L A
authors.initials: LA
authors.lastname: Liotta
authors.forename: J
authors.initials: J
authors.lastname: Blanchette
authors.forename: K
authors.initials: K
authors.lastname: Rizzo
authors.forename: E
authors.initials: E
authors.lastname: Kohn
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line / Q000187:drug effects; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010766:Phosphorylation; D014230:Triazoles / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human; D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D014230:Triazoles; D004815:Epidermal Growth Factor; C062058:carboxyamido-triazole; D010738:Type C Phospholipases; D051966:Phospholipase C gamma