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CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-γ and Hsp70/Hsc70

Oncogene volume 19, pages 4385–4395 (2000)Cite this article

Abstract

CAIR-1/BAG-3 forms an EGF-regulated ternary complex with Hsp70/Hsc70 and latent phospholipase C-γ (PLC-γ). 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-γ. PLC-γ 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-γ 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-γ, but not its N-SH2 or C-SH2 domains. Confirming the SH3 interaction, PLC-γ 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-γ as was seen in intact cells. Hsp70/Hsc70 was brought down by the PLC-γ 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.

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Abbreviations

CAI:

carboxyamido-triazole

EGF:

epidermal growth factor

PMA:

phorbol 12-myristate 13-acetate

d-ES:

d-erythrosphingosine

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Acknowledgements

The authors thank Ms V Kulpa for technical support and Dr H Krutsch for peptide production. This work was supported in part by the G Harold and Leila Y Mathers Charitable Foundation and the Florence and Max Kohn Fund.

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  1. Molecular Signaling Section, Laboratory of Pathology, National Cancer Institute, Bethesda, 20892, Maryland, MD, USA

    Howard Doong, John Price, Young Sook Kim, Christopher Gasbarre, Julie Probst, Lance A Liotta, Jay Blanchette, Kathryn Rizzo & Elise Kohn

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Doong, H., Price, J., Kim, Y. et al. CAIR-1/BAG-3 forms an EGF-regulated ternary complex with phospholipase C-γ and Hsp70/Hsc70. Oncogene 19, 4385–4395 (2000). https://doi.org/10.1038/sj.onc.1203797

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