Abstract
Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.
Highlights
Phospholipase C-3 (PLC3) is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC3
The Calmodulin Binding Site—Binding site search and analysis site The Calmodulin Target Database2 was used for computer analysis of the PLC3 protein sequence
Three putative calmodulin-binding sites were identified in the full-length PLC3 protein sequence by hydropathy, ␣-helical propensity, residue weight, residue charge, hydrophobic residue content, helical class, and occurrence of key residues
Summary
Materials—KN-62, KN-93, KN-92, W-13, and fluphenazine were obtained from Calbiochem (San Diego, CA). Positive clones in the pACT2 vector were co-transformed into the S. cerevisiae strain PL3␣ along with pBL1 (a histidine-selectable yeast expression plasmid) containing the PH-EF-PLC3 fragment subcloned in-frame with the estrogen receptor DNA-binding domain. Anti-PLC isozyme-selective polyclonal antibodies were added to soluble (cytosol) and membrane extract fractions at a 1:200 dilution and incubated at 4 °C overnight with continuous inversion. For preparation of cell lysates for CaM-Sepharose 4B binding assay, 1321N1 cells were plated at 3 ϫ 106 cells/10-cm plate and grown for 3 days at 37 °C with 5% CO2 in DMEM ϩ 10% fetal bovine serum and antibiotics. The lysate was centrifuged at 500 ϫ g, 4 °C, for 5 min to pellet nuclei and intact cells prior to incubation with CaM-Sepharose 4B beads as described below. Mitochondrial proteins are often considered to be false positives in yeast two-hybrid screens and were not pursued further
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