The role of carboxyl-terminal basic amino acids in G(q)α-dependent activation, particulate association, and nuclear localization of phospholipase C-β1

Abstract

The phospholipase C (PLC)-β isozymes differ from the PLC-γ and PLC-δ isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by α subunits of the G(q) class of G proteins, associate with the particulate subcellular fraction, and are present in the nucleus. Most of the COOH-terminal domain of PLC-β isozymes is predicted to be helical, and three regions in this domain, PLC-β1 residues 911-928 (region 1), 1055-1072 (region 2), and 1109-1126 (region 3), contain a high proportion of basic residues that are highly conserved. Projection of the sequences of these three regions in helical wheels reveals clustering of the basic residues. The role of the COOH terminus and the clustered basic residues in PLC-β1 was investigated by either truncating the entire COOH-terminal domain (mutant ΔC) or replacing two or three clustered basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The ΔC mutant no longer showed the ability to be activated by G(q)α, to translocate to the nucleus, or to associate with the particulate fraction. Substitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by G(q)α, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the cluster of lysine residues 914, 921, and 925 in region 1 had the most marked effect, reducing G(q)α-dependent activity to 10% of that of wild type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted with isoleucine, behaved like the wild-type enzyme in showing an approximately equal distribution between cytoplasm and nucleus; only 12% of the region 2 mutant was present in the nucleus. None of the basic clusters appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-β1 in the particulate fraction by some extent, suggesting that all the basic residues contribute to the association, presumably by interacting with acidic residues in the particulate fraction. Membrane localization of PLC-β isozymes is therefore likely mediated by both the COOH-terminal domain and the pleckstrin homology domain, the latter of which is known to bind phosphatidylinositol 4,5-biphosphate.