THE ROLE OF CARBOXYL-TERMINAL BASIC-AMINO-ACIDS IN G(Q)ALPHA-DEPENDENT ACTIVATION, PARTICULATE ASSOCIATION, AND NUCLEAR-LOCALIZATION OF PHOSPHOLIPASE C-BETA-1

The phospholipase C (PLC)-beta isozymes differ from the PLC-gamma and PLC-delta isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by alpha subunits of the G(q) class of G proteins, associate with the particulate subcel lular fraction, and are present in the nucleus. Most of the COOH-termi nal domain of PLC-beta isozymes is predicted to be helical, and three regions in this domain, PLC-beta 1 residues 911-928 (region 1), 1055-1 072 (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 ba sic residues. The role of the COOH terminus and the clustered basic re sidues in PLC-beta 1 was investigated by either truncating the entire COOH-terminal domain (mutant Delta C) or replacing two or three cluste red basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The Delta C mutant no longer showed the ability to be activated by G(q) alpha, to transl ocate to the nucleus, or to associate with the particulate fraction. S ubstitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by G(q) alpha, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the clus ter of lysine residues 914, 921, and 925 in region 1 had the most mark ed effect, reducing G(q) alpha-dependent activity to 10% of that of wi ld type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted wit h isoleucine, behaved like the wild-type enzyme in showing an approxim ately equal distribution between cytoplasm and nucleus; only 12% of th e region 2 mutant was present in the nucleus. None of the basic cluste rs appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-beta 1 in the particulate f raction by some extent, suggesting that all the basic residues contrib ute to the association, presumably by interacting with acidic residues in the particulate fraction, Membrane localization of PLC-beta isozym es is therefore likely mediated by both the COOH-terminal domain and t he pleckstrin homology domain, the latter of which is known to bind ph osphatidylinositol 4,5-biphosphate.