Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prost aglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma m embrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (P GE) and activated inositol phosphate (n-IP) to cyclic PIP. The K-m values o f the enzyme for the substrates PGE and n-IP are in the micromolar range. T he plasma membrane-bound synthase is activated by fluoride, by the stable G TP analog GMP-PNP by protamine or biguanide, by noradrenaline, and by insul in. The activation by protamine or biguanide and fluoride (10 mM) is additi ve, which may indicate the presence of two different types of enzyme, compa rable to phospholipase C-beta and phospholipase C-gamma. Plasma membrane-bo und cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibit or tyrphostin B46 with an IC50 of 1.7 mu M. However, the solubilized and ge l-filtrated enzyme is no longer inhibited by tyrphostin, indicating that th e activity of cyclic PIP synthase is connected with the activity of a membr ane-bound protein tyrosine kinase, Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold, Protein tyro sine phosphatase inhibitors [vanadate, fluoride (50-100mM)] stabilize the e nzyme activity, but protease inhibitors do not, indicating that inactivatio n of the enzyme is connected with protein tyrosine dephosphorylation. Cycli c PIP synthase is present in all tissues tested, like brain, heart, intesti ne, kidney, liver, lung, skeletal muscle, spleen, and testis, Apart from li tter, cyclic PIP synthase activity in most tissues is rather low, but it ca n be increased up to 5-fold when protein tyrosine phosphatase inhibitors li ke vanadate are present in the homogenization buffer. Preincubation of cycl ic PIP synthase of liver plasma membranes with the tyrosine kinase src kina se causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase:in intact cells. The data indicate that cyclic PIP synthase can be activated by two separat e mechanisms: by a G protein or by protein tyrosine phosphorylation.