of Work: In FY97 Dr. Romeo in collaboration with Dr. Rossie identified the ser/thr-directed protein phosphatase, PP5, as an arachidonic acid stimulated enzyme in brain and GH4C1 cells. We have tested the involvement of PP5 in the stimulation of BK channel activity by somatostatin through arachidonic acid metabolism in GH cells with two molecular reagents created by Dr. Romeo and tested by Dr. Wang on GH4C1 cells: a dominant negative molecule made from the amino terminal regulatory domain containing multiple tetratricopeptide repeats (TPR) of PP5 andn the green fluorescent protein; and a toxin-resistant P5 made by a point mutation in the toxin binding site on the catalytic domain of the phosphatase. Overexpression of the TPR domain prevents (completely in 25% of cells; partially in 50%; and not at all in 25%) BK channel stimulation by somatostatin in GH4 cells. In contrast, transfection of GH4C1 cells with the mutant, but not the wild type enzyme, rescues somatostatin signaling in the presence of concentrations of okadaic acid in tenfold excess of those which normally block somatostatin action completely.
