Though progesterone-induced maturation has been studied extensively in Xenopus oocytes, the mechanism whereby the prophase block arrest is released is not well understood. The current hypothesis suggests that a reduction in cAMP and subsequent inactivation of cAMP-dependent protein kinase is responsible for reentry into the cell cycle. However, several lines of evidence indicate that maturation can be induced without a concomitant reduction in cAMP. We show that the mass of diacylglycerol in whole oocytes and plasma membranes decreases 29% and 10% respectively, within the first 15 sec after the addition of progesterone. Diacylglycerol in plasma membranes further decreased 59% by 5 min. We also show that the protein kinase C inhibitors sphingosine and staurosporine can induce oocyte maturation. In addition, the synthetic diglyceride, DiC8, and microinjected PKC can inhibit or delay progesterone-induced maturation. These results together suggest that a transient decrease in protein kinase C activity may regulate entry into the cell cycle. The mechanism whereby DAG is decreased in response to progesterone is unclear. Initial studies show that progesterone leads to a decrease in IP3 suggesting that progesterone may act by reducing the hydrolysis of PIP2. On the other hand, progesterone caused a decrease in the amount of [3H]arachidonate labelling in DAG during the same time suggesting that progesterone may stimulate lipase activity. The relationship between postulated changes in the PKC pathway and those hypothesized for the PKA pathway are discussed.
Isolated membranes of soybean incorporate 32P from y-I32PIATP in vitro. The incorporation was rapid and did not require added calcium. When displayed on 10% sodium dodecyl sulfate-polyacrylamide gels, several protein bands were revealed. An apparent auxin (2,4-dichlorophenoxyacetic acid) stimulation of 32p incorporation into material from membrane vesicles insoluble in trichloroacetic acid-perchloric acid may be reflected partly in enhanced incorporation into protein bands with apparent molecular weights of 45,000 and 50,000. Additionally, a low molecular weight component was sometimes observed where incorporation was stimulated 2-to 3-fold by auxin. However, protein-bound radioactivity represented only a small fraction of the total radioactivity of the acid-insoluble material. Other labeled constituents, not retained on the gels, may contribute to the apparent, rapid (10 s or less) auxin response of the isolated membranes. Stimulation of incorporation into the low molecular weight component was given by diglyceride plus calcium, constituents known to augment protein kinase activities in other systems.
MATERIALS AND METHODSSoybean seeds were soaked in deionized H20 for 4 to 6 h, planted in moist vermiculite, and grown 4 d in the dark. The etiolated hypocotyls were harvested and used for membrane isolations.Hypocotyl segments consisting ofapproximately 2-cm sections taken 5 mm below the cotyledons were homogenized using a mechanized razor blade chopper (5) in 15 mM Tris (pH 7.2). The homogenates were filtered through Miracloth (Chicopee 10 8 Ongoing studies from our laboratory suggest that phospholipid breakdown and headgroup turnover are stimulated in isolated membrane vesicles of etiolated hypocotyls of soybean treated with the synthetic auxin, 2,4-D. Accompanying the phospholipid breakdown is the release of essentially stoichiometric amounts of membrane-bound calcium (2). In subsequent experiments to monitor incorporation of phosphate from y-[32P]ATP into phosphatidic acid, a small but rapid auxin stimulation of 32P incorporation into acid-insoluble material was observed. Subsequent examination of the acid-insoluble material after 10 s of incorporation in the presence of auxin, revealed phosphorylation of two bands of apparent mol wt of 45,000 and 50,000 and low mol wt material running just behind the dye front on 10% polyacrylamide gels with SDS. However, the materials recovered from the gels accounted for only a small fraction of the total acid precipitable radioactivity.The findings demonstrate that isolated membrane vesicles are capable of yielding a very rapid phosphorylation response of membrane proteins in the order of 10 s or less. An apparent auxin response, although variable, paralleled in magnitude the growth response to auxin in some expenments.
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