Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the postmitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

The transformed monocyte͞macrophage cell line J774.2 undergoes apoptosis when treated for 48 h with competitive inhibitors of cyclooxygenase (COX) isoenzymes 1 and 2. Many of these nonsteroid antiinf lammatory drugs (NSAIDs), but in particular diclofenac, induce during this time period a COX activity that coincides with a robust induction of COX-2 protein. Induction of this activity requires high, apoptosis-inducing concentrations of diclofenac (>100 M). Prolonged treatment of J774.2 cells with lower doses of diclofenac inhibits COX activity, indicating that diclofenac is a time-dependent, pseudoirreversible inhibitor of COX-2. It is difficult to wash out the inhibition. However, the activity evoked by high concentrations of diclofenac has a profoundly distinct COX active site that allows diclofenac, its inducer, to be washed readily from its active site. The diclofenac-induced activity also has the unusual property of being more sensitive to inhibition by acetaminophen (IC 50 ‫؍‬ 0.1-1.0 mM) than COX-2 induced with bacterial lipopolysaccharide. Moreover, relative to COX-1 or COX-2, diclofenac-induced enzyme activity shows significantly reduced sensitivity to inhibition by diclofenac or other competitively acting nonsteroid antiinf lammatory drugs (NSAIDs) and the enzyme activity is insensitive to aspirin. If the robust induction of COX-2 observed is responsible for diclofenac-induced COX enzyme activity, it is clear that COX-2 can, therefore, exist in two catalytically active states. A luciferase reporter-construct that contains part of the COX-2 structure and binds into the membrane showed that chronic diclofenac treatment of fibroblasts results in marked mobilization of the fusion protein. Such a mobilization could result in enzymatically distinct COX-2 populations in response to chronic diclofenac treatment.The cloning of cyclooxygenase-2 (COX-2) in 1991 (1-3) explained many long-standing anomalies relating to the previous assumption of a single COX enzyme. However, not all of the problems have been solved by the characterization of two COX enzymes originating from different genes. One such problem is the effect of acetaminophen, which, unlike nonsteroid antiinflammatory drugs (NSAIDs), produces analgesia and antipyresis but has little effect on inflammation. The existence of additional COX enzymes was therefore postulated (4).Acetaminophen is a weak inhibitor of COX-1 and COX-2, which displays some selectivity toward COX enzymes from different organs. Thus, it is a more potent inhibitor of COX from dog and rabbit brain than that of dog spleen (4). The activity of acetaminophen in vitro also depends on the addition of cofactors. In the presence of glutathione and hydroquinone, acetaminophen inhibited COX activity of bull seminal vesicle microsomes but, in the absence of these cofactors, stimulated that of bovine and ram seminal vesicle microsomes (5). After the recognition of COX-1 and COX-2, by using cultured whole cells, acetaminophen demonstrated a low inhibitory potency for COX enzymes. Only IC 3...

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