Effects of Inhibitors in Cell Based Experiments

The myriad biological effects of these cell-permeable inhibitors have provided many compelling demonstrations that reversible phosphorylation of serine/threonine residues is an all-pervasive mechanism of cellular control.

When protein phosphatases are inhibited, phosphate is trapped in phosphorylated substrates and accumulates if the relevant protein kinases are at least slightly active. Thus, use of protein phosphatase inhibitors in combination with protein kinase inhibitors and other effectors has provided clues about many signaling pathways. By way of example, protein phosphatase inhibitors cause sustained backward swimming of Paramecium in response to depolarizing stimuli (but only in the presence of external Ca2+ [2]), activate some and block other antifungal defense responses in plants [3], and promote apoptosis of mammalian cells [4]. Interestingly, fibroblasts that are resistant to okadaic acid-induced apopto-sis have been selected from a population of cells expressing a human cDNA library which provides a novel approach to identifying those components of the apoptotic machinery for which phosphorylation is deregulated by the inhibitor [4].

Of course, the major limitation in using protein phos-phatase inhibitors inside cells is that they inhibit many closely related enzymes, albeit with different relative potency (see Fig. 1), making dissection of cellular functions for each individual protein phosphatase difficult or impossible. The best we can do is to provisionally assign roles for PP1 when tautomycin has more potent effects than okadaic acid [5], or implicate PP2A, PP4, or PP5 when fostriecin is most effective [6].

One way around the problem of discriminating among homologous family members might be to replace the natural

Protein Kinase Drug Target

Figure 1 Naturally occurring inhibitors of PP1, PP2A, and related enzymes in the PPP family. None of these toxins is an effective inhibitor of PP2B (the rapamycin and FK506 drugs that target PP2B are not considered in this chapter). Representatives of each toxin are shown, although variants exist in Nature; for example, more than 60 congeners of microcystin exist. The toxins generally bind to the enzyme with nanomolar and subnanomolar affinities, except for cantharidin, which operates in the micromolar range.

Figure 1 Naturally occurring inhibitors of PP1, PP2A, and related enzymes in the PPP family. None of these toxins is an effective inhibitor of PP2B (the rapamycin and FK506 drugs that target PP2B are not considered in this chapter). Representatives of each toxin are shown, although variants exist in Nature; for example, more than 60 congeners of microcystin exist. The toxins generally bind to the enzyme with nanomolar and subnanomolar affinities, except for cantharidin, which operates in the micromolar range.

enzymes in cells with forms that are more or less toxin sensitive. Another would be to use the natural toxins as "leads" for the synthesis of more specific inhibitors.

As well as soothing the frustrations of cell biologists, there are also medical reasons to seek specific inhibitors. Fostriecin is reported to confer an antitumor effect [7] at concentrations that probably inhibit PP2A, PP4, and PP1 in cells. In contrast, okadaic acid and microcystin are potent tumor promoters [8]. Which actions underpin the pro- and antitumor properties of these toxins? Can more specific cancer therapies with fewer side effects than fostriecin be designed?

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