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Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

ABSTRACT: DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.

SUBMITTER: Desdouits F

PROVIDER: S-EPMC1219129 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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Project description:Protein phosphatases 1 and 2A (PP1 and PP2A) were identified in a variety of plant cells and found to be particulate or soluble depending on the species. In extracts prepared from oilseed-rape seeds these enzymes were associated with microsomes and more rapidly sedimenting fractions, whereas in wheat leaf extracts they were largely microsomal, the remainder being present in the soluble fraction. In pea leaf and carrot cell extracts PP1 and PP2A were almost entirely soluble. No PP1 or PP2A activity was associated with the membranes or stroma of chloroplasts in oilseed-rape seeds, pea leaves and wheat leaves. An Mg2(+)-dependent okadaic acid-insensitive protein phosphatase that resembles protein phosphatase 2C (PP2C) was detected in carrot cells, pea leaves and wheat leaves, but not in oilseed-rape seeds. In wheat leaf extracts PP2C was mostly present in the soluble fraction, a different location from PP1 or PP2A. The rapid inactivation of the cytosolic enzyme quinate dehydrogenase (QDH) in a fraction prepared from light-grown carrot cells was completely blocked by either okadaic acid or microcystin (two potent and specific inhibitors of PP1 and PP2A), whereas inhibitor 2 (a specific inhibitor of PP1) inhibited inactivation by only about 10%. Addition of the purified PP2A catalytic subunit from mammalian skeletal muscle increased the rate of QDH inactivation, whereas addition of mammalian PP1 did not. It is concluded that PP2A is the major enzyme responsible for dephosphorylating (inactivating) QDH in carrot cells. These observations indicate that okadaic acid and microcystin may be useful for identifying other plant processes that are controlled by phosphorylation/dephosphorylation mechanisms. Okadaic acid did not prevent the rapid inactivation of phosphoribulokinase or activation of glucose-6-phosphate dehydrogenase in a fraction prepared from light-grown pea leaves, and addition of the purified catalytic subunits of PP1 and PP2A did not accelerate either process. These observations, in conjunction with the absence of PP1 and PP2A activity in chloroplasts, suggest that these phosphatases are not involved in the regulation of chloroplast metabolism.

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Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

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