Abstract
Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr(286) phosphorylation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr(286)-autophosphorylated CaMKIIalpha. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146-493), because of the selective inhibition of the PP1gamma(1) isoform. The fraction of PP1 activity that is insensitive to Nb-(146-493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146-493) (i.e. PP1gamma(1)). However, total striatal levels of PP1gamma(1) or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1gamma(1) with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1gamma(1) interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.
Highlights
Extracts were prepared from different batches of animals 3– 4 weeks or 10 –11 months after lesion surgery to assess the consequences of long term dopamine depletion
Nb-(146 – 493) is a glutathione S-transferase (GST) fusion protein that inhibits isolated brain phosphatase 1 (PP1)␥1 catalytic subunit Ϸ20-fold more potently than it inhibits isolated brain PP1 catalytic subunit [12]. To determine whether this selectivity might be useful in discriminating PP1 isoform activities in tissue extracts, we investigated the effects of Nb-(146 – 493) (1 M) on the activities of striatal PP1␥1 and PP1 holoenzyme complexes isolated by immunoprecipitation
We show here that chronic dopamine depletion significantly decreases striatal PP1 activity apparently because of regulation of the interaction between the PP1␥1 isoform and spinophilin
Summary
6-OHDA Lesion Surgery—Male Sprague-Dawley rats (Harlan; Indianapolis, IN) were housed under a 12:12 light:dark cycle with food and water freely available. Data reported here were obtained from six batches of 6 – 8 rats sacrificed at times ranging from 3 weeks to 11 months following lesion surgery to assess the long term consequences of dopamine depletion. Protein Phosphatase Activity Assays—Phosphatase activities were assayed using [32P]phosphorylase a, [32P]casein, or Thr286-autophosphorylated CaMKII␣ substrates, as described previously [3]. For co-immunoprecipitations, intact (control) and lesioned (dopamine depleted) dorsolateral striatal punches (see above) were homogenized separately in the same buffer containing phosphatase inhibitors (50 mM sodium fluoride, 50 mM -glycerophosphate, 20 mM sodium pyrophosphate). Aliquots (0.5 ml) of diluted/precleared soluble extracts were immunoprecipitated using rabbit antibodies to PP1␥1 (5 l of affinity-purified), spinophilin (5 l of protein A-purified), or control IgG in IP Buffer containing the same phosphatase inhibitors. Statistical Analyses—Statistical comparisons were made by paired or unpaired Student’s t test or Wilcoxon signed rank test, as appropriate
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