Atsuko Horiuchi

Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2.

Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 ∼1 nm) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6–11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1–8 (I2-(9–204)) or 100–204 (I2-(1–99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9–13 (I2-(14–204)) resulted in a large decrease in inhibitory potency (IC50 ∼800 nm), whereas further COOH-terminal deletion (I2-(1–84)) caused a moderate decrease in inhibitory potency (IC50∼10 nm). Within residues 9–13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6–20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6–38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1–120) but not I2-(85–204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14–84 of inhibitor-2 and residues 6–38 of phospho-DARPP-32 share elements that are important for interaction with PP1.

Phosphorylation of Protein Phosphatase Inhibitor-1 by Cdk5

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation ofN-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.

Protein phosphatase 1 regulation by inhibitors and targeting subunits

Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli. This preparation is limited by several key differences in its properties compared with native PP1. In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, Mr 32,000). Mutations at Y272 in the β12/β13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2. Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased Km for phosphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1. Replacement of residues 274–330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS). More limited alterations in residues in β12, β13, and β14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.

Nerve growth factor controls GAP-43 mRNA stability via the phosphoprotein ARPP-19

The membrane phosphoprotein GAP-43 is involved in axon growth and synaptic plasticity. In PC12 pheochromocytoma cells, induction of a neuronal phenotype by nerve growth factor (NGF) is accompanied by a marked increase in GAP-43 levels. NGF regulates GAP-43 expression by altering the half-life of its mRNA. We report here that the phosphoprotein ARPP-19 mediates this regulation. In an NGF-dependent manner, ARPP-19 bound to a region in the 3′ end of GAP-43 mRNA previously found to be important for regulating the half-life of the mRNA. Overexpression of wild-type ARPP-19 in PC12 cells increased the NGF-dependent expression of a reporter construct linked to the critical 3′ region of GAP-43 mRNA. Mutation of serine 104, the site of phosphorylation by protein kinase A in ARPP-19, to either alanine or aspartate abolished this regulation in PC12 cells. These findings demonstrate that ARPP-19 is an important link between NGF signaling and post-transcriptional control of neuronal gene expression.

Elongation factor-2 phosphorylation and the regulation of protein synthesis by calcium.

Eukaryotic protein synthesis is a highly regulated process with many of the key proteins being controlled by phosphorylation (for reviews, see Rhoads 1993; Redpath and Proud 1994; Jefferies and Thomas 1996; Merrick and Hershey 1996; Sonenberg and Gingras 1998). Several initiation factors, as well as ribosomal proteins and aminoacyl-tRNA synthetases, are phosphoproteins. While in some cases the physiological role of phosphorylation of these factors is well characterized, in others it remains less clear. In the latter category is elongation factor-2 (eEF2), which catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome. eEF2 is phosphorylated by a highly conserved and specific Ca2+/calmodulin-dependent kinase, termed EF2 kinase (also known as CaM kinase III) at Thr56 and Thr58 (Palfrey and Nairn 1995; Nairn and Palfrey 1996). In vitro, Thr56 is more rapidly phosphorylated by EF2 kinase, and this is sufficient to result in inhibition of protein synthesis. Dephosphorylation of eEF2 by the protein phosphatase 2A (PP2A) results in reactivation of protein synthesis. By implication, similar events in vivo should regulate the rate of elongation and therefore the overall efficiency of protein svnthesis.

ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins

ARPP‐16 and ARPP‐19 are closely related cAMP‐regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP‐16 is enriched in dopamine‐responsive medium spiny neurons in the striatum, while ARPP‐19 is ubiquitously expressed. ARPP‐19 is highly homologous to α‐endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform‐specific antibodies, we now show that ARPP‐19 is composed of at least two differentially expressed isoforms (termed ARPP‐19 and ARPP‐19e/endosulfine). All ARPP‐16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP‐16 and ARPP‐19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP‐16/19/19e was used to examine the phosphorylation of ARPP‐16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP‐16 was increased in response to activation of D1‐type dopamine receptors, and decreased in response to activation of D2‐type dopamine receptors. In non‐neuronal cells, ARPP‐19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP‐16/19 proteins constitute a family of PKA‐dependent intracellular messengers that function in all cells. The high levels of ARPP‐16 in striatal neurons and its bi‐directional regulation by dopamine suggest a specific role in dopamine‐dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.

Dexamethasone coordinately inhibits plasminogen activator gene expression and enzyme activity in porcine kidney cells

The peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) enzyme activity in cultured LLC-PK1 pig kidney cells. This induction occurs as a consequence of transcriptional activation of the uPA gene. Treatment with the synthetic glucocorticoid hormone, dexamethasone, was found to inhibit calcitonin induction of uPA enzyme activity by as much as 80%. The inhibitory effect of dexamethasone was attributed to at least two mechanisms: induction of an inhibitor of uPA enzyme activity, and reduction in uPA mRNA levels. Study on reduction of uPA mRNA levels showed that dexamethasone significantly reduced the transcription rate of the calcitonin-induced uPA gene, without affecting the half-life of uPA mRNA. Although dexamethasone has been reported to induce inhibitors of plasminogen activator enzyme activity and to inhibit transcription of various genes, the system described here appears novel in that both actions are coordinated.

Preparation and characterization of recombinant protein phosphatase 1.

Publisher Summary The chapter describes preparation and characterization of recombinant protein phosphatase 1 (PP1). Various methods for preparation of PP1C in bacteria, and methods for preparation of PP1C in Sf9 cells using the baculovirus expression system are presented. The chapter discusses the preparation of phosphorylated and thiophosphorylated (dopamine- and cAMP-regulated phosphoprotein,M r 32,000)DARPP-32. Native PP1C is purified from rabbit skeletal muscle, using sequential chromatography on DEAE-cellulose, heparin-Sepharose, phenyl-Sepharose, Sephacryl S-200 and Mono-Q resins in order to recombinant PP1C. DARPP-32 and inhibitor-2 are prepared from E. coli as described. GST-tagged spinophilin and GSTPNUTS are prepared from E. coli as described. The chapter discusses Dephosphorylation of p-nitrophenyl phosphate (pNPP) and tyrosine-phosphorylated myelin basic protein. The chapter describes two different procedures that are used to prepare recombinant PP1, using the baculovirus method. Several different affinity chromatography procedures are used that utilized the His6 and GST tags attached to recombinant PP1. The chapter discusses immunoblotting of recombinant PP1C.

ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase)

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We find that the protein ARPP-16, which is highly expressed in striatal medium spiny neurons, acts as a selective inhibitor of certain forms of the serine/threonine protein phosphatase, PP2A, when phosphorylated by the kinase, MAST3. Under basal conditions, ARPP-16 is phosphorylated by MAST3 to a very high stoichiometry. However, the actions of MAST3 are antagonized by dopamine and cAMP-regulated signaling leading to disinhibition of ARPP-16 and increased PP2A action.