The

Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β‐peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen‐activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12‐M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC‐independent pathway, increased sAPP secretion mainly through a MAP kinase‐independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.

Although protein phosphatases appear to be highly controlled in intact cells, relatively little is known about the physiological regulation of their activity. a . DARPP-32 is also phosphorylated in vivo on serine residues which are phosphorylated in vitro by casein kinase I and casein kinase II (5, 6). Phosphorylation of bovine DARPP-32 by casein kinase II converts the protein into a better substrate for PKA (5). Phosphorylation, in vitro and in vivo, of rat DARPP-32 on Ser-137 by casein kinase I induces an unusual shift in the migration of the protein in SDS/PAGE which could be due to a conformational change persisting in the presence of SDS (6). This increase in electrophoretic mobility provides a convenient means to identify DARPP-32 phosphorylated by casein kinase I in vivo and in vitro. Phosphorylation of Ser-137 has no effect on the ability of DARPP-32 to serve as a substrate for PKA and casein kinase II (F.D. and J.-A.G., unpublished data), nor does it alter the ability of PKA-phosphorylated DARPP-32 to inhibit PPlc (6). Here we report that phosphorylation of DARPP-32 on Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by calcineurin. MATERIALS AND METHODSMaterials. Recombinant rat DARPP-32 was produced in Escherichia coli and purified as described (7). PKA from rabbit skeletal muscle or calf heart (8) and casein kinase I from calf thymus (9) were purified as described. Casein phosphorylated by PKA was prepared as described (10) Analysis of DARPP-32 Phosphorylation in Nigral Slices. Rat substantia nigra slices (11) were preincubated in RPMI 1640 medium for 1 hr at 25°C in the presence of 1 ,uM okadaic acid. 8-Bromo-cAMP (1 mM) or vehicle was added for a further 10 min. Stimulation was stopped by removing the medium, and slices were quickly frozen in liquid nitrogen. Tissues were homogenized in boiling 1% (wt/vol) SDS in water by sonication and subjected to SDS/PAGE (11). was analyzed by sequential immunoblotting, first with a monoclonal antibody which reacts only with DARPP-32 phosphorylated on , and then with a mixture of two monoclonal antibodies (C24-5a and C24-6a) which react with DARPP-32 independent of its state of phosphorylation (13). Immunoreactivity was detected with an enhanced chemiluminescence method (Amersham) using a horseradish peroxidasecoupled donkey anti-mouse secondary IgG antibody. The relative amounts of immunoreactive bands were quantified by computer-assisted densitometric measurement of the films. Recombinant DARPP-32 phosphorylated in vitro was used as a standard to estimate the stoichiometry of phosphorylation of DARPP-32 in nigral slices.In Vitro Phosphorylation and Dephosphorylation. In vitro phosphorylation by PKA or casein kinase I was carried out as described (6, 7). DARPP-32 was phosphorylated by PKA and by casein kinase I to a stoichiometry of -1 and -2 mol/mol, respectively, except when indicated. Stoichiometry of phosphorylation was followed by adding radioactive [y-32P]ATP.