Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.

Autophosphorylation of Thr286 in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-956). We have engineered heteromeric holoenzymes comprising distinct "kinase" and "substrate" subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated.

Characterization of the phosphorylation site of PP59, a substrate for cyclic AMP-dependent protein kinase that is enriched in the synaptic membrane fraction of rat cerebellum.

We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified 32P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.

Citizen participation and empowerment: the case of local environmental hazards.

Local environmental hazards place millions of citizens at risk of physical, emotional, and financial harm. While the discovery of such hazards can be fundamentally disempowering for individuals and communities, few scholars have examined the dynamics of empowerment in this context. We explore the relationships among forms of empowerment, citizen participation, and local environmental hazards, and offer a model of the processes of empowerment and disempowerment appropriate to a broad range of citizen issues. On the basis of this analysis we recommend a partnership approach to community decision making that is designed both to reduce the likelihood that local environmental hazards will develop and to minimize the disempowering impact of any threats that do occur.

Identification of a 59-kDa Substrate for cAMP-Dependent Protein Kinase That Is Enriched in Rat Cerebellar Membranes.

Following incubation of rat brain membranes with the catalytic subunit of cAMP-dependent protein kinase and [(32)P]ATP, a previously unreported phosphoprotein, pp59, was found to be enriched in cerebellar synaptic plasma membrane preparations, but not in those prepared from cerebral cortex, hippocampus, olfactory bulb, or striatum. This protein, which has an M(r) of 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is not phosphorylated by the cGMP-dependent protein kinase. While pp59 was consistently detected in cerebellar membranes from adult Sprague-Dawley rats, it was not detected in bovine or rabbit cerebellar membranes. Moreover, pp59 did not comigrate with any of the autophosphorylated subunits of the Ca(2+)/calmodulindependent protein kinase in rat cerebellar membranes. Extraction of pp59 from these membranes could be accomplished with 6 M urea, but not with 0.4 M NaCl or 0.5% (v/v) Triton X-100. The urea solubility suggests that pp59 is not an integral membrane protein. Acid hydrolysis of the protein phosphorylated in vitro yielded phosphoserine but no significant amount of phosphothreonine or phosphotyrosine. Further analysis of pp59 may provide new insights into the role of cAMP in modulation of synaptic function in the cerebellum.