The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4PDK3> PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

Protein-protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1-PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3-L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10 microM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1=PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist-or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC 50 (Ca 2؉ ) The nitric-oxide synthases catalyze formation of NO and L-citrulline from L-arginine and O 2 , with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS) 2 in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130 -160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca 2ϩ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca 2ϩ ) 4 -CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca 2ϩ

Sorting and packaging of regulated secretory proteins involves protein aggregation in the trans-Golgi network and secretory granules. In this work, we characterized the pH-dependent interactions of pancreatic acinar cellregulated secretory proteins (zymogens) with Muclin, a putative Golgi cargo receptor. In solution, purified Muclin co-aggregated with isolated zymogens at mildly acidic pH. In an overlay assay, All eukaryotic cells synthesize and transport both membrane and soluble proteins through the endoplasmic reticulum and the Golgi complex to the cell surface, where they are delivered by unregulated exocytosis, a process called constitutive secretion (1). Some cells also have the capacity to store proteins in secretory granules, which are exocytosed upon neural or hormonal stimulation of the cell, and this is called the regulated secretory pathway (1). Sorting and packaging of proteins in the regulated pathway involves protein selection at the trans-Golgi network (TGN) 1 (sorting-for-entry) as well as removal of residual lysosomal enzymes and constitutively secreted proteins during post-Golgi maturation of secretory granules (sorting-by-retention) (for review, see Ref.2). The underlying process operating in the regulated secretory pathway is the aggregation of regulated proteins, which excludes constitutively secreted proteins.Protein aggregation in the secretory pathway relies on a variety of mechanisms for interaction of regulated proteins. The most widespread mechanism is the pH-dependent aggregation of regulated proteins in the TGN, which has a pH of about 6.0 (3). To complete the process of granule formation and keep the content proteins aggregated, secretory granules are either mildly acidified (pancreatic zymogen granules, pH ϳ6 -6.5 (4)) or moderately acidified (neuroendocrine granules, pH ϳ5 (5)). Regulated protein storage in some cells also relies on calcium, which is at millimolar concentrations in the secretory pathway compared with submicromolar levels in the cytosol (6).Despite understanding these processes in a general way, the details of packaging of regulated secretory proteins are not well understood. The major protein of the pancreatic zymogen granule is amylase, and although this enzyme co-aggregates with other zymogens, it does not self-aggregate at mildly acidic pH (7,8). Amylase was shown to associate with an SH3 binding domain of the soluble rat zymogen granule protein ZG29p (9). There is also evidence that amylase can interact with N-glycosylated proteins (10). Whether either of these components exists in sufficient amounts in the secretory pathway to account for amylase sorting is unknown.Sulfated proteoglycans and glycoproteins are also likely to be involved in protein packaging in zymogen granules (11-13), but the nature of these interactions is not known. We previously showed that zymogen granule formation in mouse acinar cells requires O-glycosylation as well as sulfation (11). When sulfation was inhibited, regulated protein secretion was not inhibited but newly formed granules...