The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-I protein phosphatases dephosphorylate the 8-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-I and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the a-subunit of phosphorylase kinase and are insensitive to inhibitor-I and inhibitor-2.The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-I) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A.Protein phosphatase-2C (an Mg2 +-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity.Protein phosphatase-2B (a CaZ+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the a-subunit of phosphorylase kinase, inhibitor-I and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine.Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by phorylation site.The reversible phosphorylation of proteins is now recognised to be the major mechanism by which intracellular events are controlled by neural and hormonal stimuli, and more than 30 enzymes have been shown to be regulated in this manner. An important generality to have emerged is that enzymes in biodegradative pathways are activated, whereas those in biosynthetic pathways are inactivated by phosphorylation [l -31. This, in turn, implies that different metabolic pathways may be controlled by identical protein kinases and protein phosphatases, or by identical effector molecules that regulate the primary structure in the immediate vicinity of the phosdifferent protein kinases and protein phosphatases. These concepts are already well established in th...

Protein phosphorylation is a principal regulatory mechanism in the control of almost all cellular processes. The nature of the protein phosphatases that participate in these reactions has been a subject of controversy. Four enzymes, termed protein phosphatases 1, 2A, 2B, and 2C, account for virtually all of the phosphatase activity toward phosphoproteins involved in controlling glycogen metabolism, glycolysis, gluconeogenesis, fatty acid synthesis, cholesterol synthesis, and protein synthesis. The properties, physiological roles, and mechanisms for regulating the four protein phosphatases are reviewed.

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Bioactive compound(s) extracted from cinnamon potentiate insulin activity, as measured by glucose oxidation in the rat epididymal fat cell assay. Wortmannin, a potent PI 3′-kinase inhibitor, decreases the biological response to insulin and bioactive compound(s) from cinnamon similarly, indicating that cinnamon is affecting an element(s) upstream of PI 3′-kinase. Enzyme studies done in vitro show that the bioactive compound(s) can stimulate autophosphorylation of a truncated form of the insulin receptor and can inhibit PTP-1, a rat homolog of a tyrosine phosphatase (PTP-1B) that inactivates the insulin receptor. No inhibition was found with alkaline phosphate or calcineurin suggesting that the active material is not a general phosphatase inhibitor. It is suggested, then, that a cinnamon compound(s), like insulin, affects protein phosphorylation-dephosphorylation reactions in the intact adipocyte. Bioactive cinnamon compounds may find further use in studies of insulin resistance in adult-onset diabetes.

Methods were developed for quantifying protein phosphatases-I, 2A, 2B and 2C in cell extracts, and these procedures were exploited to determine their tissue and subcellular distributions. In addition, the contribution of each enzyme to the total protein phosphatase activity in skeletal muscle and liver extracts towards nine proteins involved in the control of glycogen metabolism, glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis was assessed.Each protein phosphatase was present at significant concentrations in skeletal muscle, heart muscle, liver, brain and adipose tissue, although the relative amounts differed considerably. In skeletal muscle, protein phosphatase-I was the major enzyme acting on phosphorylase, glycogen synthase and phosphorylase kinase (B-subunit), and thus was the major protein phosphatase responsible for the inactivation of glycogenolysis and stimulation of glycogen synthesis. This idea was reinforced by the observation that 50 ' %, of the protein phosphatase-I activity was associated with the protein-glycogen complex.In the liver, protein phosphatases-1, 2A and 2C each appear to play a role in the regulation of glycogen metabolism. Protein phosphatase-I accounted for a significant fraction of the total potential activity towards phosphorylase and glycogen synthase, and was the major phosphorylase kinase ([hubunit) phosphatase of this tissue. In addition, it was the only protein phosphatase present in the protein-glycogen complex. Protein phosphatase 2A was also a major phosphorylase phosphatase and glycogen synthase phosphatase in this tissue. Protein phosphatase 2C was a significant glycogen synthase phosphatase in the liver, but had negligible activity toward phosphorylase or phosphorylase kinase (p-subunit).In the absence of Ca2+, protein phosphatase 2A was the major phosphorylase kinase (a-subunit) phosphatase and the only inhibitor-1 phosphatase, in skeletal muscle or liver. In the presence of Ca2+, protein phosphatase 2B accounted for most of the activity towards these substrates.Protein phosphatase 2A was the major enzyme acting on L-pyruvate kinase, ATP-citrate lyase and acetyl-CoA carboxylase in rat liver, suggesting an important role in the regulation of glycolysis/gluconeogenesis and fatty acid synthesis. Protein phosphatase 2C was the major enzyme acting on hydroxymethylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA reductase kinase, suggesting an important role in the regulation of cholesterol synthesis. However, the observation that 20% of the protein phosphatase-I in liver was associated with the microsomal fraction suggests that this enzyme may also be involved in regulating HMG-CoA reductase, which is tightly associated with microsomes.The activity of protein phosphatase-I in dilute skeletal muscle and liver extracts was just as sensitive to inhibitor-I and inhibitor-2 as the purified enzyme. In concentrated extracts, higher concentrations of the inhibitor proteins were required and the inhibition was time-dependent. The results explain previous report...