The Protein Phosphatases Involved in Cellular Regulation. 6. Measurement of Type-1 and Type-2 Protein Phosphatases in Extracts of Mammalian Tissues; an Assessment of Their Physiological Roles

Abstract

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...

Insulin-receptor phosphotyrosyl-protein phosphatases

“…35 % (brain), 12 % (liver) and 10 % (heart) of total activity, and was > 80 % stable during incubation at 30°C for 20 min. The proportion of activity inhibited by trifluoperazine in brain extracts is similar to that reported by , although values for all three tissues are somewhat higher than those evident from the data of Ingebritsen et al (1983), presumably reflecting the use of a different substrate.…”

“…Tissue contents of calmodulin-dependent protein phosphatase are approx. 25, 12 and 5 mg/kg in both particulate and soluble phases of rat brain, liver and heart respectively (Wallace et al, 1980;Ingebritsen et al, 1983). Efficient dephosphorylation of receptors required 320 ,zg of calmodulin-dependent protein phosphatase/ml activated by the physiological activators Ca2+ and calmodulin.…”

Regulation of organelle transport in melanophores by calcineurin.

“…Anti-CN also stained a 60-kD polypeptide in a soluble extract of the dermis (Fig. 5, lane 3), indicating that CN is present in this tissue and that dermal CN is at least partially soluble, as has been previously reported for CN in other tissues (Ingebritsen, et al, 1983;Klee et al, 1988). Although a polypeptide corresponding to the B subunit could not be visualized in the whole extracts of dermis, the B subunit was apparent in soluble extracts that were partially purified by ammonium sulfate precipitation (data not shown).…”

Factors responsible for the Ca2+‐dependent inactivation of calcineurin in brain

“…Brain extracts were selected because the concentration of calcineurin ICalcineurin is the sole identified member of the protein phosphatase-2B family defined by Ingebritsen and Cohen [5]. in this tissue, determined by Western blot analysis, is ten to twenty times that of skeletal muscle [8] whereas the protein phosphatase activities of calcineurin in brain and skeletal muscle are apparently similar [6]. Endogenous inhibitory ligands could be responsible for the relatively low specific activity of calcineurin in crude brain extracts.…”