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pRB, a negative-growth regulatory protein, is a demonstrated substrate for type 1 serine/threonine protein phosphatases (PP1). In a recent report from this laboratory, we demonstrated that select forms of phosphorylated as well as hypophosphorylated pRB can be found complexed with the alpha-isotype of PP1 (PP1alpha). This complex can also be observed when PP1 is rendered catalytically dead by toxin inhibition. These data suggested to us that pRB may bind to PP1 at one or more sites other than the catalytically active one on the enzyme and that such binding may play a role other than bringing the substrate into contact with the enzyme to facilitate catalysis. To address this possibility we utilized a series of pRB deletion mutants and coprecipitation studies to map the pRB domain involved in binding to PP1. Together with competition assays using in vivo expression of SV40 T-antigen, we show here that the carboxyl-terminal region of pRB is both necessary and sufficient for physical interaction with PP1. Subsequent biochemical analyses demonstrated inhibition of PP1 catalytic activity toward the standard substrate phosphorylase a when this enzyme is bound to pRB containing this region. K(m) and V(max) calculations revealed that pRB binds to PP1 in a non-competitive manner. These data support the notion that pRB, in addition to being a substrate for PP1, also functions as a PP1 inhibitor. The significance of this finding with respect to the functional importance of this interaction is discussed.

The Mg(2+)-dependent serine/threonine protein phosphatases, also known as type 2C phosphatases (PP2C), belong to a gene family distinct from the other serine/threonine phosphatases and tyrosine phosphatases. Here we report the purification to apparent homogeneity of a novel Mg(2+)-dependent, Ca(2+)-inhibitable serine/threonine protein phosphatase from bovine brain. It is a type 2C enzyme in view of its Mg2+ requirement, resistance to okadaic acid and calyculin A, inability to use phosphorylase alpha as substrate, and a segment of amino acid sequence typical of all PP2C type phosphatases known to date. However, it differs from the other PP2C enzymes, particularly the mammalian PP2C alpha and -beta isoforms, in that its molecular weight, 76,000, is considerably larger and that it is inhibited by Ca2+, NaF, and polycations, but not by orthovanadate. The Ca2+ inhibition may not be related to its cellular regulation because of Ki values in the 20-90 microM range, but this property permits distinction of this enzyme from the other phosphatases. Although the precise physiological role of this phosphatase is not yet known, its ability to dephosphorylate a wide variety of phosphoproteins and its broad distribution, as shown by a survey of mouse tissues for its activity, suggest that it may serve an important cellular function.

In order to determine the role of fructose (Fru) 2,6-P2 in stimulation of phosphofructokinase in ischemic liver, tissue contents of Fru-2,6-P2, hexose-Ps, adenine nucleotides, and Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated during the first few minutes of ischemia. The Fru-2,6-P2 concentration in the liver changed in an oscillatory manner. Within 7 s after the initiation of ischemia, Fru-2,6-P2 increased from 6 to 21 nmol/g liver and decreased to 5 nmol/g liver within 30 s. Subsequently, it reached the maximum value at 50, 80, and 100 s and decreased to the basal concentration at 60, 90, and 120 s. Oscillatory patterns were also observed with Glc-6-P and Fru-6-P, but the ATP/ADP ratio decreased monotonically. Determination of Fru-6-P,2-kinase activity and the phosphorylation states of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase demonstrated that at 7 and 50 s, where Fru-2,6-P2 was the highest, the enzyme was activated and mostly in a dephosphorylated form. On the other hand, at 0, 30, and 300 s, the enzyme was predominantly in the phosphorylated form. The concentration of cAMP in the liver also changed in an oscillatory manner between 0.5 to 1.3 nmol/g with varying frequency of 10 to 40 s. These results indicated that: (a) Fru-2,6-P2 was important in rapid activation of phosphofructokinase in the first few seconds and up to 2-3 min, and (b) the oscillation of Fru-2,6-P2 concentration was the result of activation and inhibition of Fru-6-P,2-kinase:Fru-2,6-bisphosphatase, which was caused by changes in the phosphorylation state of the enzyme.

Kinetic studies of both liver and muscle phosphorylase a demonstrate that caffeine and glucose inhibit the binding of glucose 1-phosphate to the enzyme in a synergistic competitive and nonexclusive manner. Inhibition studies for numerous other caffeine analogs show that the muscle enzyme has a relaxed specificity for this negative effector. The liver enzyme is more discriminating by preferential binding of methylated oxypurines. Physiological concentrations of AMP and ATP, which affect the enzymic activity at a separate site, prevent glucose from effectively inhibiting the enzyme. The addition of the second synergistic ligand improves the binding of glucose. These data suggest that glucose homeostasis as regulated by phosphorylase may be dependent on a second ligand and that the role of glucose in this physiological process may have been overestimated. A structural rationalization of this synergistic response is discussed with reference to the crystal structure of the muscle enzyme.

In human platelets, the activity of total phosphorylase could be increased appreciably following incubation with MgATP at 37° for 1 hour or overnight dialysis against MgATP at 4°. The total phosphorylase activity of a 4,000 x g homogenate increased 11-fold following MgATP activation. A 105,000 x g particulate fraction when assayed for phosphorylase activity in the presence of 1 mm AMP was inactive. Following treatment with MgATP at 37° for 1 hour, the fraction gained considerable activity. Sucrose gradient analysis of a 27,000 x g charcoal-treated platelet extract revealed the presence of inactive phosphorylase species with apparent molecular weights of dimer and monomer. These were converted to phosphorylase a as well as phosphorylase b following incubation with MgATP.