The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometricaily by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by >4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: lle-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1-and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.
The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-I , has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22 835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Straphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e. g. 1 -49, 49 -92, 67 ~ 101, 108 -134, 142 -182 and 163 -197) are inactive. Digestion with clostripain generated a peptide comprising residues 25 -114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-I .In 1976, Huang and Glinsmdnn [l] identified two proteins in rabbit skeletal muscle, termed inhibitor-I (1-1) and inhibitor-2 (I-2), that were specific inhibitors of a protein phosphatase, subsequently termed protein phosphatase-1 (reviewed in [2, 31). 1-1 was only an inhibitor after it had been phosphorylated by cyclic-AMP-dependent protein kinase, whereas the activity of 1-2 was unaffected by this protein kinasc [I].1-2 can interact with protein phosphatase-I in two distinct ways. Firstly, at extremely low concentrations (Kd z 0.1 nM), it combines with the catalytic (C) subunit of protein phosphatase-I to produce an inactive 1-2 . C complex [4, 51, termed protein phosphatase-lI [6]. This form has also been termed the Mg-ATP-dependent protein phosphatase [7] because its activation requires preincubation with Mg-ATP and a protein kinase, termed factor FA [8] or glycogen synthase kinase-3 [9, 101. Activation of protein phosphatase-lI is triggered by the phosphorylation of a specific threonyl residue on 1-2 [4, 5, 11 -131 and is the first example of a protein phosphatase that is activated by a protein kinase. Secondly, at higher concentrations ( K d E S nM), 1-2 inactivates protein phosphatase-I by a different mechanism that is not reversed by preincubation with Mg-ATP and glycogen synthase kinase-3, and presumably results from the binding of 1-2 at a separate site on the enzyme [S].1-2 is also a substrate for other protein kinases, such as casein kinase-I1 [I41 and the insulin receptor [15]. Phosphorylation by casein kinase-11, which occurs on seryl residues, does not activate protein phosphatase-lI directly, but enhances the rate of phosphorylation of 1-2 and activation of protein phosphatase-1, by glycogen synthase kinase-3 [I 41. Phosphorylation by the insulin receptor occurs on a tyrosyl residue(s), but the functional significance of this modification is unknown.1-1 and 1-2 have been purified...
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