The Protein Phosphatases Involved in Cellular Regulation. 1. Classification and Substrate Specificities

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Abstract
The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only 4 enzymes which can be conveniently divided into 2 classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the .beta.-subunit of phosporylase kinase and are potently inhibited by 2 thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the .alpha.-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the 4 enzymes, i.e., protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) [from rabbit muscle and liver] were 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. 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 L 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 Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated 3 of the substrates (the .alpha.-subunit of phosphorylase kinase, inhibitor-1 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 the primary structure in the immediate vicinity of the phosphorylation site.