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

Abstract

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...

The protein phosphatases involved in cellular regulation

“…Mn2+ (1.0 mM) also had no effect or inhibited protein phosphatase-1, except when inhibitor-1 was the substrate. As noted previously [30,31], the dephosphorylation of inhibitor-1 was almost completely dependent on Mn2+, half-maximal activation occurring at about 0.2 mM Mn2+.…”

“…6). The amino acid sequence immediately following sites-3 of glycogen synthase is extremely acidic (16 out of 31 residues) [39] and it has been suggested that acidic residues C-terminal to phosphorylation sites may act as negative specificity determinants for protein phosphatases [40]. It is possible that spermine interacts with this acidic region, thereby neutralising its inhibitory effects on the dephosphorylation of sites-3.…”

“…Effect of spermine on glycogen synthase phospha e activity in skeletalmuscle extracts. Rabbit skeletal muscleextracts were prepared as in [30] and assayed in the absence of divalent cations at 150-fold final dilution. The circles show results obtained with glycogen synthase labelled in sites-3 and triangles results with glycogen synthase labelled in site-2.…”

On the 6‐phosphofructo‐1‐kinase phosphatase activity of protein phosphatase 2C and its dimeric nature

“…Ingebritsen and Cohen [3] have classified all protein phosphatases involved in the dephosphorylation of phosphoenzymes of intermediary metabolism. They arrived at the conclusion that these dephosphorylations are catalyzed by only four different protein phosphatases, termed phosphaiase 1, 2A, 2B and 2C.…”

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A recently described 6-phosphofructo-1-kinase phosphatase (PFK-phospatase [l]) shared several properties with protein phosphatase 2C [2,3], but exhibited differences with respect to molecular mass and substrate specificity. Chromatography on histone-Sepharose, gel filtration experiments on Sephacryl S-200 and Sephadex G-100 as well as sucrose density gradient centrifugation [4] show that both enzyme preparations behave identically under all experimental conditions used.

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