Classification of an eIF‐2 phosphatase as a type‐2 protein phosphatase

Stewart, Alexander A.; Crouch, D; Cohen, Philip; Safer, Brian

doi:10.1016/0014-5793(80)80988-4

Cited by 21 publications

(8 citation statements)

References 18 publications

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“…The difference in dephosphorylation between free and mRNA bound P38 is explained by a configurational change of the protein which protects some phosphorylated sites from dephosphorylation after assembly of the protein into mRNP. Similar results have been obtained for eIF-2 phosphatases from rabbit reticulocytes by Crouch and Safer [51] and Stewart et al [52]. They postulate that eIF-2 phosphatase activity is strongly influenced by the structural and physiological state of the substrate.…”

Section: Discussionsupporting

confidence: 88%

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

1987

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A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass ( M , ) of 225000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ionexchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysineagarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, CI and jl, with M , of 40000 and 75000, respectively. These values, in conjunction with the native M , and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is a2fi2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20°C or with ethanol, the enzyme released the catalytic a subunit of M , 40000. = 1.6 pM) and polylysine (Ao.s = 0.2 pM) and inhibited by ATP (10,5 = 12 pM), NaF = 3.1 mM) and pyrophosphate (fo.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type 11, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.The protein phosphatase was activated by basic proteins e.g. protamine (Ao.5 = 1 pM), histone H I Protein phosphorylation-dephosphorylation has emerged as a regulatory mechanism modifying the action of enzymes and structural proteins [I]. Rate-limiting enzymes in biodegradative pathways are usually activated by phosphorylation, whereas those in biosynthetic pathways are generally inactivated [2, 31. There is increasing evidence to support the hypothesis that different metabolic events are regulated by relatively few protein kinases and phosphatases [l, phorylate the a subunit of phosphorylase kinase and are insensitive to inhibitors-1 and -2 (protein phosphatases-2A, 2B and 2C) [17,18]. Based on enzyme-directed regulation, protein phosphatases may also be classified into four main classes: ATP/Mg-dependent protein phosphatases, calcineurin, Mg2+-dependent protein phosphatases and polycationstimulated protein phosphatases [19].Messenger RNA is associated with specific proteins to form messenger ribonucleoproteins (mRNP) [20]. It is generally accepted that the mRNA-associated proteins have a function in mRNA metabolism, e. g. replication, stability, storage, transport and translation [21-251. Phosphorylation of mRNP-associated proteins has been observed frequently and is proposed to regulate mRNA metabolism [26 -291. Furthermore, Egly and coworkers [27,30] have suggested that dephosphorylation of mRNP proteins also has a function in translation.Cryptobiotic gastrulae, a dormant phase of the embryos of the brine shrimp ...

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Section: Discussionsupporting

confidence: 88%

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

1987

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“…The question ofwhich phosphatase is actually important in the dephosphorylation of eIF-2(aP) in the lysate under conditions of translation repression is discussed below. Two groups of researchers have previously reported the purification of eIF-2(aP) phosphatases from reticulocytes [23,24]. The phosphatase isolated by Stewart et al [23] was identified as a type-2 phosphatase on the basis of its insensitivity to the inhibitor proteins and its preferential dephosphorylation of the a-subunit of phosphorylase kinase.…”

Section: Discussionmentioning

confidence: 99%

Activity of protein phosphatases against initiation factor-2 and elongation factor-2

Redpath¹,

Proud²

1990

Biochemical Journal

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The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.

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“…It is not yet clear whether the smaller forms are fragments [31] or subunits of the larger species. However all forms have the same substrate specificities [12,32,33] and sensitivity to the inhibitor proteins [12,13].…”

Section: Discussionmentioning

confidence: 99%

“…In [33] the criteria used to distinguish protein phosphatase-1 and -2 have been applied to a homogeneous protein phosphatase from reticulocytes. On the basis of the results, this enzyme is classified as a type-2 protein phosphatase.…”

Section: Discussionmentioning

confidence: 99%

The broad specificity protein phosphatase from mammalian liver

1980

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The reversible phosphorylation of enzymes is recognized to be one of the major general mechanisms by which cellular metabolism is regulated by neural and hormonal stimuli. An important generality that seems to be emerging is that enzymes in biodegradative pathways are activated and enzymes in biosynthetic pathways inactivated by phospllorylation [ 1,2]. This suggests that different metabolic pathways may be regulated in a co-ordinated manner by common protein kinases and protein phosphatases. This concept is already established in the case of cyclic AMPdependent protein kinase, which mediates the actions of hormones which use cyclic AMP as a second messenger [ 1,3].There is increasing evidence that protein phosphatases are also important targets for hormone action [ 11. The current dogma is that a single broad speciflcity catalytic subunit (MT 35 000) exists in liver, skeletal muscle and heart muscle, which is capable of reversing phosphorylation reactions catalysed by both cyclic AMP-dependent and -independent protein kinases 14-91. This enzyme, which has been termed protein phosphatase C [IO], has been obtained in highly purified form from each of these tissues using an 80% ethanol precipitation at room temperature at an early stage of the preparation. This treatment not only denatures many contaminating proteins, but is also believed to dissociate the catalytic subunit from higher M, complexes which may contain regulatory subunits [lo].In this laboratory two protein phosphatases have been implicated in the regulation of glycogen metabolism in rabbit skeletal muscle. Protein phosphatase-I Elsevier/Nor?h-Holland Biomedical Press accounts for 80-90% of the phosphorylase phosphatase activity, 70-80% of the glycogen synthase phosphatase activity, and >90% of the activity against the P-subunit of phosphorylase kinase in this tissue. Protein phosphatase-2, on the other hand, possesses >90% of the activity against the a-subunit of phosphorylase kinase, but also has some phosphorylase phosphatase and glycogen synthase phosphatase activity [ 11-l 3], The enzymes can also be distinguisiled by the use of two heat-stable proteins termed inhibitor-l and inhibitor-2, which only inhibit protein phosphathse-1 [13-151.Here, we demonstrate that protein phospllatase C isolated from either rat liver or rabbit liver is not a single enzyme. It consists of at least two different low MI protein phosphatases whose properties suggest that they are either the catalytic subunits or fragments of protein phosphatase-1 and protein phosphatase-2.The criteria used to distinguish these enzymes should be of general significance for the classification of all protein phosphatases.

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Classification of an eIF‐2 phosphatase as a type‐2 protein phosphatase

Cited by 21 publications

References 18 publications

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp.

Activity of protein phosphatases against initiation factor-2 and elongation factor-2

The broad specificity protein phosphatase from mammalian liver

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