The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.     

Basal protein phosphorylation is decreased and phosphatase activity increased by an antioxidant and a free radical trap in primary rat glia.

Reversible protein phosphorylation regulates a wide array of cellular functions. Cells respond to cytokines and various stressors via phosphorylation and thus activation of one or more of the mitogen-activated protein kinase (MAPK) pathways. Involvement of these signal transduction pathways has been implicated in numerous pathologies, including inflammation. Using a primary glia cell culture, we show here that the antioxidant N-acetylcysteine (NAC) and the nitrone-based free radical trap, alpha-phenyl-N-tert-butyl nitrone (PBN), reduce total basal protein phosphorylation in a concentration-dependent manner as assessed by phosphotyrosine analysis and by [gamma-32P]ATP transfer radioassay. In addition we show that NAC inhibits H2O2-induced phosphatase inactivation in glia cell lysate. The PBN- and NAC-induced reduction in protein phosphorylation is accompanied by an increase in phosphatase activity, suggesting that PBN and NAC reduce protein phosphorylation by globally augmenting oxidant-sensitive phosphatase activities. These results partly explain why certain antioxidants also possess anti-inflammatory actions.     

Partial characterization of a 230,000-dalton reticulocyte protein and peptides derived from it that affect the activity of a protein phosphatase

Monoclonal antibodies were raised that recognize a series of highly antigenic, protease-sensitive peptides that modulate protein phosphatase activity in reticulocyte extracts. Purified antigen peptides cause a 3-fold increase in the enzymatic activity of a homogeneous Mr congruent to 56,000 protein phosphatase. The monoclonal antibodies inhibit protein phosphatase activity in crude extracts but do not recognize the protein phosphatase itself. The antigen peptides are associated with the phosphatase throughout its purification from the postribosomal supernatant of rabbit reticulocytes but are separated from it during size exclusion high performance liquid chromatography (see accompanying article: Wollny, E., Watkins, K., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492). The series of antigenic peptides appears to be derived by proteolysis from a 230,000-Da precursor, which is relatively abundant in undegraded form in the membrane fraction of rabbit reticulocytes and is present in erythrocyte ghosts. Antigen peptides are extracted with spectrin from both sources. The Mr congruent to 230,000 peptide is not the alpha or beta subunit of spectrin or ankyrin and appears not to have been recognized previously. The name "regulin" is proposed.     

Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.     

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca²⁺/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,³²P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca²⁺/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.     

Prostaglandin E2/parathyroid hormone-induced suppression of alkaline phosphatase activity is mediated by protein kinase C

• 1.1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line.
• 2.2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity.
• 3.3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.
• 4.4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

     

Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Stress Proteins and Isozymology of Acid Phosphatase, Esterase, and Peroxidase in Phaseolus vulgaris Following Peanut Mottle Virus Infection

The present work was undertaken to study soluble protein changes and enzyme alterations in "Topcrop" bean ( Phaseolus vulgaris L.) primary leaves following inoculation with peanut mottle virus (PMV) in an attempt to elucidate the biochemical basis of the hypersensitive reaction in this host virus combination. Using the discontinuous polyacrylamide gel electrophoresis (disc-PAGE) technique, at least four apparently "novel" protein bands at Rf values of 0.80, 0.77, 0.74 and 0.68 were observed in infected tissue. The band at Rf 0.76 seems to be injury related and is shown here to be an isozyme of acid phosphatase. It is believed that these proteins are neither the cause nor the product of necrosis and may thus play a role in the active defense mechanism of the plant. In this virus host combination it was found that heating soluble protein fractions at 55 °C for 10 min before electrophoresis dramatically reduced the background and improved resolution on gels. While the biological function(s) of these proteins needs further investigations it is almost certain that none of them is an isozyme of alkaline phosphatase, acid phosphatase, esterase, or peroxidase. Zymogram analysis has additionally revealed the absence of alkaline phosphatase activity in untreated, abraded and PMV-infected primary leaves and no qualitative or quantitative changes in esterase isozymes have been observed in primary leaf tissues following abrasion or PMV–infection. By contrast, qualitative alterations in acid phosphatase after PMV-infection and both qualitative and quantitative changes in peroxidase after mechanical abrasion and PMV-infection have also been demonstrated.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes

A Mn²⁺/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase theV _max of the phosphatase but had no effect on theK _m value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn²⁺. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn²⁺ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn²⁺/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn²⁺ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn²⁺ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.     

Regulation of alternative splicing by reversible protein phosphorylation

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.     

Immobilized inhibitor-1 binds and inhibits protein phosphatase 1

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.     

Molecular characterization of catalytic-subunit cDNA sequences encoding protein phosphatases 1 and 2A and study of their roles in the gibberellin-dependent Osamy-c expression in rice

To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the -amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds.The protein phosphatase enzyme activity assays showed that there is no obvious effect of GA3 on total PP1/PP2A activities. To further understand the possible role of protein phosphatases 1 and 2A in the GA-dependent expression of Osamy-c, we isolated cDNA clones encoding protein phosphatase 1 and protein phosphatase 2A from a rice aleurone cDNA library. These were designated OsPP1c and OsPP2Ac, respectively. Comparison of the deduced amino acid sequences of OsPP1c and OsPP2Ac with the catalytic subunits of PP1 or PP2A of rabbit skeletal muscle, Arabidopsis thaliana, maize and Brassica napus showed that the catalytic subunit sequences of PP1 or PP2A among these organisms are highly conserved (73% to 90% similarity). Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice, including seed, shoot and root. We also showed by using 3 gene-specific probes of OsPP1c and OsPP2Ac cDNA, that the expression of neither gene is regulated by GA. Taken together, our results suggest that protein phosphatases PP1 or PP2A are involved in the GA-dependent expression of the rice Osamy-c gene, though the PP1 or/and PP2A enzymatic activities as well as mRNA levels do not increase upon GA3 treatment.     

Liver phosphorylase phosphatase

Summary Recent progress in studies on the properties and regulation of liver phosphorylase phosphatase can be divided into four stages. First, isolation from multiple molecular forms of phosphorylase phosphatase, of a single form of catalytic subunit (Mr = 32 000-35 000) which is active toward phosphorylase a and also toward a variety of protein substrates phosphorylated by either cyclic AMP-dependent protein kinase or other protein kinases. This was achieved by rather drastic procedures such as treatment with 80% ethanol at room temperature, incubation with 6 M urea, freeze-thawing in the presence of 0.2 M mercaptoethanol, or digestion by trypsin. These treatments caused concomitantly large enhancement of phosphorylase phosphatase activity, and the hypothesis was proposed that an inactive form of phosphorylase phosphatase existed as complexes of a catalytic subunit and inhibitory proteins. Second, it was discovered that liver and muscle extracts contain trypsin-labile proteins which, after heating at 90 °C, inhibited the catalytic subunit of phosphorylase phosphatase. Two types of protein inhibitors were identified: inhibitor-I was phosphorylated and activated by cyclic AMP-dependent protein kinase, whereas inhibitor-2 was not phosphorylated. Although inhibitor-1 has been implicated in hormonal regulation of glycogen metabolism in skeletal muscle, a similar role of protein inhibitors in the regulation of phosphorylase phosphatase in the liver has not been demonstrated and the physiological role of the inhibitor is questionable.Third, it has been demonstrated that liver phosphorylase phosphatase is reversibly inactivated and regulated by glutathione disulfide (GSSG) at the catalytic subunit level. Liver phosphorylase phosphatase contains, per mole of catalytic subunit, two sulfhydryl groups, one of which reacts with GSSG to form mixed disulfide with consequent inactivation of the enzyme. The inactivated enzyme can be reactivated by glutathione(GSH) or other sulfhydryl compounds through a reverse reaction. Injection of GSSG into the portal vein of rabbits caused a rapid increase in phosphorylase-a activity in the liver, suggesting that GSSG is involved in regulation of phosphorylase activity in vivo.Finally, current evidence suggests that liver phosphorylase phosphatase exists in the native state in a high molecular weight form which consists of the catalytic subunit and other regulatory subunits. One such enzyme species could be a 260 000-dalton protein composed of three different types of subunit, termed , and , or a 160 000-dalton protein composed of and subunits. The a subunit (Mr = 35 000) appears to be identical to the multifunctional catalytic subunit, whereas the (Mr = 69 000) and (Mr = 58 000) subunits are catalytically inactive but can modify the catalytic a subunit. It seems likely that the substrate specificity and catalytic activity of the subunit is considerably altered when it is part of larger complexes with other regulatory subunits ( and ). It has also been suggested that in addition to the native form of phosphorylase phosphatase, liver contains a considerably large amount of latent phosphorylase phosphatase, the catalytic activity of which could be revealed only by treatment with trypsin or ethanol.     

Phosphatase activity in Amoeba proteus at pH 9.0

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

The catalytic subunit of phosphatase 2A dephosphorylates phosphoopsin

Rod cell outer segments were found to contain a protein phosphatase activity toward phosphoopsin with properties very similar to those of protein phosphatase 1 or 2A. The opsin phosphatase activity was stable to ethanol precipitation, had a Mr of 35,000-38,000 as determined by gel filtration, and was not dependent on divalent cations for activity. The chromatographic properties on DEAE-cellulose of the rod outer segment protein phosphatase were also similar to those reported for protein phosphatase 1 or 2A. In order to distinguish between these two protein phosphatases, we tested homogeneous preparations of protein phosphatases 1 and 2A from skeletal muscle for activity toward phosphoopsin. Protein phosphatase 2A dephosphorylated phosphoopsin at approximately 10% of its rate toward phosphorylase a, whereas protein phosphatase 1 had no activity toward phosphoopsin. We conclude that protein phosphatase 2A is present in the rod cell outer segment and that it is a likely candidate to perform the in vivo dephosphorylation of rhodopsin in the visual cycle.     

The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.