Protein phosphatase 2A--a 'ménage à trois'.

Protein phosphorylation is probably the major regulatory mechanism employed by eukaryotic cells. Much work has been devoted to the role of protein kinases and their modulation by hormones, growth factors and neurotransmitters. It is now appreciated that protein phosphatases are also key players in actively regulating many cellular processes. In this article we discuss recent advances in our understanding of the function of protein phosphatase 2A, one of the major serine/threonine-specific protein phosphatases.

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression.

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Phosphorylation and activation of p70s6k by PDK1.

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.

Ten years of protein kinase B signalling: a hard Akt to follow.

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes, to our current understanding of the biology of PKB/Akt, and prospects for the future.

Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.

Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors.

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).

Cell polarity: Scaffold proteins par excellence.

Par proteins are involved in determining cellular asymmetry. Recent studies have identified one of these proteins, Par6, as a key regulator of cell polarity and transformation via its interactions with small GTPases and atypical forms of protein kinase C.

Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.

BACKGROUND: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling. RESULTS: The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox. CONCLUSIONS: Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.

Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.

Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation.

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.

Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation.

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.

Domain swapping used to investigate the mechanism of protein kinase B regulation by 3-phosphoinositide-dependent protein kinase 1 and Ser473 kinase.

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.

Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a).

Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state.

We have previously reported that insulin and osmotic shock stimulate an increase in glucose transport activity and translocation of the insulin-responsive glucose transporter isoform GLUT4 to the plasma membrane through distinct pathways in 3T3L1 adipocytes (D. Chen, J. S. Elmendorf, A. L. Olson, X. Li, H. S. Earp, and J. E. Pessin, J. Biol. Chem. 272:27401-27410, 1997). In investigations of the relationships between these two signaling pathways, we have now observed that these two stimuli are not additive, and, in fact, osmotic shock pretreatment was found to completely prevent any further insulin stimulation of glucose transport activity and GLUT4 protein translocation. In addition, osmotic shock inhibited the insulin stimulation of lipogenesis and glycogen synthesis. This inhibition of insulin-stimulated downstream signaling occurred without any significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, there was no effect on either the insulin-stimulated association of the p85 type I phosphatidylinositol (PI) 3-kinase regulatory subunit with IRS1 or phosphotyrosine antibody-immunoprecipitated PI 3-kinase activity. In contrast, osmotic shock pretreatment markedly inhibited the insulin stimulation of protein kinase B (PKB) and p70S6 kinase activities. In addition, the dephosphorylation of PKB was prevented by pretreatment with the phosphatase inhibitors okadaic acid and calyculin A. These data support a model in which osmotic shock-induced insulin resistance of downstream biological responses results from an inhibition of insulin-stimulated PKB activation.

Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55.

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.

Expression of the catalytic and regulatory subunits of protein phosphatase type 2A may be differentially modulated during retinoic acid-induced granulocytic differentiation of HL-60 cells.

To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions of PP1 and PP2A were examined. Treatment with 1 microM ATRA caused an 85% decrease in the PP2A activity in extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells induced by ATRA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (M(r) 55,000) regulatory subunits of PP2A were relatively unaltered. Expressions of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) were not significantly affected by ATRA treatment. Northern blot analysis revealed that mRNA levels of PP2A-C beta and A alpha regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-C alpha and B (M(r) 55,000) alpha regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-C beta preceded the granulocytic maturation induced by ATRA. Expressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentiation of HL-60 cells.

Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3-kinase-dependent mechanism.

The middle tumor antigen (middle-T) of mouse polyomavirus is responsible for the transforming potential of this virus. Middle-T has been shown to interact with a variety of cellular proteins known to mediate mitogenic signaling, like phosphatase-2A, Src family kinases, phosphatidylinositol 3-kinase (PI 3-kinase), the adapter protein SHC, phospholipase Cgamma-1 and 14-3-3 family proteins. Association with SHC and PI 3-kinase, respectively, stimulates two independent signaling pathways that are indispensible for viral oncogenicity. SHC activates the Ras/MAPK pathway via Grb2/SOS resulting in changes in early gene expression. The downstream targets of PI 3-kinase are less well studied but seem to impinge on serum response factor (SRF) which is also involved in regulating early gene expression. Recently, the protein kinase B/Akt (PKB/Akt) has been identified as a target of PI 3-kinase in receptor tyrosine kinase signaling. Here we show that PKB/Akt is a target of wild type middle-T, but not of mutants unable to activate PI 3-kinase. These data were confirmed using inhibitors of PI 3-kinase as well as dominant-negative alleles of the catalytic subunit of this lipid kinase. In addition, mutants of PKB/Akt lacking a pleckstrin homology domain and therefore unable to bind to D3 phospatidylinositides were not activated by middle-T. Taken together these data suggest that middle-T activates PKB/Akt in a PI 3-kinase-dependent manner. Furthermore, direct association with D3 phosphatidylinositides seems to be essential for activation of PKB/Akt.

Identification of the residues on cyclic GMP-dependent protein kinase that are autophosphorylated in the presence of cyclic AMP and cyclic GMP.

Autophosphorylation of cyclic GMP-dependent protein kinase (GMP:protein phosphotransferase, EC 2.7.1.37) in the presence of cyclic AMP and Mg-ATP has already been shown to result in the incorporation of up to 2.6 mol phosphate per mol subunit and decrease the A0.5 for cyclic AMP approx. 10-fold. The major sites of autophosphorylation have now been identified as serine-50, threonine-58, serine-72 and threonine-84. Serine-1 and serine-64 are phosphorylated to a minor extent. Threonine-58, which is initially phosphorylated most rapidly, is also the major site that is phosphorylated in the presence of cyclic GMP and Mg-ATP. Since autophosphorylation in the presence of cyclic GMP does not decrease the A0.5 for cyclic AMP, phosphorylation of serine-50, serine-72, or threonine-84 must be responsible for this effect.