PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth

Akt/protein kinase B critically regulates the balance between cell survival and apoptosis. Phosphorylation of Akt at two key sites, the activation loop and the hydrophobic motif, activates the kinase and promotes cell survival. The mechanism of dephosphorylation and signal termination is unknown. Here, we identify a protein phosphatase, PH domain leucine-rich repeat protein phosphatase (PHLPP), that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1), triggering apoptosis and suppressing tumor growth. The effects of PHLPP on apoptosis are prevented in cells expressing an S473D construct of Akt, revealing that the hydrophobic motif is the primary cellular target of PHLPP. PHLPP levels are markedly reduced in several colon cancer and glioblastoma cell lines that have elevated Akt phosphorylation. Reintroduction of PHLPP into a glioblastoma cell line causes a dramatic suppression of tumor growth. These data are consistent with PHLPP terminating Akt signaling by directly dephosphorylating and inactivating Akt.     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

Activation of PH-domain leucine-rich protein phosphatase 2 (PHLPP2) by agonist stimulation in cardiac myocytes expressing adenylyl cyclase type 6

The Ser/Thr-specific phosphatase PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) regulates the amplitude and duration of agonist-evoked Akt signaling by dephosphorylating the hydrophobic motif (Ser473) of Akt, therefore inactivating Akt. We recently reported that gene transfer of adenylyl cyclase type 6 (AC6) into neonatal rat cardiac myocytes was associated with increased Akt phosphorylation and activity. To determine the underlying mechanisms for AC6-associated increase in Akt activation, we determined how AC6 gene transfer regulated the activity of PHLPP2 (one of the three PHLPP family phosphatases) in neonatal rat cardiac myocytes. We found that increased Akt activity was associated with inhibition of PHLPP2 activity by AC6. AC6 was physically associated with PHLPP2, which prevents PHLPP2-mediated Akt dephosphorylation. However, isoproterenol or forskolin stimulation immediately activated PHLPP2, which resulted in markedly dephosphorylation of Akt at Ser473. Activation of PHLPP2 by isoproterenol and forskolin was cAMP-independent, but required an intact cytoplasmic domain of AC6. Mutation in the cytoplasmic domain of AC6 abolished agonist-induced PHLPP2 activation. This novel bidirectional regulation of Akt activity may contribute to the unexpected favorable effects of AC6 on the failing heart.     

PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms

Akt/protein kinase B controls cell growth, proliferation, and survival. We recently discovered a novel phosphatase PHLPP, for PH domain leucine-rich repeat protein phosphatase, which terminates Akt signaling by directly dephosphorylating and inactivating Akt. Here we describe a second family member, PHLPP2, which also inactivates Akt, inhibits cell-cycle progression, and promotes apoptosis. These phosphatases control the amplitude of Akt signaling: depletion of either isoform increases the magnitude of agonist-evoked Akt phosphorylation by almost two orders of magnitude. Although PHLPP1 and PHLPP2 both dephosphorylate the same residue (hydrophobic phosphorylation motif) on Akt, they differentially terminate Akt signaling by regulating distinct Akt isoforms. Knockdown studies reveal that PHLPP1 specifically modulates the phosphorylation of HDM2 and GSK-3alpha through Akt2, whereas PHLPP2 specifically modulates the phosphorylation of p27 through Akt3. Our data unveil a mechanism to selectively terminate Akt-signaling pathways through the differential inactivation of specific Akt isoforms by specific PHLPP isoforms.     

Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP): a new player in cell signaling

Precise balance between phosphorylation, catalyzed by protein kinases, and dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Deregulation of this balance leads to pathophysiological states that drive diseases such as cancer, heart disease, and diabetes. The recent discovery of the PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) family of Ser/Thr phosphatases adds a new player to the cast of phosphate-controlling enzymes in cell signaling. PHLPP isozymes catalyze the dephosphorylation of a conserved regulatory motif, the hydrophobic motif, on the AGC kinases Akt, PKC, and S6 kinase, as well as an inhibitory site on the kinase Mst1, to inhibit cellular proliferation and induce apoptosis. The frequent deletion of PHLPP in cancer, coupled with the development of prostate tumors in mice lacking PHLPP1, identifies PHLPP as a novel tumor suppressor. This minireview discusses the structure, function, and regulation of PHLPP, with particular focus on its role in disease.     

Invariant Leu preceding turn motif phosphorylation site controls the interaction of protein kinase C with Hsp70

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC betaII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC betaII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.     

Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.Breast cancer is diagnosed in ∼180,000 women and is the cause of 40,000 deaths each year in the U.S.² A prevalent underlying mechanism driving tumorigenesis is aberrant signal transduction pathways that result in constitutive activation of cell growth, proliferation, and survival pathways (2). A well characterized signal transduction pathway in breast cancer that promotes cellular survival, growth, and proliferation is the phosphatidylinositol 3-kinase/Akt pathway (3). This pathway is activated by a number of mechanisms, including gene amplification or gain of function mutations in upstream receptor protein-tyrosine kinases (4, 5), constitutive activation of hormone receptors (6), activating mutations in phosphatidylinositol 3-kinase and Akt (7, 8), and loss of function mutations in the regulatory phosphatase PTEN³ (phosphatase and tensin homolog on chromosome ten) (9). Thus, Akt is a major regulator of breast tumorigenesis.There are three isoforms of Akt present in humans. All three isoforms contain activating phosphorylation sites in the activation loop (Thr-308 in Akt1) and in the C-terminal hydrophobic motif (Ser-473 in Akt1) (10). Upon growth factor receptor stimulation, phosphatidylinositol 3-kinase becomes activated and phosphorylates the D3 position of, typically, phosphatidylinositol (4, 5) bisphosphate to generate phosphatidylinositol (3,4,5)-trisphosphate (11). This 3′-phosphorylated lipid recruits Akt to the plasma membrane by binding to its PH domain, resulting in conformational changes that allow access to the activation loop phosphorylation site (11). Constitutively bound phosphatidylinositol-dependent kinase-1 then phosphorylates Akt at Thr-308, accompanied by phosphorylation at Ser-473 resulting in a catalytically active kinase (12). Phosphorylation of Ser-473 depends on the mTORC2 complex (13-16). Signaling through this pathway is terminated by removal of the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate catalyzed by the phosphatase PTEN and by direct dephosphorylation of Akt by the recently-identified PHLPP family of phosphatases and protein phosphatase 2A-type phosphatases (17-20).The PHLPP family of phosphatases comprise three variants, the alternatively spliced PHLPP1α and PHLPP1β, and PHLPP2 (21). PHLPP1 and PHLPP2 specifically dephosphorylate the hydrophobic motif of specific Akt isozymes, thus decreasing Akt activity and promoting apoptosis (18, 19). PHLPP2 binds and dephosphorylates Akt1 and Akt3, whereas PHLPP1 binds and dephosphorylates Akt2 and Akt3 (18, 22). Their role in inactivating Akt suggests that both PHLPP1 and PHLPP2 could be potential tumor suppressors. Consistent with such a role, these phosphatases also dephosphorylate the hydrophobic motif of PKC, resulting in degradation of PKC. For this kinase, phosphorylation stabilizes the enzyme, so that the effect of depletion of the PHLPP phosphatases is to increase PKC protein levels (23). PKC is a well characterized oncogene, and loss of function of the PHLPP phosphatases could increase PKC protein levels and promote tumorigenesis (24). Providing further rationale that PHLPP2 could be a potential tumor suppressor, the phosphatase is located on chromosome 16q22.3, a region that encounters frequent loss of heterozygosity (LOH) in many primary and malignant breast tumors (25).Here we identify a non-synonymous polymorphism that results in an amino acid change from a Leu to a Ser at codon 1016 in the PP2C phosphatase domain of PHLPP2. Overexpression studies reveal the Ser-1016 variant has impaired phosphatase activity and is less effective at inducing apoptosis than the Leu-1016 variant. When comparing a pair-matched normal and breast cancer cell line or pair-matched normal and high grade tumor patient samples that are heterozygous, we observe preferential loss of the Leu allele in the tumor tissue or breast cancer cell line. This observation provides evidence that PHLPP2 could be one of the elusive tumor suppressor genes on chromosome 16q, and for heterozygous patients, loss of the more catalytically active Leu-1016 may promote breast tumorigenesis.     

The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation.

BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.     

Mislocalization of the E3 ligase, β-transducin repeat-containing protein 1 (β-TrCP1), in glioblastoma uncouples negative feedback between the pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) and Akt

The PH domain leucine-rich repeat protein phosphatase, PHLPP, plays a central role in controlling the amplitude of growth factor signaling by directly dephosphorylating and thereby inactivating Akt. The cellular levels of PHLPP1 have recently been shown to be enhanced by its substrate, activated Akt, via modulation of a phosphodegron recognized by the E3 ligase β-TrCP1, thus providing a negative feedback loop to tightly control cellular Akt output. Here we show that this feedback loop is lost in aggressive glioblastoma but not less aggressive astrocytoma. Overexpression and pharmacological studies reveal that loss of the feedback loop does not result from a defect in PHLPP1 protein or in the upstream kinases that control its phosphodegron. Rather, the defect arises from altered localization of β-TrCP1; in astrocytoma cell lines and in normal brain tissue the E3 ligase is predominantly cytoplasmic, whereas in glioblastoma cell lines and patient-derived tumor neurospheres, the E3 ligase is confined to the nucleus and thus spatially separated from PHLPP1, which is cytoplasmic. Restoring the localization of β-TrCP1 to the cytosol of glioblastoma cells rescues the ability of Akt to regulate PHLPP1 stability. Additionally, we show that the degradation of another β-TrCP1 substrate, β-catenin, is impaired and accumulates in the cytosol of glioblastoma cell lines. Our findings reveal that the cellular localization of β-TrCP1 is altered in glioblastoma, resulting in dysregulation of PHLPP1 and other substrates such as β-catenin.     

Phosphatase PHLPP2 regulates the cellular response to metabolic stress through AMPK

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.Subject terms: Cancer metabolism, Stress signalling     

Disruption of the interface between the pleckstrin homology (PH) and kinase domains of Akt protein is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.     

Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.     

PHLPP-mediated dephosphorylation of S6K1 inhibits protein translation and cell growth

PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Conversely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phosphorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1.     

Peptidyl-prolyl isomerase Pin1 controls down-regulation of conventional protein kinase C isozymes

The down-regulation or cellular depletion of protein kinase C (PKC) attendant to prolonged activation by phorbol esters is a widely described property of this key family of signaling enzymes. However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known. Here we show that the peptidyl-prolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes, converting the enzymes into a species that can be dephosphorylated and ubiquitinated following activation induced by either phorbol esters or natural agonists. The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of the turn motif phosphorylation site, one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes. Furthermore, the second C-terminal phosphorylation site, the hydrophobic motif, docks Pin1 to PKC. Our data are consistent with a model in which Pin1 binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus converting these PKC isozymes into species that can be efficiently down-regulated following activation.     

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.     

On the PHLPPside: Emerging roles of PHLPP phosphatases in the heart

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a family of enzymes made up of two isoforms (PHLPP1 and PHLPP2), whose actions modulate intracellular activity via the dephosphorylation of specific serine/threonine (Ser/Thr) residues on proteins such as Akt. Recent data generated in our lab, supported by findings from others, implicates the divergent roles of PHLPP1 and PHLPP2 in maintaining cellular homeostasis since dysregulation of these enzymes has been linked to various pathological states including cardiovascular disease, diabetes, ischemia/reperfusion injury, musculoskeletal disease, and cancer. Therefore, development of therapies to modulate specific isoforms of PHLPP could prove to be therapeutically beneficial in several diseases especially those targeting the cardiovascular system. This review is intended to provide a comprehensive summary of current literature detailing the role of the PHLPP isoforms in the development and progression of heart disease.     

Regulation of protein kinase C inactivation by Fas-associated protein with death domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.     

Cross-regulation of novel protein kinase C (PKC) isoform function in cardiomyocytes. Role of PKC epsilon in activation loop phosphorylations and PKC delta in hydrophobic motif phosphorylations

Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.     

蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

CSTP1, a Novel Protein Phosphatase,Blocks Cell Cycle,Promotes Cell Apoptosis,and Suppresses Tumor Growth of Bladder Cancer by Directly Dephosphorylating Akt at Ser473 Site

Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.