Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.     

Prostaglandin E2 selectively antagonizes prostaglandin F2alpha-stimulated T-cell factor/beta-catenin signaling pathway by the FPB prostanoid receptor

FP prostanoid receptors are G-protein-coupled receptors that consist of two isoforms named FPA and FPB. Both isoforms activate inositol phosphate second messenger signaling pathways by their endogenous ligand prostaglandin F2alpha (PGF2alpha). Previously we have shown that both isoforms undergo Rho-mediated cell rounding following treatment with PGF2alpha. Following the removal of PGF2alpha, however, FPA-expressing cells return to their original morphology, whereas FPB-expressing cells do not. It was also found that PGF2alpha-could activate T-cell factor (Tcf)/beta-catenin signaling in cells expressing the FPB isoform but not in cells expressing the FPA isoform. We now show that prostaglandin E2 (PGE2) can induce cell rounding and stimulate the formation of inositol phosphates to the same extent as PGF2alpha in cells expressing either the FPA or FPB isoforms. However, PGE2 has much lower efficacy as compared with PGF2alpha for the activation of Tcf/beta-catenin signaling in FPB-expressing cells, and the cell rounding is reversible. Interestingly, pretreatment of FPB-expressing cells with PGE2-attenuated PGF2alpha-stimulated Tcf/beta-catenin signaling in a dose-dependent manner while having no effect on PGF2alpha-stimulated inositol phosphates formation. Thus, the ratio of endogenous PGE2 and PGF2alpha has the potential to selectively regulate one signaling pathway over another. This represents a novel mechanism for the regulation of cell signaling that is distinct from regulation occurring at the level of the receptor and its effector pathways.     

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1

A previous study demonstrated that calcineurin preparations contain variable amounts of endogenous phosphate. This observation suggests that calcineurin may be regulated by protein phosphorylation. In this study we have used calcineurin as a potential substrate for eight different protein kinases and significant phosphorylation was observed only with glycogen synthase (casein) kinase-1 (CK-1). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that only subunit A of calcineurin was phosphorylated. The incorporation of 32P into calcineurin catalyzed by CK-1 ranged from 0.4 to 1.5 mol, depending on the preparation of the substrate used. Peptide mapping revealed that two major sites on calcineurin were phosphorylated. No change in calcineurin activity was observed as a result of phosphorylation. The results of this study suggest that CK-1 may be responsible for phosphorylating calcineurin in vivo.     

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.     

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle: Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3

Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase     

Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway. Implications for neuroprotection

We hypothesize that in neurodegenerative disorders such as Alzheimer's disease and human immunodeficiency virus encephalitis the neuroprotective activity of fibroblast growth factor 1 (FGF1) against several neurotoxic agents might involve regulation of glycogen synthase kinase-3beta (GSK3beta), a pathway important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a time-dependent inactivation of GSK3beta by phosphorylation at serine 9. Blocking FGF1 receptors with heparinase reduced this effect. The effects of FGF1 on GSK3beta were dependent on phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) because inhibitors of this pathway or infection with dominant negative Akt adenovirus blocked inactivation. Furthermore, treatment of neuronal cells with FGF1 resulted in ERK-independent Akt phosphorylation and beta-catenin translocation into the nucleus. On the other hand, infection with wild-type GSK3beta recombinant adenovirus-associated virus increased activity of GSK3beta and cell death, both of which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity was dependent on GSK3beta inactivation by the PI3K-Akt but was independent of ERK. Taken together these results suggest that neuroprotective effects of FGF1 might involve inactivation of GSK3beta by a pathway involving activation of the PI3K-Akt cascades.     

HaCaT keratinocyte migration is dependent on epidermal growth factor receptor signaling and glycogen synthase kinase-3alpha

After epithelial disruption by tissue injury, keratinocytes migrate from the wound edge into a provisional matrix. This process is stimulated by growth factors that signal through epidermal growth factor (EGF) receptor, including EGF, heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha), and by for example keratinocyte growth factor (KGF) and TGF-beta1 that function through different receptors. We have previously shown that keratinocyte migration induced by EGF or staurosporine is dependent on the activity of glycogen synthase kinase-3 (GSK-3). In the present study, we show that keratinocyte migration induced by TGF-beta1, KGF, EGF, TGF-alpha and staurosporine depends on EGFR signaling, involves autocrine HB-EGF expression and is potently blocked by GSK-3 inhibitors SB-415286 and LiCl. Inhibition of GSK-3 also retards wound reepithelialization in vivo in mice. Moreover, inhibition of GSK-3 activity prevented cell rounding that is an early event in EGFR-mediated keratinocyte migration. Isoform-specific GSK-3alpha and GSK-3beta knockdown and overexpression experiments with siRNAs and adenoviral constructs, respectively, revealed that GSK-3alpha is required for keratinocyte migration, whereas excessive activity of GSK-3beta is inhibitory. Thus, induction of keratinocyte migration is conveyed through EGFR, promoted by endogenous HB-EGF and requires GSK-3alpha activity.     

Regulation of angiogenesis by glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK3beta) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3beta signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3beta was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKbeta promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3beta promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3beta signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3beta activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3beta signaling enhanced capillary formation. These data suggest that GSK3beta functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.     

Phospholipase C-independent activation of glycogen synthase kinase-3beta and C-terminal Src kinase by Galphaq

It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.     

Canine prostaglandin E2 synthase (PGES) and its receptors (EP2 and EP4): expression in the corpus luteum during dioestrus

In the dog CL are the only source of the progesterone in cyclic and pregnant animals. From a high expression of cyclooxygenase 2 (Cox2) at the beginning of the dioestrus and a low one at the end it was suggested that prostanoids may play a role in the formation of the CL. This led to the hypothesis that also in the dog PGE2 of luteal origin might act as paracrine/autocrine factor. Hence, expression of the prostaglandin E2 synthase (PGES) and its receptors (EP2 and EP4) was determined during the course of dioestrus in canine CL from days 5, 15, 25, 35, 45, 65 after ovulation, following cloning of PGES using SMART RACE PCR, which revealed a high homology (82-94%) with other species. Real Time (TaqMan) PCR showed a high PGES and EP2 expression in the early CL-phase with a significant decrease thereafter. EP4 revealed a constant expression pattern throughout the life span of the CL. In situ hybridization co-localized PGES, EP2 and EP4 in the cytoplasm of the luteal cells only. In conclusion, our data suggest that in the dog PGE2 of luteal origin acts by autocrine mechanism as a luteotropic factor through its EP2 and EP4 receptors during the phase of CL-formation.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

Functional significance of glycogen synthase kinase-3 regulation by serotonin

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3β in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3β, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3β in the hippocampus. Blocking phosphorylation of GSK3α and GSK3β diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3β was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.