Inhibition of glycogen synthase kinase-3beta is sufficient for airway smooth muscle hypertrophy

We examined the role of glycogen synthase kinase-3beta (GSK-3beta) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3beta, each of which inhibit GSK-3beta activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-beta, a proasthmatic cytokine. GSK-3beta inhibition increased mRNA expression of alpha-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bepsilon (eIF2Bepsilon) attenuated LiCl- and SB216763-induced protein synthesis and expression of alpha-actin and SM22, indicating that eIF2B is required for GSK-3beta-mediated airway smooth muscle hypertrophy. eIF2Bepsilon siRNA also blocked CT-1- but not TGF-beta-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3beta-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3beta, blocked protein synthesis and alpha-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-beta. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased alpha-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3beta phosphorylation. These data suggest that inhibition of the GSK-3beta/eIF2Bepsilon translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-beta-induced hypertrophy does not depend on GSK-3beta/eIF2B signaling.     

Direct and novel regulation of cAMP-dependent protein kinase by Mck1p, a yeast glycogen synthase kinase-3

The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.     

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.     

Macrocyclic bisindolylmaleimides as inhibitors of protein kinase C and glycogen synthase kinase-3

Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.     

Heparin-activated protein kinase from rabbit muscle: relationship to enzymes of the glycogen synthase kinase-3 category

A heparin-activated protein kinase has been previously identified in rabbit skeletal muscle extracts (Z. Ahmad et al. (1985) FEBS Lett. 179, 96-100). Further study has indicated that this enzyme phosphorylates rabbit muscle glycogen synthase in the same tryptic peptide(s) as the protein kinase FA/GSK-3 (glycogen synthase kinase-3) and is able to activate the ATP-Mg2+-dependent protein phosphatase. These results indicate similarities in properties between the two protein kinases. Exposure of the heparin-activated enzyme to trypsin resulted in loss of heparin activation, from 3-fold to 1.3-fold. One hypothesis suggested by this result is that the enzyme FA/GSK-3 could be a derivative of the heparin-activated enzyme that has lost heparin sensitivity. The conceptual importance of this hypothesis is that it may provide a clue to the mode of regulation of this important class of protein kinases.     

R-Ras controls axon specification upstream of glycogen synthase kinase-3beta through integrin-linked kinase

The initial event in establishing a polarized neuron is the specification of a single axon. Spatially regulated glycogen synthase kinase-3beta (GSK-3beta) activity is critical for specifying axon-dendrite fate; however, the upstream signaling of GSK-3beta in the determination of neuronal polarity still remains obscure. Here, we found that, in cultured hippocampal neurons, the small GTPase R-Ras selectively localized in a single neurite of stage 2 neurons and that its activity increased after plating and peaked between stages 2 and 3. Ectopic expression of R-Ras induced global inactivation of GSK-3beta and formation of multiple axons, whereas knockdown of endogenous R-Ras by RNA interference blocked GSK-3beta inactivation and axon formation. GSK-3beta inactivation and axon formation by R-Ras required integrin-linked kinase (ILK), and subcellular localization of ILK was strictly regulated by R-Ras-mediated phosphatidylinositol 3-kinase activity. In addition, membrane targeting of ILK was sufficient to inactivate GSK-3beta and to form multiple axons. Our study demonstrates a novel role of R-Ras and ILK upstream of GSK-3beta in the regulation of neuronal polarity.     

Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3

The glycogen-binding (G) subunit of protein phosphatase-1G is phosphorylated stoichiometrically by glycogen synthase kinase-3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP-dependent protein kinase (A-kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS-, while the C-terminal serine (site 1) is one of the two residues phosphorylated by A-kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C-terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase-2.     

Enhanced glycogen synthase kinase-3beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism

Hypoxia triggers apoptosis in a number of different cell types largely through a mitochondrial cell death pathway, which can be abrogated for the most part by enhanced glucose metabolism. The purpose of the current study was to identify intracellular signaling mechanisms that mediate hypoxia-induced apoptosis and are regulated by glucose metabolism. Hypoxia-induced apoptosis in vascular smooth muscle cells and COS-7 cells was accompanied by a significant reduction in Akt and glycogen synthase kinase-3 (GSK-3) phosphorylation resulting in increased GSK-3 activity. Morphologic features of apoptosis, as well as caspases 3 and 9 activation, were prevented by GSK-3 inhibition with either LiCl or SB216763. Phosphorylation of Akt and GSK-3 was enhanced by glucose metabolism or overexpression of the glucose transporter, GLUT1, and was prevented by glycolytic inhibition. These findings indicate that GSK-3 is an important mediator of hypoxia-induced apoptosis and that GSK-3-mediated apoptotic effects occur via activation of the mitochondrial death pathway. Moreover, the results suggest that prevention of hypoxia-mediated apoptosis by enhanced glucose transport and metabolism results, in part, from inhibition of GSK-3 activation.     

Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory

Neurofibrillary tangles (NFTs) consisting of the hyperphosphorylated microtubule-associated protein tau are a defining pathological characteristic of Alzheimer's disease (AD). Hyperphosphorylation of tau is hypothesized to impair the microtubule stabilizing function of tau, leading to the formation of paired helical filaments and neuronal death. Glycogen synthase kinase-3 (GSK-3) has been shown to be one of several kinases that mediate tau hyperphosphorylation in vitro. However, molecular mechanisms underlying overactivation of GSK-3 and its potential linkage to AD-like pathologies in vivo remain unclear. Here, we demonstrate that injection of wortmannin (a specific inhibitor of phosphoinositol-3 kinase) or GF-109203X (a specific inhibitor of protein kinase C) into the left ventricle of rat brains leads to overactivation of GSK-3, hyperphosphorylation of tau at Ser 396/404/199/202 and, most significantly, impaired spatial memory. The effects of wortmannin and GF-109203X are additive. Significantly, specific inhibition of GSK-3 activity by LiCl prevents hyperphosphorylation of tau, and spatial memory impairment resulting from PI3K and PKC inhibition. These results indicate that in vivo inhibition of phosphoinositol-3 kinase and protein kinase C results in overactivation of GSK-3 and tau hyperphosphorylation and support a direct role of GSK-3 in the formation of AD-like cognitive deficits.     

Knockdown of apoptosis signal-regulating kinase 1 modulates basal glycogen synthase kinase-3β kinase activity and regulates cell migration

GSK-3β is a basally active kinase. Axin forms a complex with GSK-3β and β-catenin; this complex promotes the GSK-3β-dependent phosphorylation of β-catenin, thereby inducing its degradation. However, the inhibition of GSK-3β provokes cell migration via the dysregulation of β-catenin. In this study, we determined that the level of apoptosis signal-regulating kinase 1 (ASK1) was lower in a metastatic breast cancer cell line, compared to that of non-metastatic cancer cell lines and the knockdown of ASK1 not only induces β-catenin activation via the inhibition of GSK-3β and collapsing the subsequent protein complex by regulating Axin dynamics, but also stimulates cell migration. Together, the blockage of the GSK-3β-β-catenin pathway resulting from the knockdown of ASK1 modulates the migration of breast cancer cells.     

Serine-threonine kinase 38 is regulated by glycogen synthase kinase-3 and modulates oxidative stress-induced cell death

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated the regulatory mechanism of STK38 and assessed its role in the cellular stress response. Among various environmental stresses, STK38 was specifically activated by H(2)O(2), and the phosphatidylinositol 3-kinase inhibitor wortmannin or AKT inhibitor IV suppressed this activation. STK38 was also activated by a constitutively active AKT1 or by GSK-3β inhibitor VII. The phosphorylation level of GSK-3β was correlated with the STK38 activity, in response to various stimuli and in different cell lines. Co-immunoprecipitation analysis revealed that GSK-3β physically interacted with STK38 in cells. GSK-3β overexpression inhibited the H(2)O(2)-stimulated STK38 activity. GSK-3β phosphorylated STK38 on residues S6 and T7 in vitro, depending largely on a PKA-mediated priming phosphorylation of STK38 on residues S10 and S11, respectively. STK38's H(2)O(2)-stimulated activity was enhanced by alanine substitution at its priming sites and/or at S6 and T7, and it was partially reduced by a phosphomimetic mutation at S6 or T7. STK38 knockdown enhanced the H(2)O(2)-induced JNK phosphorylation and cell death. Our results indicate that that GSK-3β inhibits STK38's full activation, and suggest that STK38 activation is required to prevent cell death in response to oxidative stress.     

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM alpha-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14-22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G(s) activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.