Abstract: ROCKs, or Rho kinases, are serine/threonine kinases that are involved in many aspects of cell motility, from smooth-muscle contraction to cell migration and neurite outgrowth. Recent experiments have defined new functions of ROCKs in cells, including centrosome positioning and cell-size regulation, which might contribute to various physiological and pathological states.

Abstract: Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.

Abstract: A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

Abstract: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

Abstract: The protein tyrosine kinase focal adhesion kinase (FAK) plays a prominent role in integrin signaling. FAK activation, demonstrated by an increase in phosphorylation of Tyr397 as well as other sites in the protein, is best understood in the context of the engagement of integrins at the cell surface. Activation of FAK results in recruitment of a number of SH2-domain- and SH3-domain-containing proteins, which mediate signaling to several downstream pathways. FAK-dependent activation of these pathways has been implicated in a diverse array of cellular processes, including cell migration, growth factor signaling, cell cycle progression and cell survival.

Abstract: Mutations in MeCP2, which encodes a protein that has been proposed to function as a global transcriptional repressor, are the cause of Rett syndrome (RT T), an X-linked progressive neurological disorder Although the selective inactivation of MeCP2 in neurons is sufficient to confer a Rett-like phenotype in mice, the specific functions of MeCP2 in postmitotic neurons are not known We find that MeCP2 binds selectively to BDNF promoter III and functions to repress expression of the BDNF gene Membrane depolarization triggers the calcium-dependent phosphorylation and release of MeCP2 from BDNF promoter III, thereby facilitating transcription These studies indicate that MeCP2 plays a key role in the control of neuronal activity-dependent gene regulation and suggest that the deregulation of this process may underlie the pathology of RT T

Abstract: The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to environmental stress, including UV radiation. Gene disruption studies demonstrate that JNK is essential for UV-stimulated apoptosis mediated by the mitochondrial pathway by a Bax/Bak-dependent mechanism. Here, we demonstrate that JNK phosphorylates two members of the BH3-only subgroup of Bcl2-related proteins (Bim and Bmf) that are normally sequestered by binding to dynein and myosin V motor complexes. Phosphorylation by JNK causes release from the motor complexes. These proapoptotic BH3-only proteins therefore provide a molecular link between the JNK signal transduction pathway and the Bax/Bak-dependent mitochondrial apoptotic machinery.

Abstract: The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca(2+) levels, an increase in Ca(2+) alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser(1177)) and a threonine residue (Thr(495)) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser(1177) and Thr(495) in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

Abstract: The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

Abstract: Interleukin (IL)-1β is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1β and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1β affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1β (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of ∼45%. This effect occurred only when neurons were pretreated with IL-1β, whereas it was absent if IL-1β and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1β was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 μm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1β. Finally, 0.05 ng/ml IL-1β increased by ∼30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1β increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.

Abstract: Phosphorylation plays a central role in regulating the activation and signalling lifetime of protein kinases A, B (also known as Akt) and C. These kinases share three conserved phosphorylation motifs: the activation loop segment, the turn motif and the hydrophobic motif. This review focuses on how phosphorylation at each of these sites regulates the maturation, signalling and down-regulation of PKC as a paradigm for how these sites control the function of the ABC kinases.

Abstract: Glycolysis and apoptosis are considered major but independent pathways that are critical for cell survival1,2,3,4. The activity of BAD, a pro-apoptotic BCL-2 family member, is regulated by phosphorylation in response to growth/survival factors5,6,7,8. Here we undertook a proteomic analysis to assess whether BAD might also participate in mitochondrial physiology. In liver mitochondria, BAD resides in a functional holoenzyme complex together with protein kinase A7 and protein phosphatase 1 (PP1) catalytic units9, Wiskott–Aldrich family member WAVE-1 as an A kinase anchoring protein10, and glucokinase (hexokinase IV)11. BAD is required to assemble the complex in that Bad-deficient hepatocytes lack this complex, resulting in diminished mitochondria-based glucokinase activity and blunted mitochondrial respiration in response to glucose. Glucose deprivation results in dephosphorylation of BAD, and BAD-dependent cell death. Moreover, the phosphorylation status of BAD helps regulate glucokinase activity. Mice deficient for BAD or bearing a non-phosphorylatable BAD(3SA) mutant12 display abnormal glucose homeostasis including profound defects in glucose tolerance. This combination of proteomics, genetics and physiology indicates an unanticipated role for BAD in integrating pathways of glucose metabolism and apoptosis.

Abstract: Gibberellin (GA) regulates growth and development in plants. We isolated and characterized a rice GA-insensitive dwarf mutant, gid2. The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay. In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type. We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCF(GID2)-proteasome pathway.

Abstract: The serine/threonine kinase Akt is a component of many receptor signal transduction pathways and can prevent cell death following growth factor withdrawal. Here, we show that Akt inhibition of cell death is not dependent on new protein translation. Instead, Akt inhibition of cell death requires glucose hydrolysis through glycolysis. Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that included localization of the glucose transporter, Glut1, to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. To test the role of glucose uptake and phosphorylation in growth factor-independent survival, cells were transfected with Glut1 and hexokinase 1 (Glut1/HK1) cells. Glut1/HK1 cells accumulated Glut1 on the cell surface and had high glucose uptake capacity similar to that of cells with constitutively active Akt (mAkt). Unlike mAkt-expressing cells, however, they did not consume more glucose, did not maintain prolonged phosphofructokinase-1 protein levels and activity, and did not maintain pentose phosphate shuttle activity in the absence of growth factor. Nevertheless, expression of Glut1 and HK1 promoted increased cytosolic NADH and NADPH levels relative to those of the control cells upon growth factor withdrawal, prevented activation of Bax, and promoted growth factor-independent survival. These data indicate that Bax conformation is sensitive to glucose metabolism and that maintaining glucose uptake and phosphorylation can promote cell survival in the absence of growth factor. Furthermore, Akt required glucose and the ability to perform glycolysis to prevent Bax activation. The prevention of Bax activation by posttranscriptional regulation of glucose metabolism may, therefore, be a required aspect of the ability of Akt to maintain long-term cell survival in the absence of growth factors.

Abstract: Photosynthetic organisms adapt to changes in light quality by redistributing light excitation energy between two photosystems through state transition. This reorganization of antenna systems leads to an enhanced photosynthetic yield. Using a genetic approach in Chlamydomonas reinhardtii to dissect the signal transduction pathway of state transition, we identified a chloroplast thylakoid-associated serine-threonine protein kinase, Stt7, that has homologs in land plants. Stt7 is required for the phosphorylation of the major light-harvesting protein (LHCII) and for state transition.

Abstract: Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.

Abstract: Recent studies have demonstrated that transgenic (TG) expression of either Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδ B , both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIδ B but also a cytoplasmic isoform, CaMKIIδ C . In the present study, we demonstrate that expression of the δ C isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic δ C isoform of CaMKII can lead to phosphorylation of Ca 2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δ C isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca 2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIδ C can mediate phosphorylation of Ca 2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδ C activation in the pathogenesis of dilated cardiomyopathy and heart failure.

Abstract: Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.

Abstract: The presence of tangles of abnormally phosphorylated tau is a characteristic of Alzheimer's disease (AD), and the loss of synapses correlates with the degree of dementia. In addition, the overexpression of interleukin-1 (IL-1) has been implicated in tangle formation in AD. As a direct test of the requirement for IL-1 in tau phosphorylation and synaptophysin expression, IL-1 actions in neuron-microglia cocultures were manipulated. Activation of microglia with secreted beta-amyloid precursor protein or lipopolysaccharide elevated their expression of IL-1alpha, IL-1beta, and tumor necrosis factor alpha (TNFalpha) mRNA. When such activated microglia were placed in coculture with primary neocortical neurons, a significant increase in the phosphorylation of neuronal tau was accompanied by a decline in synaptophysin levels. Similar effects were evoked by treatment of neurons with recombinant IL-1beta. IL-1 receptor antagonist (IL-1ra) as well as anti-IL-1beta antibody attenuated the influence of activated microglia on neuronal tau and synaptophysin, but anti-TNFalpha antibody was ineffective. Some effects of microglial activation on neurons appear to be mediated by activation of p38 mitogen-activated protein kinase (p38-MAPK), because activated microglia stimulated p38-MAPK phosphorylation in neurons, and an inhibitor of p38-MAPK reversed the influence of IL-1beta on tau phosphorylation and synaptophysin levels. Our results, together with previous observations, suggest that activated microglia may contribute to neurofibrillary pathology in AD through their production of IL-1, activation of neuronal p38-MAPK, and resultant changes in neuronal cytoskeletal and synaptic elements.

Abstract: Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.

Abstract: Numerous reports suggest that IL-6 promotes survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell signaling protein, STAT3. Thus, agents that suppress STAT3 phosphorylation have potential for the treatment of MM. In the present report, we demonstrate that curcumin (diferuloylmethane), a pharmacologically safe agent in humans, inhibited IL-6–induced STAT3 phosphorylation and consequent STAT3 nuclear translocation. Curcumin had no effect on STAT5 phosphorylation, but inhibited the IFN-α-induced STAT1 phosphorylation. The constitutive phosphorylation of STAT3 found in certain MM cells was also abrogated by treatment with curcumin. Curcumin-induced inhibition of STAT3 phosphorylation was reversible. Compared with AG490, a well-characterized Janus kinase 2 inhibitor, curcumin was a more rapid (30 min vs 8 h) and more potent (10 μM vs 100 μM) inhibitor of STAT3 phosphorylation. In a similar manner, the dose of curcumin completely suppressed proliferation of MM cells; the same dose of AG490 had no effect. In contrast, a cell-permeable STAT3 inhibitor peptide that can inhibit the STAT3 phosphorylation mediated by Src blocked the constitutive phosphorylation of STAT3 and also suppressed the growth of myeloma cells. TNF-α and lymphotoxin also induced the proliferation of MM cells, but through a mechanism independent of STAT3 phosphorylation. In addition, dexamethasone-resistant MM cells were found to be sensitive to curcumin. Overall, our results demonstrated that curcumin was a potent inhibitor of STAT3 phosphorylation, and this plays a role in the suppression of MM proliferation.

Abstract: Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.