CD28 signal transduction: tyrosine phosphorylation and receptor association of phosphoinositide-3 kinase correlate with Ca2+-independent costimulatory activity

The interaction of CD28 with its counter-receptor, B7, induces a cosignal in T cells required to prevent clonal anergy and to promote antigen-dependent interleukin-2 production. The molecular basis of the CD28 cosignal is not well understood but involves the activation of protein tyrosine kinase(s) (PTK). In this report we demonstrate that CD28 cross-linking on Jurkat T leukemic cells causes the activation of at least two PTK pathways. A CD28-induced, p56^lck kinase-independent pathway causes tyrosine-phosphorylation of a 110-kDa substrate while recruitment of p56^lck kinase activity is apparently required for CD28-induced tyrosine-phosphorylation of 97- and 68-kDa substrates as well as CD28-induced increases in intracellular calcium. The tyrosine phosphorylation of p110, but not p97 or p68, correlated with CD28 calcium-independent costimulatory activity. The pp110 molecule was tentatively identified as the catalytic subunit of phosphoinositide (PI)-3 kinase based upon its coimmunoprecipitation with the p85 regulatory subunit of PI-3 kinase. PI-3 kinase protein and catalytic activity were found complexed with the CD28 receptor if the receptor was “activated” by cross-linking on the surface of intact cells prior to detergent solubilization. The kinetics of association of PI-3 kinase with the “activated” CD28 receptor was rapid, occurring within 30 s of receptor cross-linking and was stable for at least 30 min. Analysis of the CD28 cytoplasmic peptide sequence revealed a putative PI-3 kinase src homology 2 binding motif and CD28 tyrosine phosphorylation site, DYMNM. Tyrosine phosphorylation of CD28 was detected in pervanadate-treated Jurkat B2.7 cells, but not untreated cells. Pervanadate-induced tyrosine phosphorylation of CD28 correlated with receptor association of PI-3 kinase in the absence of CD28 cross-linking, suggesting that CD28 association with PI-3 kinase uses a tyrosine phosphorylation-dependent mechanism. These data provide a model for CD28 signal transduction and support a role for PI-3 kinase in mediating the CD28 calcium-independent, cyclosporin A-insensitive costimulatory signal.     

Phosphorylation at the hydrophobic site of protein kinase C Apl II is increased during intermediate term facilitation

In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.     

Toll-like receptor 2-mediated sequential activation of MyD88 and MAPKs contributes to lipopolysaccharide-induced sp-a gene expression in human alveolar epithelial cells

Surfactant proteins (SPs) produced by pulmonary epithelial cells participate in the regulation of sepsis-induced acute lung injury. Our previous study has shown that lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, can regulate sp-a gene expression in human lung carcinoma type II epithelial A549 cells. This study was further designed to evaluate the signal-transducing mechanisms of LPS-induced sp-a gene expression. Exposure of A549 cells to LPS induced SP-A mRNA and protein production in time-dependent manners. Application of toll-like receptor 2 (TLR2) siRNA into A549 cells decreased the levels of this receptor and simultaneously inhibited LPS-induced SP-A mRNA expression. Sequentially, LPS enhanced phosphorylation of mitogen-activated protein kinase (MEK) 4 and c-Jun NH₂ terminal kinase 1 (JNK1) in time-dependent manners. Application of TLR2 siRNA decreased LPS-enhanced phosphorylation of MEK4 and JNK1. After knocking-down the translation of MyD88 by RNA interference, the LPS-triggered MEK4 phosphorylation was attenuated. Consequently, LPS augmented the translocation of c-Jun from the cytoplasm to nuclei without affecting c-Fos. Pretreatment of A549 cells with SP600125, an inhibitor of JNK1, significantly lowered LPS-induced SP-A mRNA production. Analyses of an electrophoretic mobility shift assay and a reporter gene further showed that LPS increased the transactivation activity of AP-1 in A549 cells. Therefore, the present study demonstrates that LPS can induce sp-a gene expression in human type II epithelial A549 cells through TLR2-mediated sequential activation of MyD88-MEK4-JNK1-AP-1.     

Mutation of a putative AMPK phosphorylation site abolishes the repressor activity but not the nuclear targeting of the fungal glucose regulator CRE1

In filamentous ascomycetes, glucose repression is mediated by CRE1, a zinc-finger protein related to Mig1p from yeast. Five putative AMPK phosphorylation motifs identified in the glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum were mutated in a GFP::CRE1 translational fusion. Complementation experiments in Aspergillus nidulans and fluorescence microscopy analyses showed that mutation of one site (Ser₂₆₆) abolishes the repressor activity of the fusion protein but not its nuclear targeting, suggesting that an AMPK protein kinase may be involved in the function of the fungal glucose repressor. Received: 23 November 1999 / 25 January 2000     

Okadaic Acid,a Phosphatase Inhibitor,Enhances the Phorbol Ester-Induced Interleukin-1B Expression via an AP-1-Mediated Mechanism

Protein kinase C (PKC)-activating phorbol esters are known to induce the expression of several genes in monocytic cells. As the effect of serine-threonine kinases, such as PKC, is often counteracted by specific protein phosphatases, we have now examined the role of phosphatases in the regulation of the phorbol ester (PM A)-induced interleukin-B(IL-1 B) gene expression in theTHP-1 monocytic leukaemia cell line. Okadaic acid (OA) is a potent tumour promoter, the function of which is based on its activity to inhibit the serine/threonine specific phosphatases 1 and 2A (PPI and PP2A, respectively). Thus, it mimicks or potentiates the action of PKC activators in several cell types. Our data demonstrate that alone OA induced a very weak expression of IL-B mRNA, but it strongly enhanced the PMA-induced IL-1BS expression. To analyse the site of action of OA, the cells were transiently transfected with a chloramphenicol acetyl transferase (CAT) –reporter plasmid containing the AP-1 binding site as the enhancer. Alone, OA was a weak inducer of CAT–activity in these cells, but again it strongly enhanced the PMA-induced response. Similar data were obtained with cells transfected with a reporter plasmid containing the PMA-responsive element (containing a putative AP-1 binding site) of the IL-B gene. Thus, these data indicate that the PMA-induced AP-1 enhancer activity, which is required for the expression of the IL–lB gene, is controlled in these cells by PPI and/or PP2A. As OA did not synergize with PMA in the induction of expression of genes encoding the AP-1 proteins (c–fos, c–jun, junB), it is likely that OA potentiates the AP-1 enhancer activity by its effect on protein phosphorylation.     

Modeling ATM mutant proteins from missense changes confirms retained kinase activity

Ataxia‐telangiectasia mutated (ATM) is the gene mutated in the cancer‐predisposing disorder ataxia‐telangiectasia (A‐T). We modeled ATM sequence variants identified in UK A‐T patients to determine the stability and kinase activity of the resulting proteins as well as the distribution of these mutations across the coding region. Of 20 missense changes modeled, 10 proteins showed ATM kinase activity and 10 showed none. In the majority of cases the mutant ATM protein was unstable, although this was variable. Reduction in ATM kinase activity can result either from the presence of low levels of unstable mutant protein with relatively normal specific kinase activity or from stable mutant protein with deficient ATM kinase activation. Indeed, ATM mutant proteins without kinase activity toward downstream targets were still able to autophosphorylate on serine 1981, although in a much less efficient manner, suggesting that this was not sufficient for ATM activation. In terms of function, green fluorescent protein (GFP)‐tagged kinase inactive ATM proteins could form ionizing radiation (IR)‐induced foci (IRIF), at least temporarily, which colocalized with the DNA double‐strand break (DSB) marker γH2AX. Consistent with this, both kinase active and inactive mutant ATM proteins were able to interfere with phosphorylation of targets by endogenous ATM. Since the majority of missense mutations occurred C‐terminal to aa1966, including all 10 mutations with absence of kinase activity, the implication was that mutations N‐terminal to this, with exceptions, are less likely to result in loss of kinase activity and therefore, are less likely to be identified in A‐T patients. Hum Mutat 30:1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Mutational analysis of the SH2-kinase linker region of Bruton's tyrosine kinase defines alternative modes of regulation for cytoplasmic tyrosine kinase families

Bruton's tyrosine kinase (Btk) plays critical roles in B cell development and activation. Mutations of Btk cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. An Src homology domain 2-kinase linker region exists in all Src, Abl, ZAP70/Syk and Btk/Tec non-receptor tyrosine kinase families. Missense mutations in the Btk linker region can cause XLA, supporting an essential role for this protein segment. We investigated the regulatory role of the linker region in Btk function by mutational analysis. XLA-causing mutations L369F and R372G abolished Btk-mediated calcium response without affecting Btk protein stability and kinase activity significantly. Although mutation of a well-conserved tryptophan (W260A) in the linker region of the Src family kinase Hck has been shown to cause a hyperactive kinase, an analogous mutation in Btk (W395A) dramatically decreased Btk kinase activity. Tyrosine phosphorylation in the linker region was previously shown to regulate the function of Abl and ZAP70/Syk kinases. Even though tyrosine phosphorylation was detected on tyrosine 375 in the Btk linker region, no significant alteration was observed in Btk-signaling activity and biological function when this tyrosine was mutated in DT-40 cells or in Y375F knock-in mice. Our data and previous studies suggest that each cytoplasmic tyrosine kinase family has evolved a unique strategy to utilize the linker region to regulate the function of the enzyme.     

Ataxia telangiectasia: more variation at clinical and cellular levels

Ataxia telangiectasia (A‐T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A‐T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A‐T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect.     

The slowpoke channel binding protein Slob from Drosophila melanogaster exhibits regulatable protein kinase activity

The slowpoke channel binding protein Slob from Drosophila melanogaster contains a putative protein kinase domain within its amino acid sequence. We find that Slob exhibits weak and barely detectable protein kinase activity in vitro, as evidenced by autophosphorylation and by phosphorylation of exogenously added histone as substrate. The phosphorylation of histone is enhanced markedly when Slob is pretreated with the catalytic subunit of cyclic AMP-dependent protein kinase (PKAc). Mass spectrometric and mutational analysis demonstrates that the major site of phosphorylation by PKAc within Slob is serine 54. The enhancement of Slob kinase activity by PKAc pretreatment is eliminated when serine 54 in Slob is mutated to alanine (S54A). Furthermore, Slob kinase activity is enhanced in an S54E mutant that mimics phosphorylation at serine 54, and there is no further enhancement of S54E Slob kinase activity by pretreatment with PKAc. The results are consistent with the hypothesis that Slob exhibits regulatable protein kinase activity, whose activity is enhanced by phosphorylation at serine 54.     

Roscovitine inhibits thyroid hormone‐induced tail regression of the frog tadpole and reveals a role for cyclin C/Cdk8 in the establishment of the metamorphic gene expression program

The involvement of phosphorylation signaling pathways in postembryonic development of the frog is poorly understood. The thyroid hormone, 3, 5, 3′‐triiodothyronine (T₃), is essential for inducing tadpole metamorphosis and we show that the cyclin‐dependent kinase (Cdk) inhibitor, roscovitine, prevents T₃‐dependent regression of cultured tail tips from Rana catesbeiana tadpoles. Using tail tips from precociously induced and naturally metamorphosing tadpoles, our data suggest that protein phosphorylation is important in the establishment of the T₃‐dependent proapoptotic gene expression program. Our evidence indicates that Cdk8 is the most likely candidate for this proapoptotic activity with the expression of its regulatory subunit, cyclin C, identified as a novel T₃‐responsive gene. We suggest that this activity is crucial for the genetic reprogramming required for tail regression and demonstrate that protein phosphorylation is important in T₃‐induced apoptosis in normal cells during development. Developmental Dynamics 237:3787–3797, 2008. © 2008 Wiley‐Liss, Inc.     

Basic Fibroblast Growth Factor Stimulates Glomerular Mesangial Cell Proliferation Through a Protein Kinase C-Independent Pathway

Abstract

Basic Fibroblast Growth Factor (bFGF) is shown to be a potent mitogen for cultured glomerular mesangial cells. bFGF induces an increase in cell number and stimulates DNA synthesis measured by [³H]thymidine incorportion in normal as well as in protein kinase C-depleted cells. The ED₅₀ observed in both cases are nearly identical (approximately 0.04 nM) and maximal responses are obtained at 1 nM. Staurosporine, a potent protein kinase C inhibitor, does not prevent bFGF from inducing mitogenesis. On the contrary, the tumour promoting phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) and the bradykinin derivative Des-Arg⁹bradykinin that we have previously shown as mitogens for mesangial cells, fail to trigger DNA synthesis or cell proliferation upon staurosporine treatment or in protein kinase C-depleted cells. bFGF is unable to induce the association of the enzyme to membranes, the so-called translocation process, although the growth factor induces a slight production of diacylglycerol. Using a highly resolutive two-dimensional electrophoresis, we show that bFGF, in contrast to TPA, is unable to stimulate the phosphorylation of a M_r 80, 000/pI 4.5 protein, a major and specific protein kinase C substrate. By contrast, bFGF stimulates the phosphorylation of a M, 28, 000/pI 5.7-5.9 protein in normal as well as in protein kinase C-depleted cells while TPA induces this protein phosphorylation only in normal cells. Our results suggest that bFGF exerts its proliferative action on mesangial cells through a protein kinase C-independent pathway and that the growth factor does not activate anyway the enzyme in this cell type.     

Protein kinase A inhibits intermediate conductance Ca2+-activated K+ channels expressed in Xenopus oocytes

Intermediate-conductance (IK) Ca²⁺-activated K⁺ channels are expressed in many different cell types where they perform a variety of functions including cell volume regulation, transepithelial secretion, lymphocyte activation and cell cycle progression. IK channels are thought to be regulated by phosphorylation; however, whether kinases act directly on the channel is unclear. Using IK channels heterologously expressed in Xenopus oocytes, we demonstrate that IK channels are potently inhibited (60%) by the catalytic subunit of protein kinase A (PKA). Inhibition of IK channel current by PKA is abolished by mutation of four phosphorylation residues (S312, T327, S332, and T348) in the putative calmodulin-binding region of the channel. Evidence for direct modulation of the IK channel by PKA was further demonstrated using GST fusion proteins. The major site of phosphorylation was found to be serine 332; however, other residues were also phosphorylated. We conclude that IK channels can be directly regulated by the cAMP second-messenger system. The mechanism appears to involve direct phosphorylation by PKA of a modulatory locus in the cytoplasmic region of the channel, the site at which calmodulin is thought to interact. Modulation of IK channels by protein kinases may be an important mechanism regulating cell function.     

Protein Kinase C In Larval Brain of Wild-type and Dunce Memory-Mutant Drosophila

Protein kinase C activity has been measured in extracts of larval brain of Drosophila melanogaster, with the synthetic nonapeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide. Protein kinase C activity in such extracts is abolished in a Ca²⁺-dependent manner at 18°C, and partly converted to a form independent of effectors. The decay of protein kinase C activity can be prevented by leupeptin or a crude calpastatin preparation isolated from fly heads, indicating the presence of the Ca²⁺-dependent neutral protease, calpain, in larval brains. The total protein kinase C levels were nearly the same in wild type and three different dunce “memory-mutant” strains. In contrast, the soluble/particulate activity ratios were different: wild-type, 0.91; dunceM11, 0.46; dunceM11/Df(1)dm75,49, 1.23; dunce², 0.88. These data suggest that the membrane adherence of protein kinase C in larval brain is governed by the actor of genes other than dunce.     

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin‐mediated MAPK and PKB phosphorylation

Aim: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. Methods: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). Results: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen‐activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal‐regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin‐stimulated PI 3‐kinase activity increased with differentiation. Insulin‐mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. Conclusion: Components of the insulin‐signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.     

Identification and Phylogeny of a Protein Kinase Gene of White Spot Syndrome Virus

White spot syndrome virus (WSSV) is a virus infecting shrimp and other crustaceans, which is unclassified taxonomically. A 2193 bp long open reading frame, encoding a putative protein kinase (PK), was found on a 8.4 kb EcoRI fragment of WSSV proximal to the gene for the major envelope protein (VP28). The identified PK shows a high degree of homology to other viral and eukaryotic PK genes. Homology in the catalytic domains suggests that this PK is a serine/threonine protein kinase. All of the conserved PK domains are present in the WSSV PK gene product and this allowed the alignment with PK proteins from other large DNA viruses, which encode one or more PK proteins. An unrooted parsonimous phylogenetic tree was constructed and indicated that the PK gene is well conserved in all DNA virus families and hence can be used as a phylogenetic marker. Baculoviruses to date contain only a single PK gene, which is present in a separate well bootstrap-supported branch in the tree. The WSSV PK is not present in the baculovirus clade and therefore is clearly separated phylogenetically from the baculovirus PK genes. Furthermore, the WSSV PK gene does not share a most recent common ancestor with any known PK gene from other viruses. This provides further and independent evidence for the unique position of WSSV in a newly proposed genus named Whispovirus.     

Changes in protein kinase C activity during histamine release from activated rat mast cells

Rat mast cells were challenged with compound 48/80 or calcium ionophore A23187, and protein kinase C activity in the cell pellets and the amount of histamine release into the supernatant were measured. After stopping the reaction, rat mast cells were lysed in a medium which prevents alterations in phosphorylation and dephosphorylation during sample processing. Histamine was significantly released from compound 48/80-stimulated mast cells at 30 s after the stimulation. In parallel with this, protein kinase C activity in the cell pellets increased at 30 s and 1 min and returned to basal value 3 min after the stimulation. When mast cells were incubated with various concentrations of 48/80 for 30 s, the amount of histamine release and protein kinase C activity increased dependently on the concentration of 48/80. Significant histamine release from A23187-stimulated mast cells was found at 1 min after the stimulation. Also protein kinase C activity in the cell pellets increased at 1 min and returned to basal value 5 min after the stimulation. A reduction of cytosolic protein kinase C activity was observed upon 48/80 treatment in a time- and concentration-dependent manner. Further, staurosporine, a potent inhibitor of protein kinase C, inhibited 48/80-induced histamine release in parallel with the inhibition of protein kinase C activity. These findings suggest that transient increase of protein kinase C activity may be involved in the mast cell activation process.