Search

A large number of studies reported an association between elevated circulating and tissue lipid content and metabolic disorders in obesity, type 2 diabetes (T2D) and aging. This state of uncontrolled tissue lipid accumulation has been called lipotoxicity. It was later shown that excess lipid flux is mainly neutralized within lipid droplets as triglycerides, while several bioactive lipid species such as diacylglycerols (DAGs), ceramides and their derivatives have been mechanistically linked to the pathogenesis of insulin resistance (IR) by antagonizing insulin signaling and action in metabolic organs such as the liver and skeletal muscle. Skeletal muscle and the liver are the main sites of glucose disposal in the body and IR in these tissues plays a pivotal role in the development of T2D. In this review, we critically examine recent literature supporting a causal role of DAGs and ceramides in the development of IR. A particular emphasis is placed on transgenic mouse models with modulation of total DAG and ceramide pools, as well as on modulation of specific subspecies, in relation to insulin sensitivity. Collectively, although a wide number of studies converge towards the conclusion that both DAGs and ceramides cause IR in metabolic organs, there are still some uncertainties on their mechanisms of action. Recent studies reveal that subcellular localization and acyl chain composition are determinants in the biological activity of these lipotoxic lipids and should be further examined. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:21 issue:17 ispartof: location:Switzerland status: published

OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif.

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

The insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for hormonal control of metabolism. Tissues from insulin-resistant and diabetic humans exhibit defects in IRS-dependent signalling, implicating their dysregulation in the initiation and progression of metabolic disease. However, IRS1 and IRS2 are regulated through a complex mechanism involving phosphorylation of >50 serine/threonine residues (S/T) within their long, unstructured tail regions. In cultured cells, insulin-stimulated kinases (including atypical PKC, AKT, SIK2, mTOR, S6K1, ERK1/2 and ROCK1) mediate feedback (autologous) S/T phosphorylation of IRS, with both positive and negative effects on insulin sensitivity. Additionally, insulin-independent (heterologous) kinases can phosphorylate IRS1/2 under basal conditions (AMPK, GSK3) or in response to sympathetic activation and lipid/inflammatory mediators, which are present at elevated levels in metabolic disease (GRK2, novel and conventional PKCs, JNK, IKKβ, mPLK). An emerging view is that the positive/negative regulation of IRS by autologous pathways is subverted/co-opted in disease by increased basal and other temporally inappropriate S/T phosphorylation. Compensatory hyperinsulinaemia may contribute strongly to this dysregulation. Here, we examine the links between altered patterns of IRS S/T phosphorylation and the emergence of insulin resistance and diabetes.

Objective To investigate the role of serine–threonine phosphorylation on the activity of human P450c17. Design In vitro study. Setting Academic basic research laboratory. Patient(s) None. Intervention(s) P450c17 expression constructs with a FLAG-tag on either the C-terminus or N-terminus of the protein were generated. Human C-terminal FLAG-tagged P450c17 chromosomal DNA was subjected to site-directed mutagenesis. Serine 258 and threonine 260 each were mutated to alanine and aspartic acid. The mutant P450c17s were expressed in COS-7 cells, and the enzymatic activities were measured. Main Outcome Measure(s) 17α-Hydroxylase and C 17–20 lyase activities of human P450c17. Result(s) C-terminal FLAG-tagged P450c17 functioned indistinguishably from the wild-type P450c17. Mutants S258A, S258D, and T260D had significantly less 17α-hydroxylase and C 17–20 lyase activities than the wild type. Conclusion(s) Adding an epitope tag to the C-terminus of the P450c17 protein does not interfere with its activities and will be a useful tool to isolate human P450c17 protein from cultured cells. Phosphorylation of serine 258 but not threonine 260 may act as a physiologic regulator of both enzymatic activities through interaction with obligatory redox partners.

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the Ser/Thr protein kinases which phosphorylate insulin receptor substrates and the major Ser sites that are phosphorylated, as key elements in the uncoupling of insulin signalling and the induction of an insulin resistance state.

Background & Aims : Cyclic AMP (cAMP) inhibits bile salt-induced hepatocyte apoptosis; the underlying mechanisms are unclear. Methods : The effects of cAMP on taurolithocholate-3-sulfate-(TLCS)- or glycochenodesoxycholate (GCDC)-induced CD95 (Fas/APO-1) activation and apoptosis were studied in 24-hour cultured rat hepatocytes and in perfused rat liver. Results : TLCS induced a rapid oxidative stress response, c-Jun-N-terminal kinase (JNK) and epidermal growth factor (EGF) receptor (EGF-R) activation, subsequent EGF-R/CD95 association and CD95 tyrosine phosphorylation, CD95 membrane targeting, death-inducing signal complex (DISC) formation and hepatocyte apoptosis. None of these responses was triggered by cAMP; however, cAMP induced H89-sensitive serine/threonine phosphorylation of CD95. Similar data were obtained with GCDC, another proapoptotic bile acid. cAMP did not prevent the TLCS-induced oxidative stress response, JNK activation and EGF-R/CD95 association, however abolished EGF-R activation and subsequent CD95 tyrosine phosphorylation, CD95 membrane trafficking, and DISC formation in a H89-sensitive way. Also in presence of TLCS, cAMP induced rapid Ser/Thr phosphorylation of CD95 within 10 min. The effects of cAMP on the various steps of CD95 activation were also found in the intact perfused rat liver. Evidence is given that a cAMP-induced Ser/Thr phosphorylation favors internalization of CD95. Conclusions : Inhibition of bile salt-induced apoptosis by cAMP involves both PKA-dependent Ser/Thr phosphorylation of the CD95 and inhibition of EGF-R activation, which results in an inhibition of CD95 tyrosine phosphorylation, CD95 membrane targeting, and DISC formation. CD95 regulation involves complex phosphorylations with CD95-tyrosine phosphorylation favoring CD95 membrane trafficking and DISC formation, whereas CD95 Ser/Thr phosphorylation inhibits these processes.

Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in the head. The aim of the present study is to examine viability and protein phosphorylation patterns of cAMP-dependently agglutinated boar spermatozoa. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, a cell-permeable cAMP analog) at 38.5 degrees C up to 180 minutes. Aliquots of the sperm suspensions were recovered after various incubation periods and then used to examine the state of agglutination, the viability by SYBR14-PI staining and motility assay, and the state of protein phosphorylation by Western blotting and indirect immunofluorescence. In the control samples incubated without cBiMPS for 180 minutes, less than 30% of the total spermatozoa were agglutinated with one another at the heads, and more than 70% of the agglutinated spermatozoa were propidium iodide (PI)-positive (dead). However, the incubation with cBiMPS rapidly increased the percentages of head-to-head agglutinated spermatozoa to approximately 60% within 30 minutes, but did not significantly change them thereafter. In the samples incubated with cBiMPS for 180 minutes, moreover, the percentages of PI-positive cells of the agglutinated spermatozoa (approximately 30%) were significantly lower than those obtained in the control samples (more than 70%). This result was supported by the observation that the percentages of motile cells of the agglutinated spermatozoa were much higher in the samples incubated with cBiMPS for 180 minutes than in the control samples incubated without cBiMPS. As revealed by Western blotting and indirect immunofluorescence, cBiMPS-induced serine/threonine phosphorylation of the proteins (eg, >220 kd, 220 kd, 180 kd, 84 kd, and 54 kd) appeared mainly in the connecting and principal pieces of both agglutinated and free spermatozoa within 30 minutes, and additional phosphorylation occurred in the middle piece later than 30 minutes. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kd, 190 kd, 93 kd, 59 kd, 54 kd, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. These findings are consistent with the following suggestions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; and slow (later than 30 minutes) protein tyrosine phosphorylation in the connecting, middle, and principal pieces of the cAMP-dependently agglutinated spermatozoa. Based on these suggestions, we conclude that many of cAMP-dependently agglutinated spermatozoa are live cells in which cAMP-signaling cascades leading to protein serine/threonine and tyrosine phosphorylation are activated in the whole flagellum.

Insulin receptor substrates (IRS) 1 and 2 are phosphorylated on serine/threonine (Ser/Thr) residues in quiescent cells (basal phosphorylation), and phosphorylation on both Ser/Thr and tyrosine residues is increased upon insulin stimulation. To determine whether basal Ser/Thr phosphorylation of IRS proteins influences insulin receptor catalyzed tyrosine phosphorylation, recombinant FLAG epitope-tagged IRS-1 (F-IRS-1) and IRS-2 (F-IRS-2) were expressed, purified, and subjected to both dephosphorylation and hyperphosphorylation prior to phosphorylation by the insulin receptor kinase. As expected, hyperphosphorylation of F-IRS-1 and F-IRS-2 by GSK3beta decreased their subsequent phosphorylation on tyrosine residues by the insulin receptor. Surprisingly, however, dephosphorylation of the basal Ser/Thr phosphorylation sites impaired subsequent phosphorylation on tyrosine, suggesting that basal Ser/Thr phosphorylation of F-IRS-1 and F-IRS-2 plays a positive role in phosphorylation by the insulin receptor tyrosine kinase. Dephosphorylation of basal Ser/Thr sites on F-IRS-1 also significantly reduced tyrosine phosphorylation by the IGF-1 receptor. However, dephosphorylation of F-IRS-2 significantly increased phosphorylation by the IGF-1 receptor, suggesting that basal phosphorylation of IRS-2 has divergent effects on its interaction with the insulin and IGF-1 receptors. Phosphorylation of endogenous IRS-1 and IRS-2 from 3T3-L1 adipocytes was modulated in a similar manner. IRS-1 and IRS-2 from serum-fed cells were hyperphosphorylated, and dephosphorylation induced either by serum deprivation or by alkaline phosphatase treatment after immunoprecipitation led to an increase in tyrosine phosphorylation by the insulin receptor. Dephosphorylation of IRS-1 and IRS-2 immunoprecipitated from serum-deprived cells, however, resulted in inhibition of tyrosine phosphorylation by the insulin receptor. These data suggest that Ser/Thr phosphorylation can have both a positive and a negative regulatory role on tyrosine phosphorylation of IRS-1 and IRS-2 by insulin and IGF-1 receptors.

Epidermal growth factor (EGF)-stimulated proliferation of renal epithelial cells plays an important role in the recovery of kidney tubule epithelia following exposure to insult. Numerous studies have demonstrated that tyrosine phosphorylation of the focal adhesion protein paxillin mediates in part the effects of growth factors on cell growth, migration, and organization of the actin-based cytoskeleton. The experiments in this report were designed to determine the effect of EGF on paxillin phosphorylation in normal rat kidney (NRK) epithelial cells. Interestingly, treatment of NRK cells with EGF stimulated paxillin serine/threonine phosphorylation, which caused a reduction in the mobility of paxillin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The EGF-stimulated mobility shift of paxillin was independent of an intact cytoskeleton, phosphatidylinositol 3-kinase (PI 3-kinase) activation, protein kinase C (PKC) activation, and cellular adhesion. However, inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase abrogated the EGF-stimulated change in paxillin mobility. In addition, the EGF-stimulated change in paxillin serine/threonine phosphorylation was not accompanied by a profound reorganization of the actin cytoskeleton. These results identify paxillin as a component EGF signaling in renal epithelial cells and implicate members of the MAP kinase pathway as critical regulators of paxillin serine/threonine phosphorylation.

IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb(Irs1)). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)(Irs1)) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302(Irs1), Ser(P)-307(Irs1), and four others) correlated significantly with impaired insulin-stimulated Tyr(P)(Irs1). Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)(Irs1) in CHO(IR)/IRS1 cells.

Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.

Platelet-endothelial cell adhesion molecule (PECAM)-1 is a 130-kDa glycoprotein commonly used as an endothelium-specific marker. Evidence to date suggests that PECAM-1 is more than just an endothelial cell marker but is intimately involved in signal transduction pathways. This is mediated in part by phosphorylation of specific tyrosine residues within the ITAM domain of PECAM-1 and by recruitment of adapter and signaling molecules. Recently we demonstrated that PECAM-1/beta-catenin association functions to regulate beta-catenin localization and, moreover, to modulate beta-catenin tyrosine phosphorylation levels. Here we show that: 1) not only beta-catenin, but also gamma-catenin is associated with PECAM-1 in vitro and in vivo; 2) PKC enzyme directly phosphorylates purified PECAM-1; 3) PKC-derived PECAM-1 serine/threonine phosphorylation inversely correlates with gamma-catenin association; 4) PECAM-1 recruits gamma-catenin to cell-cell junctions in transfected SW480 cells; and 5) gamma-catenin may recruit PECAM-1 into an insoluble cytoskeletal fraction. These data further support the concept that PECAM-1 functions as a binder and modulator of catenins and provides a molecular mechanism for previously reported PECAM-1/cytoskeleton interactions.

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85α and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) ζ activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC ζ, the PKC ζ pathway contributing to glucose transport induced by insulin in fetal brown adipocytes., This work was supported by a grant PM98-0082 from the Ministerio de Educación y Cultura, Spain.

Fischer rat airway smooth muscle (ASM) models two potential risk factors for asthma: hyperresponsiveness to contractile agonists and to growth stimuli. The aim of this study was to identify the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not be accounted for by phospholipase C, mitogen-activated protein kinases, or tyrosine kinase activities as assessed by pharmacological inhibition and Western blotting. In contrast, depletion of phorbol ester-sensitive isoforms of the serine/threonine kinase protein kinase C (PKC) removed the difference in growth response between the rat strains. Additionally, FBS selectively induced serine/threonine phosphorylation of a 115-kDa protein in Fischer ASM cells. Enhanced activation of PKC-βI and decreased activation of PKC-δ in Fischer compared with Lewis cells following FBS stimulation were suggested by Western blotting of membrane and cytosolic fractions. The data are consistent with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.

Protein serine/threonine (ser/thr) phosphorylation is an early signaling event in macrophage activation. We investigated the changes in stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats challenged with intratracheal endotoxin. Animals were sacrificed 90 min post intratracheal lipopolysaccharide (LPS, 100 microg/rat) challenge. AMs were incubated with or without phosphatase inhibitors at 37 degrees C for 30 min. Phagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs were determined. LPS challenge activated SAPK/JNK activity and enhanced phagocytosis of AMs without altering phosphatase activity in these cells. Inhibition of phosphatase 1 and 2A activity with okadaic acid and calyculin A exerted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concentration of 1 microM increased the mean channel fluorescence intensity (MCF) and the percentage of cells engaged in phagocytosis (percent phagocytosis) in AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 microM enhanced both the MCF and percent phagocytosis of AMs from LPS-challenged rats. Calyculin A at a concentration of 10 nM increased the MCF phagocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagocytosis of AMs in both saline and LPS groups. AM CD18 expression was not altered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (calyculin A) of AM CD18 expression. These results suggest that ser/thr phosphorylation and dephosphorylation participate in mediating the phagocytic response of AMs to LPS.

We have previously shown that several protein kinases are present in higher activity levels in multidrug resistant cell lines, such as KB-V1. We have now isolated a gene that codes for a putative protein kinase, PKY, of over 130 kDa that is expressed at higher levels in multidrug-resistant cells. RNA from KB-V1 multidrug-resistant cells was reverse-transcribed and amplified by using primers derived from consensus regions of serine threonine kinases and amplified fragments were used to recover overlapping clones from a KB-V1 cDNA library. An open reading frame of 3648 bp of DNA sequence predicting 1215 aa, has been identified. This cDNA hybridizes to a mRNA of about 7 kb which is expressed at high levels in human heart and muscle tissue and overexpressed in drug-resistant KB-V1 and HL60/ADR cells. Because its closest homolog is the yeast serine/threonine kinase, Yak1, we have called this gene PKY. PKY is also related to the protein kinase family that includes Cdks, Gsk-3, and MAPK proline-directed protein kinases. This protein represents the first of its type known in mammals and may be involved in growth control pathways similar to those described for Yak1, as well as possibly playing a role in multidrug resistance.

We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors. In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system. The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS). The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues. Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample. DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS. Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations. Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well.

The serine/threonine phosphatase inhibitor, okadaic acid (OA), exerted several insulin-like effects in rat adipose cells and was, in part, synergestic with insulin. OA stimulated glucose transport activity, altered the electrophoretic mobility of IRS-1, increased the phosphorylation of the MAP-kinases ERK 1 and 2 on tyrosine sites, markedly increased MAP kinase activity and also acted synergistically with insulin in activating these enzymes. However, OA did not increase PI 3-kinase activity or the tyrosine phosphorylation of key upstream proteins in insulin's signaling cascade. Staurosporine virtually completely inhibited the insulin-stimulated glucose transport and MAP kinase activation in spite of a maintained high PI 3-kinase activity. In contrast, the effects of OA alone or in the presence of insulin were less, or not at all, affected. These data suggest that OA exerts an insulin-like effect through a serine/threonine-related pathway which is distinct from, but converges with, that of insulin downstream PI 3-kinase and upon which staurosporine exerts an inhibitory effect.

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.

We have previously shown, in rat-1 fibroblasts which stably overexpress high levels of human insulin receptor (HIR), that high glucose levels induce an inhibition of insulin receptor tyrosine kinase (IRK) activity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., Seffer, E., Seedorf, K., Haring, H. (1994) J. Biol. Chem. 269, 3381-3386]. This effect appears to be mediated through activation of protein kinase C and phosphorylation of the receptor beta-subunit on threonine or serine residues. The aim of the present study was to determine whether the juxtamembrane region or the C-terminus tail of the receptor are involved in the IRK modulation by glucose. In these domains increased serine and threonine phosphorylation was observed after phorbol ester or insulin stimulation of cells, and a regulatory function for IRK activity seems conceivable. We used an antibody directed against one potential regulatory site in the C-terminus tail, i.e. PSer1315, to study the effect of glucose. An increased signal was detected in HIR from rat-1 fibroblasts treated with phorbol 12-myristate 13-acetate or glucose (25 mM). To investigate whether this site in the C-terminus is essential for glucose-dependent IRK inhibition, rat-1 fibroblasts stably overexpressing a C-terminus-truncated human insulin receptor lacking 43 amino acids (HIR delta CT) were studied in parallel with cells expressing the wild-type receptor. As described earlier, HIR delta CT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibited the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of Ser1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtamembrane region of the insulin receptor are involved in the inhibitory effect of glucose, 293 cells transiently transfected either with wild-type HIR or HIR with a juxtamembrane deletion spanning amino acids 954-965 [des-(954-965)-HIR] were studied in parallel. As described earlier, the des-(954-965)-HIR has lost the ability to stimulate PI-3 kinase. However, 25 mM glucose equally inhibited the insulin effect on tyrosine phosphorylation of the receptor. Together, the data suggest that the regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C-terminus nor in the juxtamembrane region of the insulin receptor beta subunit.

Background The failure of cytotoxic cancer regimens to cure the most drug-resistant, well-differentiated solid tumors has been attributed to the heterogeneity of cell types that differ in their capacities for growth, differentiation, and metastases. We investigated the effect of LB1, a small molecule inhibitor of serine/threonine protein phosphatase 2A (PP2A), on its ability to inhibit a low growth fraction and highly drug-resistant solid neuroendocrine tumor, such as metastatic pheochromocytoma (PHEO). Subsequently, we evaluated the increased efficacy of chemotherapy combined with LB1. Methodology/Principal Findings The effect of LB1 and temozolomide (TMZ), a standard chemotherapeutic agent that alone only transiently suppressed the growth and regression of metastatic PHEO, was evaluated in vitro on a single PHEO cell line and in vivo on mouse model of metastatic PHEO. In the present study, we show that metastatic PHEO, for which there is currently no cure, can be eliminated by combining LB1, thereby inhibiting PP2A, with TMZ. This new treatment approach resulted in long term, disease-free survival of up to 40% of animals bearing multiple intrahepatic metastases, a disease state that the majority of patients die from. Inhibition of PP2A was associated with prevention of G1/S phase arrest by p53 and of mitotic arrest mediated by polo-like kinase 1 (Plk-1). Conclusions/Significance The elimination of DNA damage-induced defense mechanisms, through transient pharmacologic inhibition of PP2A, is proposed as a new approach for enhancing the efficacy of non-specific cancer chemotherapy regimens against a broad spectrum of low growth fraction tumors very commonly resistant to cytotoxic drugs.

Vibrio cholerae-derived zonula occludins toxin (Zot) is a multifunctional protein that reversibly disassembles intestinal tight junctions (tjs). Zot structure-function analysis has mapped this activity to aa 288–293, named AT1002. AT1002 reduced transepithelial electrical resistance across rat small intestine, ex vivo, as did Zot and its processed mature form, ΔG. AT1002 increased in vivo permeability to sugar tracers, whereas scrambled control peptides did not. Binding and barrier assays in proteinase activated receptor (PAR)2-expressing and PAR2-null cells established AT1002 activity to be PAR2 dependent. Coincident with the increased intestinal permeability, confocal microscopy of AT1002-exposed rat intestinal IEC6 cells revealed displacement of ZO-1 and occludin from intercellular boundaries. In coimmunoprecipitation assays, AT1002 decreased ZO-1-occludin and ZO-1-claudin 1 interactions coincident with PKCα-dependent ZO-1 serine/threonine phosphorylation. Further, AT1002 increased serine phosphorylation of myosin 1C and, at the same time, transiently diminished its association with ZO-1. The COOH-terminal domain of ZO-1 was required for its association with myosin 1C. These data indicate that the NH2-terminal portion of active Zot contains a PAR2-activating motif, FCIGRL, that increases PKCα-dependent ZO-1 and myosin 1C serine/threonine phosphorylation. These modifications provoke selective disengagement of ZO-1 from its binding partners, occludin, claudin 1, and myosin 1C, coincident with opening of tjs.—Goldblum, S. E., Rai, U., Tripathi, A., Thakar, M., De Leo, L., Di Toro, N., Not, T., Ramachandran, R., Puche, A. C., Hollenberg, M. D., Fasano, A. The active Zot domain (aa 288–293) increases ZO-1 and myosin 1C serine/threonine phosphorylation, alters interaction between ZO-1 and its binding partners, and induces tight junction disassembly through proteinase activated receptor 2 activation.

In prokaryotes and eukaryotes, phosphotransfer represents a common mechanism to regulate cellular functions. Recent work revealed that modulation of cellular processes by eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) are widespread in bacteria. During the last two years, first evidence on the role of Ser/Thr phosphorylation/dephosphorylation in Staphylococcus aureus has emerged leading to the identification of a functional STK and corresponding STP. Due to homology to known STKs/STPs in other bacterial species the kinase was designated PknB or alternatively Stk/Stk1, and the phosphatase Stp. The role of these enzymes in S. aureus has been examined by use of knock-out mutants and a kinase-overexpressing strain. These studies uncovered PknB/Stk and Stp as modulators of cell wall structure and susceptibility to cell wall-acting antibiotics such as certain beta-lactams and tunicamycin. By utilizing transcriptional profile analysis a strong regulatory impact of PknB/Stk on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis could be identified. Moreover, PknB/Stk is able to phosphorylate MgrA, thereby regulating activity of the efflux pump NorA. In a mouse pyelonephritis model PknB/Stk has been shown to play a role in virulence. Overall, Ser/Thr phosphorylation/dephosphorylation is a common theme in regulation of cellular functions determining metabolic activity and virulence also in the major human pathogen S. aureus.

Iridovirus is an important pathogen causing serious diseases among wild, cultured and ornamental fish. Previous studies have shown that Singapore grouper iridovirus (SGIV) contains 162 open reading frames (ORFs) from which 51 viral proteins have been confirmed by proteomics studies. ORF018R, which is conserved among vertebrate iridoviruses, is an abundant virion protein identified from SGIV. Here, immunofluorescence staining showed that ORF018R occurred at high abundance throughout SGIV-infected cells. The function of ORF018R was explored using antisense morpholino oligonucleotides (asMOs). Knockdown of ORF018R expression resulted in a reduction in the expression of viral late genes, distortion of viral particle assembly and inhibition of SGIV infection in grouper embryonic cells. Western blotting with phosphoserine-specific antibody indicated that serine phosphorylation was significantly enhanced for proteins of molecular masss 17–32 kDa by SDS-PAGE when ORF018R expression was eliminated. These proteins were analysed further by two-dimensional gel electrophoresis, and numerous protein spots were found to shift to a lower pI and higher molecular mass as a result of the loss of ORF018R function. Five proteins with enhanced phosphorylation were identified by matrix-assisted laser desorption/ionization time-of-flight (TOF)-TOF mass spectrometry, including three viral proteins: ORF049L (dUTPase), ORF075R and ORF086R, and two host proteins: subunit 12 of eukaryotic translation factor 3 and natural killer enhancing factor. These findings suggest that ORF018R is involved in serine/threonine phosphorylation in SGIV-infected late-stage cells and plays an important role in expression of viral late genes and virion assembly.

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time-dependent and dose-dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S-methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8-Br-cGMP, a cGMP agonist, and found that 8-Br-cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp-8-Br-pCPT-cGMP, an inhibitor of cGMP, reduced NO donor-stimulated prolidase activity to control levels. To test wheher the MAPK pathway is involved in this NO-dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG-cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.

The purpose of the present study is to explore whether a poly-L-arginine (poly-L-Arg)-induced increase in tight junctions (TJ) permeability of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) is associated with the Ca2+-dependent signaling and occurs following the phosphorylation/dephosphorylation of TJ proteins.Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of FD-4 transport and membrane conductance (Gt) in the presence of various inhibitors that are involved in the Ca2+-dependent pathway and the phosphorylation/dephosphorylation of TJ proteins. The resultant distribution of TJ proteins was observed using confocal laser scanning microscopy (CLSM) in an immunostaining.The increase in TJ permeability of FD4 induced by 0.2 mg/ml poly-L-Arg was not altered by treatment with inhibitors (of possible Ca2+ mobilization pathways followed by exposure of poly-L-Arg, suggesting that the promoting effect of poly-L-Arg is independent of Ca2+-related signaling. On the other hand, the protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress the increase in TJ permeability by poly-L-Arg, indicating that serine/threonine phosphorylation by way of Ca2+-independent PKC and tyrosine dephosphorylation of junction proteins may have occurred. Furthermore, immunofluorescent monitoring of ZO-1, a TJ associated protein, and occludin, an integral membrane protein localizing at TJ, after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-L-Arg treatment has shown that the internalization of ZO-1 and occludin occurred by way of serine/threonine phosphorylation by PKC activation and by way of tyrosine dephosphorylation, respectively, providing TJ disassembly.We conclude that poly-L-Arg enhances the paracellular permeability of FD-4 (i.e., macromolecules), at least, by way of both serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.

p120-catenin (p120) was originally identified as a tyrosine kinase substrate, and subsequently shown to regulate cadherin-mediated cell-cell adhesion. Binding of the p120 Arm domain to E-cadherin appears to be necessary to maintain adequate cadherin levels for strong adhesion. In contrast, the sequence amino-terminal to the Arm domain confers a negative regulatory function that is likely to be modulated by phosphorylation. Several agents that induce rapid changes in cell-cell adhesion, including PDBu, histamine, thrombin, and LPA, result in significant changes in p120 S/T phosphorylation. In some cases, these changes are PKC-dependent, but the relationship among adhesion, PKC activation, and p120 phosphorylation is unclear, in part because the relevant p120 phosphorylation sites are unknown. As a crucial step toward directly identifying the function of these modifications in adhesion, we have used two-dimensional tryptic mapping and site-directed mutagenesis to pinpoint the constitutive and PKC-modulated sites of p120 S/T phosphorylation. Of eight sites that have been identified, two were selectively phosphorylated in vitro by GSK3 beta, but in vivo treatment of cells with GSK3 beta inhibitors did not eliminate these sites. PKC stimulation in vivo induced potent dephosphorylation at S268, and partial dephosphorylation of several additional sites. Surprisingly, PKC also strongly induced phosphorylation at S873. These data directly link PKC activation to specific changes in p120 phosphorylation, and identify the target sites associated with the mechanism of PKC-dependent adhesive changes induced by agents such as histamine and PDBu.

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post-translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR alpha and beta chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR alpha and beta chains was accomplished using site-directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL-2 in response to substrate-immobilized antibody to TCR. However, mutation of serine-88 in the protein kinase A phosphorylation site of the TCR beta chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR-associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine-88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three-dimensional structure of the complex of SEB and TCR, are consistent with the view that serine-88 is important for the contact of the TCR beta chain with SEB.

Nitric oxide (NO), endogenously synthesized by inducible NO synthase (iNOS), serves antioxidant and antiapoptotic functions in settings characterized by oxidative stress and proinflammatory cytokines such as sepsis and shock. However, the redox-sensitive mechanisms regulating hepatocyte expression of iNOS are largely unknown. In interleukin-1beta (IL-1beta)-stimulated hepatocytes exposed to superoxide, we demonstrate that hepatocyte nuclear factor-4alpha (HNF-4alpha) acts as an activator of redox-associated hepatocyte iNOS expression at the level of protein, mRNA, and promoter activation. In the absence of HNF-4alpha, this redox-mediated enhancement is ablated. HNF-4alpha functional activity is associated with a unique serine/threonine kinase-mediated phosphorylation pattern. This suggests that a redox-sensitive kinase pathway targets HNF-4alpha to augment hepatocyte iNOS expression. Previous studies have not addressed a redox-dependent kinase signaling pathway that targets HNF-4alpha and enhances hepatocyte iNOS gene transcription. A unique pattern of phosphorylation determines HNF-4alpha activity as a trans-activator of IL-1beta-mediated hepatocyte iNOS expression in the presence of oxidative stress.

Over the past decade, the involvement of tyrosine kinases in signal transduction pathways evoked by cytokines has been intensively investigated. Only relatively recently have the roles of serine/threonine kinases in cytokine-induced signal transduction and anti-apoptotic pathways been examined. Cytokine receptors without intrinsic kinase activity such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and the interferons were thought to transmit their regulatory signals primarily by the receptor-associated Jak family of tyrosine kinases. This family of tyrosine kinases activates STAT transcription factors, which subsequently transduced their signals into the nucleus to modulate gene expression. Cytokine receptors with intrinsic tyrosine kinase activity such as c-Kit were initially thought to transduce their signals independently of serine/threonine kinase cascades. Recently, both of these types of receptor signaling pathways have been shown to interact with serine/threonine kinase pathways as maximal activation of these tyrosine kinase regulated cascades involve serine/threonine phosphorylation modulated by, for example MAP kinases. A common intermediate pathway initiating from cytokine receptors is the Ras/Raf/MEK/ERK (MAPK) cascade, which can result in the phosphorylation and activation of additional downstream kinases and transcription factors such as p90Rsk, CREB, Elk and Egr-1. Serine/threonine phosphorylation is also involved in the regulation of the apoptosis-controlling Bcl-2 protein, as certain phosphorylation events induced by cytokines such as IL-3 are anti-apoptotic, whereas other phosphorylation events triggered by chemotherapeutic drugs such as Paclitaxel are associated with cell death. Serine/threonine phosphorylation is implicated in the etiology of certain human cancers as constitutive serine phosphorylation of STATs 1 and 3 is observed in chronic lymphocytic leukemia and can be inhibited by the chemotherapeutic drug fludarabine. Serine/threonine phosphorylation also plays a role in the etiology of immunodeficiencies. Activated STAT5 proteins are detected in reduced levels in lymphocytes recovered from HIV-infected individuals and immunocompromised mice. Serine/threonine phosphorylation may be an important target of certain chemotherapeutic drugs which recognize the activated proteins. This meeting report and mini-review will discuss the interactions of serine/threonine kinases with signal transduction and apoptotic molecules and how some of these pathways can be controlled by chemotherapeutic drugs. Leukemia (2000) 14, 9-21.

In the Xenopus egg and oocyte system, oncogenic Ras protein can induce cell cycle arrest. The effect of oncogenic Ras on the cell cycle seems to be mediated by the Raf-Mek-Erk pathway of Ras signal transduction since constitutively active Raf, Mek, or Erk can mimic the effect of oncogenic Ras protein and since specific inhibition of these kinases can block the effect of oncogenic Ras. Using activated Xenopus egg extracts, we previously reported that the cell cycle arrest induced by oncogenic Ras correlates with the activation of a 96 kDa protein that phosphorylates histone h2b in vitro. This result raised the possibility that the 96 kDa kinase (designated as p96h2bk) is a potential target of the Raf-Mek-Erk pathway that links the pathway to the control of the cell cycle. We report here that constitutively active Mek1 could activate p96h2bk in the absence of oncogenic Ras. Moreover, inhibition of endogenous Mek by a specific inhibitor, PD 098059, suppressed the activation of p96h2bk by oncogenic Ras. These results are consistent with the concept that p96h2bk is a component or target of the Raf-Mek-Erk pathway. Furthermore, we have shown that activation of p96h2bk requires serine/threonine phosphorylation of p96h2bk.

The effect of a beta-adrenergic agonist (isoproterenol) on the tyrosine kinase activity of the insulin receptor was studied in intact adipocytes. Isoproterenol treatment rapidly (5 min) inhibited the insulin-induced autophosphorylation of the insulin receptor on tyrosine residues in intact adipocytes. The effect of insulin on the phosphorylation of cellular proteins on tyrosine residues was also inhibited by isoproterenol. In order to understand the mechanism responsible for this inhibition, two-dimensional phosphopeptide mapping of the insulin receptor was performed. The pattern of phosphorylation of the insulin receptor in freshly isolated adipocytes showed marked differences from that previously observed in cultured cells overexpressing insulin receptors. These differences include a larger proportion of receptors being phosphorylated on the three tyrosines from the kinase domain and no apparent phosphorylation of the two tyrosines close to the C-terminus after insulin stimulation. Isoproterenol markedly inhibited the effect of insulin on the phosphorylation of the three tyrosines from the kinase domain. However, this inhibition was not associated with an increase in the phosphorylation of serine/threonine peptides. Thus, this direct analysis of insulin receptor phosphorylation sites in intact adipocytes does no support the idea that beta-adrenegic agents inhibit the tyrosine kinase activity of the receptor through a serine/threonine phosphorylation-dependent mechanism.

Cycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3'UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule-1 (ICAM-1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4-10 (lymphoid endothelial cell), and ICAM-1-transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM-1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stabilization of the ICAM-1 mRNA as indicated by the half-life analysis. To determine whether this effect is dependent on the 3'UTR containing the AUUUA sequences, L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form lacking the AUUUA sequences in the 3'UTR (ICAM-1Delta3). There was no discernible difference in the effect of cycloheximide on ICAM-1 mRNA accumulation or half-life between the two types of transfected cells. The effect of cycloheximide on ICAM-1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H-7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM-1 as well as de novo synthesis of ICAM-1 in SVEC4-10 and the ICAM-1-transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM-1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect.

Rabbit antisera were raised against synthetic phosphopeptides corresponding to defined or putative sites of insulin receptor serine/threonine phosphorylation (Ser-1305, Ser-1327, Thr-1348). All of these antibodies bound specifically to the immunogenic phosphopeptide but not to the non-phosphorylated form of the peptide or to other phosphopeptides, in a microtitre plate competition enzyme-linked immunosorbent assay. Anti-PS1327 antibody reacted well with native insulin receptor prepared from phorbol ester-treated transfected CHO.T cells, but showed little reaction with receptor from untreated cells. Anti-PT1348 antibody in crude form reacted substantially with receptor from both phorbol 12-myristate 13-acetate-treated and untreated cells, but displayed specificity for phosphoreceptor after adsorption to remove antibodies reactive with dephosphopeptide. The ability to discriminate between receptor from cells treated with or without phorbol ester was retained when these antibodies were used to probe denatured receptor on Western blots. Thus anti-PS1327 and anti-PT1348 react with insulin receptor in a site-specific and phosphorylation-state-dependent manner. Anti-PT1348, but not anti-PS1327, also showed increased reactivity with receptor prepared from insulin-treated cells. The third antibody, anti-PS1305, did not react with intact insulin receptor under any conditions. It is concluded that serine 1327 is a major, previously unrecognized, site of phorbol ester-induced receptor phosphorylation, and that anti-phosphopeptide antibodies will be valuable reagents with which to examine the serine/threonine phosphorylation state of receptor extracted from tissues.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.

Treatment of cells with okadaic acid, a protein phosphatase inhibitor, leads to an insulin-resistant state without modification in the tyrosine kinase activity of the receptor toward exogenous substrates. In 3T3-L1 adipocytes, okadaic acid induced a similar dose-dependent inhibition of the insulin effect on deoxyglucose uptake, phosphatidylinositol 3-kinase (PI 3-kinase) activation, and insulin receptor substrate (IRS) 1 tyrosine phosphorylation. Simultaneously, in okadaic acid-treated 3T3-L1 adipocytes, the reduced IRS 1 tyrosine phosphorylation was linked to a decrease in its electrophoretic mobility due to phosphorylation on serine/threonine residues. This phosphorylation appeared to result from the activation of cytosolic kinase(s). Furthermore, using in vitro reconstitution, we show that, compared to IRS 1 immunopurified from untreated cells, the IRS 1 obtained from okadaic acid-treated cells had a reduced capacity to be phosphorylated by insulin receptors and, concomitantly, to bind PI 3-kinase. Taken together these data suggest that serine/threonine phosphorylation of IRS 1 induced by okadaic acid reduces the ability of the insulin receptor to phosphorylate IRS 1 and to dock one of its interacting molecules, i.e. PI 3-kinase. Finally, the inhibitory effect of okadaic acid on the stimulatory action of insulin on glucose transport suggests that the serine/threonine phosphorylation of IRS 1 might represent a key regulatory mechanism of insulin action.