A NEW PROCEDURE FOR THE PHOSPHORYLATION OF NUCLEOSIDES: APPLICATION TO THE DISCOVERY OF INHIBITORS OF HIV INTEGRASE

A new phosphorylating agent for nucleosides, 2-O-(4,4′-dimethoxytrityl) ethylsulfonylethan-2′-yl-phosphate (1), has been developed by us. In the many examples studied by us, phosphorylation yields were found to be very high (about 90%). The procedure appears to be remarkably general and can be utilized for the phosphorylation of many biomolecules. Successful application of this phosphorylation method has contributed to the discovery of inhibitors of HIV integrase in our laboratory.     

GSK-3beta directly phosphorylates and activates MARK2/PAR-1

In Alzheimer disease (AD), the microtubule-associated protein tau is found hyperphosphorylated in paired helical filaments. Among many phosphorylated sites in tau, Ser-262 is the major site for abnormal phosphorylation of tau in AD brain. The kinase known to phosphorylate this particular site is MARK2, whose activation mechanism is yet to be studied. Our first finding that treatment of cells with LiCl, a selective inhibitor of another major tau kinase, glycogen synthase kinase-3beta (GSK-3beta), inhibits phosphorylation of Ser-262 of tau led us to investigate the possible involvement of GSK-3beta in MARK2 activation. In vitro kinase reaction revealed that recombinant GSK-3beta indeed phosphorylates MARK2, whereas it failed to phosphorylate Ser-262 of tau. Our further findings led us to conclude that GSK-3beta phosphorylates MARK2 on Ser-212, one of the two reported phosphorylation sites (Thr-208 and Ser-212) found in the activation loop of MARK2. Down-regulation of either GSK-3beta or MARK2 by small interfering RNAs suppressed the level of phosphorylation on Ser-262. These results, respectively, indicated that GSK-3beta is responsible for phosphorylating Ser-262 of tau through phosphorylation and activation of MARK2 and that the phosphorylation of tau at this particular site is predominantly mediated by a GSK-3beta-MARK2 pathway. These findings are of interest in the context of the pathogenesis of AD.     

Comprehensive phosphoproteome analysis of INS-1 pancreatic beta-cells using various digestion strategies coupled with liquid chromatography-tandem mass spectrometry

Type 2 diabetes results from aberrant regulation of the phosphorylation cascade in beta-cells. Phosphorylation in pancreatic beta-cells has not been examined extensively, except with regard to subcellular phosphoproteomes using mitochondria. Thus, robust, comprehensive analytical strategies are needed to characterize the many phosphorylated proteins that exist, because of their low abundance, the low stoichiometry of phosphorylation, and the dynamic regulation of phosphoproteins. In this study, we attempted to generate data on a large-scale phosphoproteome from the INS-1 rat pancreatic beta-cell line using linear ion trap MS/MS. To profile the phosphoproteome in-depth, we used comprehensive phosphoproteomic strategies, including detergent-based protein extraction (SDS and SDC), differential sample preparation (in-gel, in-solution digestion, and FASP), TiO2 enrichment, and MS replicate analyses (MS2-only and multiple-stage activation). All spectra were processed and validated by stringent multiple filtering using target and decoy databases. We identified 2467 distinct phosphorylation sites on 1419 phosphoproteins using 4 mg of INS-1 cell lysate in 24 LC-MS/MS runs, of which 683 (27.7%) were considered novel phosphorylation sites that have not been characterized in human, mouse, or rat homologues. Our informatics data constitute a rich bioinformatics resource for investigating the function of reversible phosphorylation in pancreatic beta-cells. In particular, novel phosphorylation sites on proteins that mediate the pathology of type 2 diabetes, such as Pdx-1, Nkx.2, and Srebf1, will be valuable targets in ongoing phosphoproteomics studies.     

Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.     

The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells

Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476-11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis.     

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.     

Cross-talk between PDGF and S1P signalling elucidates the inhibitory effect and potential antifibrotic action of the immunomodulator FTY720 in activated HSC-cultures

Platelet-derived growth factor (PDGF) has been shown to be essential in the activation of hepatic stellate cells (HSCs), contributing to the onset and development of hepatic fibrosis. Recently, sphingosine-1-phosphate (S1P) has been shown to be a mitogen and stimulator of chemotaxis also for HSCs. Since it has been demonstrated in several cell types that cross-talk between PDGF and S1P signalling pathways occurs, our aim was to investigate the potential antifibrotic effect of FTY720, whose phosphorylated form acts as a potent S1P receptor (S1PR) modulator, on HSCs. FTY720 inhibits cell proliferation and migration after PDGF stimulation on HSCs in a concentration range between 0.1 and 1 muM. By using compounds that block S1P signalling (PTX and VPC23019), we assessed that FTY720 also acts in an S1P receptor-independent way by decreasing the level of tyrosine phosphorylation of PDGF receptor, with subsequent inhibition of the PDGF signalling pathway. In addition, inhibition of sphingosine kinase2 (SphK2), which is responsible for FTY720 phosphorylation, by DMS/siRNA unveils a mechanism of action irrespective of its phosphorylation, in particular decreasing the level of S1P(1) on the plasma membrane. These findings led us to hypothesize a potential use of FTY720 as a potential antifibrotic drug for further clinical application.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.     

Regulation of Bcl-2 family proteins by posttranslational modifications

Like many proteins, function and abundance of Bcl-2 family proteins are influenced by posttranslational modifications. These modifications include phosphorylation, proteolytic cleavage, ubiquitination, and proteosomal degradation. These modifications, depending on cellular context and the proteins involved, can result either in a promotion of inhibition of apoptosis. Many of these modifications are governed by the activity of enzymes. As modulation of enzymatic activity can be achieved fairly efficiently using small molecules, understanding the effects of posttranslational modifications may allow for the therapeutic inhibition or promotion of apoptosis.     

ROCKII Ser1366 phosphorylation reflects the activation status

ROCK (Rho-associated protein kinase), a downstream effector of RhoA, plays an important role in many cellular processes. Accumulating evidence has shown the involvement of ROCK activation in the pathogenesis of many diseases. However, a reagent capable of detecting ROCK activation directly is lacking. In the present study, we show autophosphorylation of ROCKII in an in vitro kinase reaction. The phosphorylation sites were identified by MS, and the major phosphorylation site was found to be at the highly conserved residue Ser1366. A phospho-specific antibody was generated that can specifically recognize ROCKII Ser1366 phosphorylation. We found that the extent of Ser1366 phosphorylation of endogenous ROCKII is correlated with that of myosin light chain phosphorylation in cells in response to RhoA stimulation, showing that Ser1366 phosphorylation reflects its kinase activity. In addition, ROCKII Ser1366 phosphorylation could be detected in human breast tumours by immunohistochemical staining. The present study provides a new approach for revealing the ROCKII activation status by probing ROCKII Ser1366 phosphorylation directly in cells or tissues.     

Major Role of Epidermal Growth Factor Receptor and Src Kinases in Promoting Oxidative Stress-dependent Loss of Adhesion and Apoptosis in Epithelial Cells

A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H₂O₂-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H₂O₂ alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.     

A cell surface phosphoprotein of 48 kDa specific for myoblast fusion

The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.     

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

A peptide sequence controls the physical properties of nanoparticles formed by peptide-polymer conjugates that respond to a protein kinase a signal

We previously reported that poly(N-isopropylacrylamide) grafted with Peptide 1 (-GLRRASLG) and poly(ethylene glycol) changed its physical properties in response to an intracellular protein phosphorylation signal, protein kinase A (PKA) (Katayama, Y. et al. (2001) Macromolecules 34, 905). In this study, we investigated the effect of changing peptide structure on the lower critical solution temperature (LCST) of peptide-polymer conjugates, before and after phosphorylation with PKA. For Peptide 2 (Ac-LRRASL-), which has a formal net charge of +2 at physiological pH, the LCST of the conjugate decreased on phosphorylation. In contrast, the LCSTs of the conjugates with Peptide 3 (-ALRRASLE) and Peptide 4 (Ac-DWDALRRASL-), which have neutral net charges, were greatly increased. This suggests that the LCST of the polymer was mainly governed by two factors: the change in hydration around the polymer chain and the interpeptide electrostatic repulsion, resulting from phosphorylation. These polymers have potential for use as drug capsules that respond to cellular conditions.     

A systematic approach to the analysis of protein phosphorylation

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.