The mechanism of phospholipase C-gamma1 regulation

Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.     

Characterization of Protein Complexes Containing Nucleoside Diphosphate Kinase with Characteristics of Light Signal Transduction through Phytochrome in Etiolated Pea Seedlings

Abstract— At 5 days after sowing of pea seeds in darkness, intact seedlings were either irradiated with red light for 40 s at 50 μmol/m²/s at the third internode or with red light as above and then with far-red light for 180 s at 0.4 μmol/m²/s, and the stems were sectioned from below the hook (mainly the third internodes) and placed in liquid N₂ in a mortar. The samples were well ground, and after the addition of extraction buffer, homogenates were centrifuged to prepare the crude membrane and soluble fractions. Red-light irradiation increased the phosphorylation of an 18 kDa protein, while far-red-light irradiation decreased it. The 18 kDa protein (formerly 15 kDa protein) was identified as nucleoside diphosphate kinase (EC 2.4.6) (NDP kinase) by western blotting using an NDP kinase-specific antibody. The membrane and the soluble fractions of the red-light-irradiated samples were separated by native polyacrylamide gel electrophoresis. The protein complexes prepared from the membrane and soluble fractions differed in their mobilities, as determined by two-dimensional electrophoresis and nonequilibrium pH gradient electrophoresis. The major protein spots from both samples were cut out from the gel and tested for NDP kinase and protein kinase activity. Both protein preparations showed NDP kinase activity and changes from nucleoside diphosphates and deoxynucleoside diphosphates to nucleoside triphosphates and deoxynucleoside triphosphates in the presence of [γ-³²P]ATP. Both preparations showed protein kinase phosphorylation of myelin basic protein (MBP) rather than histone H1 as protein substrates, suggesting that NDP kinase possesses a function similar to that of MAP kinase.     

Light-Induced Uncaging for Time-Resolved Observations of Biochemical Reactions by MAS NMR Spectroscopy

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time ³¹P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.     

Photodynamic Treatment with Benzoporphyrin Derivative Monoacid Ring A Produces Protein Tyrosine Phosphorylation Events and DNA Fragmentation in Murine P815 Cells

Treatment with benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of >200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the >200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photo-dynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.     

80 kDa mouse sperm protein as a substrate of protein kinase C.

Calcium and phospholipid-dependent protein kinase (PKC) activity was detected mainly in the cytosol of the mouse sperm. The PKC in the cytosol fraction was partially purified by ion-exchange chromatography. Using the partially purified PKC, the phosphorylation of PKC substrates was examined in vitro. The phosphorylation of the 80 kDa protein was enhanced by phorbol ester treatment in vitro as well as in vivo. The partial amino acid sequence of this protein was homologous with that of guanosine 5'-cyclic monophosphate (cGMP)-dependent protein kinase and myosin light chain kinase, both of which are related to ligand-receptor-transduction. The present data suggest that the activation of PKC and subsequent specific protein phosphorylation might be involved in the regulation of the zona pellucida-induced acrosome reaction.     

Prunus yedoensis Matsum. stimulates glucose uptake in L6 rat skeletal muscle cells by activating AMP-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways

Prunus yedoensis Matsum. is used as a medicinal plant to alleviate symptoms of diabetes; however, the molecular mechanism underlying its antihyperglycaemic activity is unknown. In this study, we investigated the antihyperglycaemic effects of P. yedoensis and its molecular mechanism. Prunus yedoensis leaf extract (PLE) increased the glucose uptake of phosphorylatinginsulin receptor substrate (IRS)-1, 3'-phosphoinositide-dependent kinase (PDK)-1 and Akt PLE, and also increased the phosphorylation of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK). PLE-stimulated glucose uptake was blocked by an AMPK inhibitor (Compound C) and a p38 MAPK inhibitor (SB203580). Inhibition of AMPK activity reduced p38 MAPK phosphorylation, whereas the inhibition of p38 MAPK activity did not affect AMPK phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 and Compound C reduced PLE-stimulated glucose uptake. Our results demonstrate that PLE stimulated glucose uptake by activating both insulin signalling and AMPK-p38 MAPK pathways. PLE shows potential as a natural antihyperglycaemic agent.     

PHYTOCHROME and PROTEIN PHOSPHORYLATION

The molecular mode of signal transduction triggered by phytochrome is unknown. One characteristic structural/topographic feature of the physiologically active form (Pfr) of phytochrome is that its tetrapyrrole chromophore becomes preferentially exposed in the Pfr form (compared to the Pr form). Phytochrome in its Pfr form appears to affect phosphorylation of cellular proteins. The literature on the phytochrome-mediated protein phosphorylation has been reviewed in an attempt to search for the role of the chromophore topography of phytochrome in the signal transduction process. In order to initiate a dephosphorylation-phosphorylation cascade as a possible step for the signal transduction, it may interact with a cellular protein kinase to inhibit its activity. This hypothesis has been reviewed with results from phosphorylation inhibition assays by the Pfr form of phytochrome and in light of the inhibition of protein kinase activity by tetrapyrroles in general.     

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidylcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.     

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent

BACKGROUND: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimer's disease (cross-reacting with Alzheimer's-specific AT100 antibodies). CONCLUSIONS: The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.     

Involvement of protein kinase C epsilon in thyrotropin-releasing hormone-stimulated phosphorylation of the myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells

We have shown previously that novel protein kinase Cepsilon (nPKCepsilon) plays a key role in the basal and thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion in rat pituitary GH4C1 cells (Akita et al., J. Biol. Chem. 1994, 269, 4653-4660). Here we examined the region downstream of nPKCepsilon activation in order to understand the molecular mechanism by which nPKCepsilon mediates TRH-induced signal transduction. Exposure of GH4C1 cells to TRH causes a stimulation of the phosphorylation of a p80 (Mr approximately 80 000, pI approximately 4.3) and two p19 (p19a and b; Mr approximately 19 000, pI approximately 5.6 and 5.5, respectively). Phorbol ester, a potent activator of protein kinase C (PKC), also enhances these phosphorylations, whereas bisindolylmaleimide I, a specific inhibitor of PKC, clearly inhibits the phosphorylation of p80. p80 and p19 were identified as myristoylated alanine-rich C kinase substrate (MARCKS) and stathmin, respectively, as assessed by their two-dimensional gel electrophoretic profiles and their stabilities to heat and acid treatment. In nPKCepsilon-overexpressing stable clones, the phosphorylated level of MARCKS but not stathmin was high in the resting state, and enhanced and sustained upon TRH stimulation, correlating with the increased activation of nPKCepsilon. TRH stimulates the release of MARCKS from the membrane/cytoskeletal fraction to the cytosol fraction. These results, taken together with previous data concerning PRL secretion, suggest that MARCKS, a regulatory component of the cytoskeletal architecture, is the major substrate of nPKCepsilon in vivo, and that its phosphorylation may regulate TRH-stimulated PRL secretion.     

Effects of isoflavone compounds on the activation of phospholipase C.

The effect of isoflavone compounds, genistein and daidzein, on the breakdown of inositol phospholipids in 3T3 cells was studied. Genistein (100 micrograms/ml) inhibited the stimulation of the production of inositol phosphates by bombesin. The stimulated production of inositol phosphates by AlF-4 was also inhibited by genistein (IC50 = 0.6 micrograms/ml) and daidzein (IC50 = 2 micrograms/ml). However, the catalytic activity of phospholipase-C (PLC) in 3T3 cell extracts was not inhibited by these isoflavones. These results suggest that the isoflavones inhibited the activation of PLC at the G-protein or downstream of the sequences in signal transduction. In permeabilized 3T3 cells, the inhibition of AlF-4 plus adenosine triphosphate (ATP)-dependent PLC was recovered by increasing ATP but not AlF-4. Genistein also inhibited the activity of adenosine 5'-[3-O-thiotriphosphate] (ATP[S])-dependent PLC. The effect of genistein and other inhibitors of protein tyrosine kinases and phosphatases suggests that protein tyrosine phosphorylation is not involved in the activation of PLC in 3T3 cells and that AlF-4- and ATP[S]-mediated activation of PLC involves a different mechanism from the tyrosine kinase-mediated activation of PLC. Daidzein and genistein seem to interrupt the ATP-dependent step of PLC activation by a putative G-protein.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

Singlet oxygen-induced activation of Akt/protein kinase B is independent of growth factor receptors

Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of 1O2 to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to 1O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by 1O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by 1O2 may contribute to fibroblast survival under this form of oxidative stress. 1O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by 1O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the 1O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by 1O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after 1O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by 1O2 does not involve activation of growth factor receptors, FAK or Ras protein.     

Kaempferol 3-Rhamnoside on Glutamate Release from Rat Cerebrocortical Nerve Terminals Involves P/Q-Type Ca2+ Channel and Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Pathway Suppression

Excess synaptic glutamate release has pathological consequences, and the inhibition of glutamate release is crucial for neuroprotection. Kaempferol 3-rhamnoside (KR) is a flavonoid isolated from Schima superba with neuroprotective properties, and its effecton the release of glutamate from rat cerebrocortical nerve terminals was investigated. KR produced a concentration-dependent inhibition of 4-aminopyridine (4-AP)-evoked glutamate release with half-maximal inhibitory concentration value of 17 µM. The inhibition of glutamate release by KR was completely abolished by the omission of external Ca²⁺ or the depletion of glutamate in synaptic vesicles, and it was unaffected by blocking carrier-mediated release. In addition, KR reduced the 4-AP-evoked increase in Ca²⁺ concentration, while it did not affect 4-AP-evoked membrane potential depolarization. The application of selective antagonists of voltage-dependent Ca²⁺ channels revealed that the KR-mediated inhibition of glutamate release involved the suppression of P/Q-type Ca²⁺ channel activity. Furthermore, the inhibition of release was abolished by the calmodulin antagonist, W7, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN62, but not by the protein kinase A (PKA) inhibitor, H89, or the protein kinase C (PKC) inhibitor, GF109203X. We also found that KR reduced the 4-AP-induced increase in phosphorylation of CaMKII and its substrate synapsin I. Thus, the effect of KR on evoked glutamate release is likely linked to a decrease in P/Q-type Ca²⁺ channel activity, as well as to the consequent reduction in the CaMKII/synapsin I pathway.     

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN

Abstract
We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol. 92 , 179–185). By incubating pea stem sections in ³²P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°C. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.     

Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).     

Dysferlin in a hyperCKaemic patient with caveolin 3 mutation and in C2C12 cells after p38 MAP kinase inhibition

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies.     

THE EFFECTS OF PHOTOACTIVATED PROTOPORPHYRIN ON RETICULOCYTE MEMBRANES, INTRACELLULAR ACTIVITIES and HEMOGLOBIN PRECIPITATION

Abstract— Photoactivated protoporphyrin effects were studied on reticulocyte membranes and distinct intracellular activities. Membrane bound (Na ₊ -K ₊)-ATPase activity and incorporation of ₅₅Fe into heme were almost 80% inhibited at a low concentration of protoporphyrin (3 fiM). On the other hand, a much higher protoporphyrin concentration (15 nM) was needed to cause 80% inhibition of protein synthesis. By 15 JXM protoporphyrin and treatment with light, an initial leak of hemoglobin out of the cells was observed. Electron microscopic examination showed that the lytic effects seem to be a result of membrane damage which appeared as holes in the membrane. Heinz-body-like particles of condensed hemoglobin were observed in the protoporphyrin-treated cells. The condensed hemoglobin spheres were shown to be bound to disrupted membranes prepared from photoactivated protoporphyrin-treated reticulocytes     

Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry

Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The technology is especially appropriate for profiling steady-state and dynamic phosphorylation on a proteome-wide scale, as demonstrated through detection of the native phosphorylation of cardiac mitochondrial phosphoproteins and changes in this profile arising from the activity of a protein kinase. For example, Pro-Q Diamond phosphoprotein gel stain was employed to demonstrate that among the 46 subunits of the mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase (complex I), a 42 kDa subunit is phosphorylated in the steady-state. However, exposure of mitochondria to cAMP-dependent protein kinase increases phosphorylation of this 42 kDa subunit and results in de novo phosphorylation of an 18 kDa subunit as well. Since Pro-Q Diamond dye binds to phosphorylated residues noncovalently, the staining technology is fully compatible with modern microchemical analysis procedures, such as peptide mass profiling by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and post-source decay analysis of peptide phosphorylation.