Novel phospho-specific monoclonal antibodies reveal differential regulation of tyrosine phosphorylation within the immunoreceptor tyrosine-based activation motif of the Fc receptor γ subunit leading to fine tuning of Syk activation.

The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation.

DNA microarray and signal transduction analysis in pulmonary artery smooth muscle cells from heritable and idiopathic pulmonary arterial hypertension subjects.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular smooth muscle contraction and proliferation. Here, we analyze genome-wide mRNA expression in human pulmonary arterial smooth muscle cells (HPASMC) isolated from three control, three hereditary (HPAH), and three idiopathic PAH (IPAH) subjects using the Affymetrix Human Gene ST 1.0 chip. The microarray analysis reveals the expression of 537 genes in HPAH and 1024 genes in IPAH changed compared with control HPASMC. Among those genes, 227 genes show similar directionality of expression in both HPAH and IPAH HPASMC. Ingenuity™ Pathway Analysis (IPA) suggests that many of those genes are involved in cellular growth/proliferation and cell cycle regulation and that signaling pathways such as the mitotic activators, polo-like kinases, ATM signaling are activated under PAH conditions. Furthermore, the analysis demonstrates downregulated mRNA expression of certain vasoactive receptors such as bradykinin receptor B2 (BKB2R). Using real time PCR, we verified the downregulated BKB2R expression in the PAH cells. Bradykinin-stimulated calcium influx is also decreased in PAH PASMC. IPA also identified transcriptional factors such p53 and Rb as downregulated, and FoxM1 and Myc as upregulated in both HPAH and IPAH HPASMC. The decreased level of phospho-p53 in PAH cells was confirmed with a phospho-protein array; and we experimentally show a dysregulated proliferation of both HPAH and IPAH PASMC. Together, the microarray experiments and bioinformatics analysis highlight an aberrant proliferation and cell cycle regulation in HPASMC from PAH subjects. These newly identified pathways may provide new targets for the treatment of both hereditary and idiopathic PAH.

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes.

Myotubularin-related 2 (MTMR2) is a 3-phosphoinositide lipid phosphatase with specificity towards the D-3 position of phosphoinositol 3-phosphate [PI(3)P] and phosphoinositol 3,5-bisphosphate lipids enriched on endosomal structures. Recently, we have shown that phosphorylation of MTMR2 on Ser58 is responsible for its cytoplasmic sequestration and that a phosphorylation-deficient variant (S58A) targets MTMR2 to Rab5-positive endosomes resulting in PI(3)P depletion and an increase in endosomal signaling, including a significant increase in ERK1/2 activation. Using in vitro kinase assays, cellular MAPK inhibitors, siRNA knockdown and a phosphospecific-Ser58 antibody, we now provide evidence that ERK1/2 is the kinase responsible for phosphorylating MTMR2 at position Ser58, which suggests that the endosomal targeting of MTMR2 is regulated through an ERK1/2 negative feedback mechanism. Surprisingly, treatment with multiple MAPK inhibitors resulted in a MTMR2 localization shift from Rab5-positive endosomes to the more proximal APPL1-positive endosomes. This MTMR2 localization shift was recapitulated when a double phosphorylation-deficient mutant (MTMR2 S58A/S631A) was characterized. Moreover, expression of this double phosphorylation-deficient MTMR2 variant led to a more sustained and pronounced increase in ERK1/2 activation compared with MTMR2 S58A. Further analysis of combinatorial phospho-mimetic mutants demonstrated that it is the phosphorylation status of Ser58 that regulates general endosomal binding and that the phosphorylation status of Ser631 mediates the endosomal shuttling between Rab5 and APPL1 subtypes. Taken together, these results reveal that MTMR2 compartmentalization and potential subsequent effects on endosome maturation and endosome signaling are dynamically regulated through MAPK-mediated differential phosphorylation events.

Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.

The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites-the global pattern of phosphorylation or 'phospho-form'-giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2(n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods-western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)-on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²âº concentration in boar spermatozoa during cryopreservation.

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

Phosphorylation of MeCP2 at Serine 80 regulates its chromatin association and neurological function.

Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain unclear. Although neuronal activity-induced phosphorylation of MeCP2 at serine 421 (S421) has been reported, the full spectrum of MeCP2 phosphorylation together with the in vivo function of such modifications are yet to be revealed. Here, we report the identification of several MeCP2 phosphorylation sites in normal and epileptic brains from multiple species. We demonstrate that serine 80 (S80) phosphorylation of MeCP2 is critical as its mutation into alanine (S80A) in transgenic knock-in mice leads to locomotor deficits. S80A mutation attenuates MeCP2 chromatin association at several gene promoters in resting neurons and leads to transcription changes of a small number of genes. Calcium influx in neurons causes dephosphorylation at S80, potentially contributing to its dissociation from the chromatin. We postulate that phosphorylation of MeCP2 modulates its dynamic function in neurons transiting between resting and active states within neural circuits that underlie behaviors.

Tyrosine 656 in topoisomerase IIß is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.

Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

Study of the in vivo phosphorylation of E2F1 on Ser403.

Multiple E2F1 phosphorylation sites have been described as targets of different kinases, yet their in vivo implication is uncertain. We previously reported that GSK3beta is able to phosphorylate E2F1 in vitro at Ser403 and Ser433. Recently, it has been shown that both residues are also direct targets of p38 MAP kinase. In order to determine whether Ser403 phosphorylation occurs in vivo and to elucidate its role in E2F1 transcription activity, we developed a phospho-E2F1(Ser403) antibody for use in in vivo detection studies. Our results demonstrate that endogenous E2F1 is phosphorylated in vivo on Ser403, however neither GSK3beta nor p38 MAP kinase are responsible for this event. E2F1 phosphorylation on Ser403 is induced after treatment with doxorubicin in a dose response manner. The transcriptional response of E2F1 to doxorubicin is lower in an E2F1 Ser/Ala403 mutated construct relative to the wild type, suggesting a role for Ser403 phosphorylation in DNA damage conditions. Comparative study between the expression of the bcl2 gene family induced by the wild type and E2F1 Ser/Ala403 mutant revealed a statistically different pattern between both conditions. These results suggest that phosphorylation of Ser403 could influence the selection and regulation of E2F1 target genes.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Identification and characterization of neuronal mitogen-activated protein kinase substrates using a specific phosphomotif antibody.

Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in delta-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.

c-Fos expression in preoptic nuclei as a marker of sleep rebound in the rat.

Thermoregulation is known to interfere with sleep, possibly due to a functional interaction at the level of the preoptic area (POA). Exposure to low ambient temperature (T(a)) induces sleep deprivation, which is followed by sleep rebound after a return to laboratory T(a). As two POA subregions, the ventrolateral preoptic nucleus (VLPO) and the median preoptic nucleus (MnPO), have been proposed to have a role in sleep-related processes, the expression of c-Fos and the phosphorylated form of the cAMP/Ca(2+)-responsive element-binding protein (P-CREB) was investigated in these nuclei during prolonged exposure to a T(a) of -10 degrees C and in the early phase of the recovery period. Moreover, the dynamics of the sleep rebound during recovery were studied in a separate group of animals. The results show that c-Fos expression increased in both the VLPO and the MnPO during cold exposure, but not in a specific subregion within the VLPO cluster counting grid (VLPO T-cluster). During the recovery, concomitantly with a large rapid eye movement sleep (REMS) rebound and an increase in delta power during non-rapid eye movement sleep (NREMS), c-Fos expression was high in both the VLPO and the MnPO and, specifically, in the VLPO T-cluster. In both nuclei, P-CREB expression showed spontaneous variations in basal conditions. During cold exposure, an increase in expression was observed in the MnPO, but not in the VLPO, and a decrease was observed in both nuclei during recovery. Dissociation in the changes observed between c-Fos expression and P-CREB levels, which were apparently subject to state-related non-regulatory modulation, suggests that the sleep-related changes observed in c-Fos expression do not depend on a P-CREB-mediated pathway.

Identification of four novel phosphorylation sites in estrogen receptor alpha: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2.

BACKGROUND: Estrogen receptor alpha (ERalpha) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERalpha phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERalpha phosphorylation sites exist, COS-1 cells expressing human ERalpha were labeled with [32P]H3PO4 in vivo and ERalpha tryptic phosphopeptides were isolated to identify phosphorylation sites. RESULTS: Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERalpha in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERalpha as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERalpha at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. CONCLUSION: These novel ERalpha phosphorylation sites represent new means for modulation of ERalpha activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts.

Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.

Protein tyrosine kinase characterization based on fully automated synthesis of (phospho) peptide arrays in microplates.

In view of the importance of information transfer mediated throughout the cell by recognition, phosphorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The process can be easily automated using robotic liquid handlers and is extremely rapid, sensitive, and economical. Optionally, the method can be upgraded to a higher throughput level using more powerful workstations with greater capacity, such as the Biomek FX, or any similar robotics capable of transfer-from-file logic to guide synthesis cycles.

Development of phosphorylation site-specific antibodies to nuclear receptors.

Protein phosphorylation is a versatile posttranslational modification that can regulate nuclear receptor function. Although the precise role of receptor phosphorylation is not fully understood, it appears that it functions to direct or refine receptor activity in response to particular physiological requirements. Identifying and characterizing specific nuclear receptor phosphorylation sites is an important step in elucidating the role(s) receptor phosphorylation plays in function. Although traditional methods of metabolic labeling and in vitro protein phosphorylation have been informative, receptor phosphorylation site-specific antibodies are simple and reliable tools to study receptor phosphorylation. This chapter will discuss how to develop nuclear receptor phosphorylation site-specific antibodies to elucidate function.

Fibronectin-replating experiment: procedure and analysis.

In this review protocols are described for studying protein tyrosine kinase signalling upon integrin-mediated cell adhesion. We have outlined detailed procedures for fibronectin-replating experiment, biochemical examination of the phosphotyrosine content of cellular proteins by immunoblotting using phosphorylation-specific antibodies or immunoprecipitation and analysis with general phosphotyrosine antibodies. Despite great advances that were made toward optimizing the described procedures, all these methods still remain in many respects an art, given the plentiful of variables and the extent to which the optimum conditions vary from one experimental condition to the other. Examples of performed experiments using the described procedures thus also include notes regarding variability of approaches based on experimental conditions.

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1.

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

Identification of novel in vivo Raf-1 phosphorylation sites mediating positive feedback Raf-1 regulation by extracellular signal-regulated kinase.

The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.

Selection and characterization of FcεRI phospho-ITAM specific antibodies.

Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three ß-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both ß and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.