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PubMed2000

Transforming growth factor-alpha-mediated growth pathways in human gastro-intestinal cell lines in relation to the gastrin autocrine pathway.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF-stimulated tyrosine kinase. Our aims were to compare EGF/TGF-alpha interactions in 2 human gastro-intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF-alpha gene. MGLVA1 expressed TGF-alpha protein as determined by immuno-cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF-alpha, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti-sera against TGF-alpha, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti-sera. Neutralisation of TGF-alpha resulted in undetectable cell-associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 x 10(6) cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 microg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 microg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC(50) 1.3 microM) and C170HM2 (9.5 microM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60(c-src) kinase, reduced the basal growth of MGLVA1 (0.67 microM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine-phosphorylated proteins and pp60(c-src) within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF.

Blotting, Southern↗

PubMed2025

Tonic signaling of the B-cell antigen-specific receptor is a common functional hallmark in chronic lymphocytic leukemia cell phosphoproteomes at early disease stages.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by highly heterogeneous genomic alterations and altered signaling pathways, with limited studies on its proteome. Our study presents a comprehensive analysis of the proteome and phosphoproteome in B-CLL and CLL-like monoclonal B-cell lymphocytosis (MBL) primary cells. Using high-resolution mass spectrometry, we identified 2970 proteins and 316 phosphoproteins across five tumor samples, including 55 newly identified phosphopeptides (ProteomeXchange-PXD005997). Our multifaceted approach also integrated protein microarrays and western blotting for further data validation in a new patient cohort of 14 patients. Despite sharing 73% of their proteomes, the phosphoproteomes varied significantly among samples, independent of cytogenetic alterations and immunoglobulin heavy variable cluster (IGHV) mutational status. We identified common functional hallmarks in B-CLL and MBL phosphoproteomes, notably tonic signaling (low-level, constitutive signaling) of the B-cell antigen-specific receptor (BCR) and nuclear factor NF-kappa-B (NF-kβ)/signal transducer and activator of transcription 3 (STAT3) pathways. Nine phosphoproteins involved in BCR signaling were further validated, showing a high correlation with early disease stages. Our study advances the field by providing a detailed perspective on the proteome and phosphoproteome of B-CLL cells, revealing signaling pathways crucial for disease development and progression. Integrating diverse proteomics techniques and identifying novel phosphopeptides offers new insights into CLL biology, potentially informing future therapeutic strategies and biomarker development for early diagnosis and personalized treatment.

Humans↗

PubMed1991

Interleukin 2 stimulates serine phosphorylation of CD45 in CTLL-2.4 cells.

Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.

Animals↗

PubMed1994

Interleukin-7 induces the association of phosphatidylinositol 3-kinase with the alpha chain of the interleukin-7 receptor.

The recently characterized receptor for interleukin (IL)-7 (IL-7R) includes a unique alpha chain as well as a common gamma chain shared with the receptors for IL-2 and IL-4. Engagement of the IL-7R activates the intracellular enzyme phosphatidylinositol (PtdIns) 3-kinase but the mechanism of PtdIns 3-kinase activation and the molecular basis of its interaction with IL-7R are not known. Here we show that IL-7 causes the 85-kDa regulatory subunit of PtdIns 3-kinase (p85), and PtdIns 3-kinase activity, to associate with the IL-7R. This interaction can be ascribed to ligand-induced phosphorylation of a single Tyr residue in the receptor's unique alpha chain. Herbimycin A, a specific protein tyrosine kinase inhibitor, suppresses not only tyrosine phosphorylation of the IL-7R but also its association with p85. A phosphopeptide corresponding to the sequence surrounding Tyr449 in the cytoplasmic tail of the IL-7R alpha chain, but not its non-phosphorylated analogue or phosphopeptides coincident with the sequences surrounding other alpha chain Tyr residues, efficiently competes out p85 binding. Replacement of Tyr449 with Phe results in a loss of p85 binding. Finally, soluble forms of the src homology 2 domains of p85, which bind directly to phosphotyrosyl peptides, specifically inhibit the association of p85 with the IL-7R. Thus, PtdIns 3-kinase recruitment occurs through a single, phosphotyrosine dependent recognition motif surrounding Tyr449 in the IL-7R alpha chain. This motif corresponds to a canonical sequence for p85 binding, Tyr(P)-X-X-Met. Since the closely related IL-2R and IL-4R also activate PtdIns 3-kinase but are devoid of such canonical motifs, our results suggest that the mechanism by which IL-7R recruits and activates PtdIns 3-kinase differs fundamentally from that used by the other receptors. PtdIns 3-kinase may, therefore, play a unique and important role in the biological response to IL-7.

Amino Acid Sequence↗

PubMed2004

Photopolymerized microtips for sample preparation in proteomic analysis.

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.

Disposable Equipment↗

PubMed1989

Myc oncoproteins are phosphorylated by casein kinase II.

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.

Amino Acid Sequence↗

PubMed1991

Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

Amino Acid Sequence↗

PubMed1993

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.

Alanine↗

PubMed1994

Distinct sub-populations of the retinoblastoma protein show a distinct pattern of phosphorylation.

Phosphorylation of the retinoblastoma protein (pRB) is assumed to regulate its growth-controlling function. Moreover, hypophosphorylated and hyperphosphorylated forms of pRB can be distinguished by virtue of the distinct affinities with which they bind to the cell nucleus. This property allows the identification of individual cell nuclei that contain pRB in one or the other form. We show here that after cells emerge from a quiescent (G0) state, conversion of their complement of pRB into a hyperphosphorylated form occurs in late G1, preceding entry into S phase by several hours. Thus, contrary to earlier reports, pRB phosphorylation is not co-ordinated with the G1-S transition and may not directly regulate it. A distinct set of phosphopeptides is found exclusively in those forms of pRB that show the loose nuclear association characteristic of the hyperphosphorylated form of pRB. Another set of phosphopeptides is found with both hypophosphorylated and hyperphosphorylated forms. This suggests the existence of distinct patterns of phosphorylation that are associated with different subsets of pRB molecules. We conclude that substantial phosphorylation of pRB exists in G1 even prior to the hyperphosphorylation point. Cyclin-dependent kinases can cause a liberation of pRB from cell nuclei in vitro. Phosphorylation by members of this kinase family is therefore likely to be directly involved in the change in nuclear affinity in vivo and the associated changes in pRB functioning.

Adenovirus E1A Proteins↗

PubMed1985

Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II.

When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.

Animals↗

PubMed2004

Intregrated analysis of the human cardiac transcriptome, proteome and phosphoproteome.

Altered expression of different classes of genes has been shown to differentiate between failing and nonfailing human hearts. However, characterization of proteins and the post-translational modifications that regulate their functions is required for understanding both the physiology of cardiac muscle and the mechanisms leading to pathological states associated with cardiac diseases. We present in this paper, an analysis of the human cardiac transcriptome, proteome and phosphoproteome. Data from two sources (i) experiments performed in our laboratory and (ii) bioinformatics searches of public databases (SWISS-PROT, NCBI, Cardiac Gene Expression Knowledge Base, Gene Ontology Consortium and Affymetrix) are reported in a relational database that allows user-designed specific queries. Microarray experiments were performed with Affymetrix Hu95Av2. Cardiac proteins were digested with trypsin. An 11 step cation exchange procedure produced fractions for analysis in separate reversed phase high-performance liquid chromatography-tandem mass spectrometry (MS/MS) experiments. Immobilized metal affinity chromatography was used to select the phosphopeptides from the same tryptic peptide mixture. They were then further investigated by MS/MS. Gel-free approaches were used to detect 267 proteins and 47 phosphopeptides. Our human cardiac database contains 447 entries. We propose the use of this platform, built with data derived from nonfailing hearts, as a template for initiating the effort to characterize the human cardiac proteome and its associated post-translational modifications.

Chromatography, Affinity↗

PubMed2006

CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii.

Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.

Acclimatization↗

PubMed2007

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis.

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.

Animals↗

PubMed2007

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2.

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.

Alanine↗

PubMed2005

Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.

A versatile integrated system has been developed for the automated enrichment and analysis of phosphopeptides by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This system utilizes two independently controlled high-performance liquid chromatography (HPLC) pumps, an autosampler and microvalves to prepare and elute samples into an ion trap mass spectrometer. The use of robust reversed-phase HPLC columns with integrated ESI emitter tips enables the reproducible detection and identification of low-femtomole quantities of phosphopeptides. The entire system is coordinated through a simple user interface by customized software. The ruggedness of the system is demonstrated by highly reproducible analyses of single and multi-protein digests, while its utility is demonstrated by the thorough evaluation of the relative immunoprecipitation efficiencies of several commercially available anti-phosphotyrosine antibodies.

Amino Acid Sequence↗

PubMed2005

Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry.

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.

Affinity Labels↗

PubMed2003

Analysis of class I phosphoinositide 3-kinase autophosphorylation sites by mass spectrometry.

This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms beta and gamma by mass spectrometry. Since discrimination and suppression effects prevented the immediate detection and sequencing of phosphopeptides in complex mixtures, a strategy was applied that involved (32)P-radiolabeling of the phosphoproteins, cleavage of the phosphoproteins with several proteases and/or cyanogen bromide, separation of the resulting peptide mixtures by micro-reversed-phase liquid chromatography, and mass spectrometric analysis of fractions containing phosphopeptides. As a result the primary autophosphorylation sites of phosphoinositide 3-kinase p110beta and p110gamma subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively.

Amino Acid Sequence↗

PubMed1998

Competitive and slow-binding inhibition of calcineurin by drug x immunophilin complexes.

The calcium- and calmodulin-activated protein phosphatase calcineurin (CN) is the target for the immunosuppressive drugs FK506 and cyclosporin A (CsA) when bound to their intracellular receptor proteins, the immunophilins known as FK506-binding protein (FKBP) and cyclophilin A (CypA), respectively. Investigation of the reaction kinetics for inhibition of CN using progress curves of [33P]phosphopeptide hydrolysis revealed slow-binding inhibition by the FK506 . FKBP complex. Final steady-state velocities were extracted by curve fitting over a range of substrate and inhibitor concentrations; the data fit well to a simple competitive inhibition model with a Ki of 14 nM for the FK506 . FKBP complex. The FKBP complex with L-732,531, an analog of FK506 containing a hydroxyethylindole substituent, was significantly more potent than FK506 x FKBP and was investigated in greater detail. The hyperbolic dependencies of the initial velocities and the first-order rate constants for the approach to steady state upon the concentration of L-732,531 x FKBP were consistent with a two-step inhibition mechanism in which the initial E x I complex slowly isomerizes to a more stable E x I* form. The reverse isomerization rate constant with L-732,531 . FKBP was markedly slower than that with FK506 x FKBP and is likely responsible for the higher affinity of the former for CN. Inhibition of CN by the CsA x CypA complex was not time-dependent, but the data did conform to a competitive inhibition model like FK506 x FKBP. These results are consistent with the hypothesis that both classes of drug x immunophilin complexes interact with a common locus on CN which excludes phosphopeptide binding in the enzyme's active site.

Amino Acid Sequence↗