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PubMed2004

Performance in long-term memory tasks is augmented by a phosphorylated growth factor receptor fragment.

To elucidate the role of enhanced phosphoinositide-3-kinase (PI3-kinase) activity in memory, a synthetic phosphopeptide (TAT-YPMDM) containing the p85 regulatory subunit receptor-binding motif (YXXM) coupled to the cell transduction domain of HIV-TAT protein was employed. This phosphopeptide bound the p85 subunit of PI3-kinase, and was internalized by both granule and pyramidal neurons when injected into the hippocampus. Increased lipid kinase activity and enhanced phosphorylation of the PI3-kinase substrates Akt (protein kinase B) and ribosomal S6 kinase were associated with TAT-YPMDM administration. Bilateral infusion of the phosphopeptide into the dorsal hippocampus after training improved performance in three hippocampus-dependent memory tasks: contextual fear conditioning, trace fear conditioning, and the Morris water maze. Both the biochemical and behavioral effects of the TAT-YPMDM phosphopeptide could be blocked by wortmannin. No effect was observed when a nonphosphorylated peptide (TAT-YMDM), or a second, unrelated phosphopeptide (TAT-YPLDL) was utilized. In addition, infusion of the TAT-YPMDM phosphopeptide did not interfere with memory acquisition or 4 hr memory. In addition, pretesting administration did not affect the ability to recall a previously established long-term memory. These findings suggest that stimulation of PI3-kinase activity by phosphorylated receptor fragments containing the YMDM motif augments long-term memory.

Amino Acid Motifs↗

PubMed2003

Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition.

A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.

Amino Acid Sequence↗

PubMed2006

Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry.

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.

Amino Acid Sequence↗

PubMed1993

Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding.

We have developed a competition binding assay to quantify relative affinities of isolated Src-homology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMXM or YVXM recognition motifs bound to a PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines of the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, and polyoma middle T antigens; matched, unphosphorylated sequences did not bind. A scrambled YMXM phosphopeptide or sequences corresponding to the GAP or PLC-gamma SH2 domain binding motifs of the PDGF, FGF, and EGF receptors bound to the p85 SH2 domain with 30-100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed with an SH2 domain from PLC-gamma: a phosphopeptide corresponding to the site surrounding PDGF receptor Tyr1021 binds with approximately 40-fold higher affinity than a YMXM-phosphopeptide. We conclude that essential features of specific phosphoprotein/SH2 domain interactions can be reconstituted using truncated versions of both the phosphoprotein (a phosphopeptide) and cognate SH2 domain-containing protein (the SH2 domain). SH2 domain binding specificity results from differences in affinity conferred by the linear sequence surrounding phosphotyrosine.

Amino Acid Sequence↗

PubMed1993

Insulin receptor autophosphorylation. II. Determination of autophosphorylation sites by chemical sequence analysis and identification of the juxtamembrane sites.

Autophosphorylation sites of the human insulin receptor were identified by microsequence analysis of 19 discrete tryptic [32P]phosphopeptides, purified from the autophosphorylated cytoplasmic kinase domain (CKD). Seventeen phosphopeptides were generated by cleavage at Arg and/or Lys, but two phosphopeptides were generated reproducibly by anomalous cleavages. Two new sites were identified in the juxtamembrane region of the intact insulin receptor beta-subunit (the amino terminus of the CKD), including phosphotyrosines 965 and 972. Three sites in the central region, including phosphotyrosines 1158, 1162, and 1163, were identified from six phosphopeptides; tyrosine at 1158 was the least phosphorylated. Monophosphopeptides contained phosphotyrosine at either residue 1158 or 1163, but not at 1162. Bisphosphorylation included phosphotyrosines only at 1162 and 1163. The two autophosphorylation sites near the carboxy terminus were found in seven phosphopeptides, including phosphotyrosines at 1328 and 1334. When mapped by reverse-phase high-performance liquid chromatography, these phosphopeptides eluted in the order central domain, first; carboxy-terminal region, second; and juxtamembrane (amino-terminal) domain, last.

Adenosine Triphosphate↗

PubMed1996

Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor.

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed1985

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.

Amino Acid Sequence↗

PubMed1997

Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.

Amino Acid Sequence↗

PubMed1993

Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase.

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.

Amino Acid Sequence↗

PubMed1995

c-Src phosphorylates epidermal growth factor receptor on tyrosine 845.

In the previous study [Sato et al. (1995) Biochem. Biophys. Res. Commun. 210, 844-851], we found that c-Src was associated with epidermal growth factor (EGF) receptor and activated upon EGF treatment in A431 cells. In the present study, we investigated the phosphorylation of EGF receptor by c-Src in the c-Src-EGF receptor complex. We have focused our attention to tyrosine residue 845 (Y845) of EGF receptor as a candidate for the phosphorylation site. A synthetic peptide containing Y845, named Y845 peptide, which corresponds to residue 837 to 856 of EGF receptor, was found to be phosphorylated by c-Src and used to provide the standard phosphopeptide. In addition to the autophosphorylated peptide of 25 kDa, a phosphopeptide of 7 kDa was detected in the cyanogen bromide-digested fragments of the c-Src-associated EGF receptor phosphorylated in vitro in an EGF-dependent manner. In phosphopeptide mapping, tryptic digest of the 7-kDa phosphopeptide was shown to co-migrate with that of the phosphorylated Y845 peptide. The 7-kDa phosphopeptide was found to be phosphorylated exclusively on tyrosine. These results suggest that c-Src can phosphorylate EGF receptor on Y845 in an EGF-dependent manner. Furthermore, we confirmed that the same site of the c-Src-associated EGF receptor was phosphorylated in EGF-treated A431 cells.

Amino Acid Sequence↗

PubMed2000

Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A.

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of (32)P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization-tandem mass spectrometry revealed that Ser(2194) was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK. Substitution for Ser(2194) with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser(2194) and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser(2194) may be an important mechanism for modulating NS5A biological functions.

Amino Acid Sequence↗

PubMed1991

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells.

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coom-massie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.

Animals↗

PubMed1996

Recovery from acute challenge with noradrenaline, vasopressin and angiotensin II in isolated rat hepatocytes.

We have previously established that the eleven cytosolic peptides phosphorylated in response to acute glucagon challenge in isolated rat hepatocytes undergo rapid dephosphorylation following transfer to medium free of 32PO4(3-). This dephosphorylation, far from being a simple process, is complex and asynchronous. This novel finding of asynchrony raises the question of whether, by analogy to glucagon, protein dephosphorylation is asynchronous during the recovery phase from acute challenge with noradrenaline, vasopressin or angiotensin II. One-dimensional SDS-PAGE of hepatocyte extracts indicates that noradrenaline stimulates the phosphorylation of ten cytosolic peptides, whereas vasopressin and angiotensin II stimulate the phosphorylation of six cytosolic peptides. Transfer of the hormone-challenged hepatocytes to medium devoid of 32PO4(3-) and hormone led to the rapid net dephosphorylation of the 32P-labelled phosphopeptides, albeit at different rates. In all instances, the most rapidly dephosphorylated phosphopeptide was glycogen phosphorylase. Statistical analysis indicates that during recovery from noradrenaline challenge three distinct groups of phosphopeptides can be delineated on the basis of their rates of dephosphorylation. Despite the fact that vasopressin and angiotensin II stimulate the phosphorylation of the same sub-set of phosphopeptides, there were differences in the rates of dephosphorylation of these phosphopeptides during the recovery phase from acute hormonal challenge.

Adenosine Triphosphate↗

PubMed2004

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

Alkaline Phosphatase↗

PubMed1993

Phosphorylation of ribonucleotide reductase R2 protein: in vivo and in vitro evidence of a role for p34cdc2 and CDK2 protein kinases.

Ribonucleotide reductase is responsible for supplying the deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two dissimilar protein components called R1 and R2. Immunoprecipitation of R1 and R2 proteins from [32P]orthophosphate-labeled exponentially growing mouse L cells showed that the R2 protein but not the R1 protein of ribonucleotide reductase could be phosphorylated in vivo. Two-dimensional phosphopeptide mapping experiments of trypsin-digested R2 protein showed a major spot containing more than 90% of the total radioactivity and a minor spot with the remaining radioactivity. Phosphoamino acid analysis of R2 phosphorylated protein indicated that phosphorylation occurred exclusively on serine. Protein kinase C, cAMP-dependent protein kinase, p34cdc2, and CDK2 were capable of phosphorylating the R2 protein in vitro, whereas casein kinase II was not. To determine whether any of these enzymes could phosphorylate peptides observed to be phosphorylated in actively growing cells, tryptic phosphopeptide maps of R2 that had been phosphorylated in vitro were compared with maps of R2 that had been isolated from [32P]-labeled cells. Only the phosphopeptide maps obtained with p34cdc2 and CDK2 matched the pattern found in [32P]-labeled cells. Experiments in which tryptic digests from different samples were mixed prior to two-dimensional separation demonstrated comigration of phosphopeptides obtained by in vivo phosphorylation with phosphopeptides derived from p34cdc2 or CDK2 obtained by in vitro phosphorylations. These studies indicate that protein R2 phosphorylation may play an important role in the regulation of ribonucleotide reduction, DNA synthesis, and cell cycle progression, and suggest a potentially important p34cdc2 and/or CDK2 regulation point in DNA replication.

Amino Acid Sequence↗

PubMed1998

Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese.

To examine the requirements for activation of the p21-activated protein kinase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTPgammaS), autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein kinase activity were examined. Autophosphorylation with ATP(Mg) alone was minimal with negligible protein kinase activity; the rate of autophosphorylation was increased 3-4-fold upon binding of Cdc42(GTPgammaS), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein substrates. The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTPgammaS). However, gamma-PAK autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS) did not phosphorylate peptide or protein substrates in the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) for autophosphorylation but not for phosphorylation of exogenous substrates. Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) alone showed 3 phosphopeptides, while with Cdc42(GTPgammaS) a total of 9 major phosphopeptides was observed. When gamma-PAK was autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS), 7 major phosphopeptides were observed, which were identical to peptides obtained with Cdc42(GTPgammaS) and ATP(Mg). Utilizing a recombinant mutant of gamma-PAK with alanine replacing threonine 402 in the catalytic region (T402A), it was determined that the two additional phosphopeptides observed in active PAK (peptides 7 and 8) were due to phosphorylation of threonine 402. These results show that Mn sustains autophosphorylation on serine but does not support autophosphorylation of threonine 402, which is required for activity toward exogenous substrates, or phosphorylation of these substrates.

Adenosine Triphosphate↗

PubMed2006

Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications.

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.

Amino Acid Sequence↗

PubMed1994

Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor.

Phorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of the human insulin receptor (IR) was characterized and compared in two cell types of different lineage: normal rat kidney epithelial (NRK) cells and Chinese hamster ovary (CHO) fibroblasts. PMA stimulation increased IR beta-subunit phosphorylation to 252 +/- 43 and 25- +/- 47% (+/- S.D.) of the unstimulated control in NRK and CHO cells respectively. Tryptic phosphopeptide analysis by Tricine/SDS/PAGE revealed significant differences in the PMA-stimulated phosphorylation of the IR in these two cell types. This phosphorylation of the IR was predominantly located in two tryptic phosphopeptides, and these phosphopeptides were absent in an IR mutant truncated by 43 C-terminal amino acids. The major PMA-stimulated tryptic phosphopeptide from in vivo-labelled CHO/IR was immunoprecipitated with an antibody against residues Ser1315 to Lys1329, and this precipitation was blocked with excess unlabelled peptide containing this sequence. Radiosequencing by manual Edman degradation revealed that this tryptic phosphopeptide was phosphorylated at Ser1315. This PMA-stimulated phosphorylation did not inhibit autophosphorylation of the IR in vivo. These results demonstrate that PMA-stimulated phosphorylation of the IR can exhibit significant differences when expressed in different cell types, and that Ser1315 is a major PMA-stimulated phosphorylation site on the human IR.

Amino Acid Sequence↗