Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [gamma-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the beta-subunit digested with trypsin revealed one minor and two major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the beta-subunit of the insulin-stimulated receptor labeled in vivo. In contrast, the elution position of one of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Our results indicate that: tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessibility of the phosphorylation sites are different in vivo and in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.     

Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites

We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.     

Phosphorylation of the alpha subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687

The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.     

Multiple in vivo tyrosine phosphorylation sites in EphB receptors

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.     

Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites.

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCalpha in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCalpha in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory "loop" region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.     

A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor

We identified the major autophosphorylation sites in the insulin receptor and correlated their phosphorylation with the phosphotransferase activity of the receptor on synthetic peptides. The receptor, purified from Fao hepatoma cells on immobilized wheat germ agglutinin, undergoes autophosphorylation at several tyrosine residues in its beta-subunit; however, anti-phosphotyrosine antibody (alpha-PY) inhibited most of the phosphorylation by trapping the initial sites in an inactive complex. Exhaustive trypsin digestion of the inhibited beta-subunit yielded two peptides derived from the Tyr-1150 domain (Ullrich, A, Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) called pY4 and pY5. Both peptides contained 2 phosphotyrosyl residues (2Tyr(P], one corresponding to Tyr-1146 and the other to Tyr-1150 or Tyr-1151. In the absence of the alpha-PY additional sites were phosphorylated. The C-terminal domain of the beta-subunit contained phosphotyrosine at Tyr-1316 and Tyr-1322. Removal of the C-terminal domain by mild trypsinolysis did not affect the phosphotransferase activity of the beta-subunit suggesting that these sites did not play a regulatory role. Full activation of the insulin receptor during in vitro assay correlated with the appearance of two phosphopeptides in the tryptic digest of the beta-subunit, pY1 and pY1a, that were inhibited by the alpha-PY. Structural analysis suggested that pY1 and pY1a were derived from the Tyr-1150 domain and contained 3 phosphotyrosyl residues (3Tyr(P] corresponding to Tyr-1146, Tyr-1150, and Tyr-1151. The phosphotransferase of the receptor that was phosphorylated in the presence of alpha-PY at 2 tyrosyl residues in the Tyr-1150 domain was not fully activated during kinase assays carried out with saturating substrate concentrations which inhibited further autophosphorylation. During insulin stimulation of the intact cell, the 3Tyr(P) form of the Tyr-1150 domain was barely detected, whereas the 2Tyr(P) form predominated. We conclude that 1) autophosphorylation of the insulin receptor begins by phosphorylation of Tyr-1146 and either Tyr-1150 or Tyr-1151; 2) progression of the cascade to phosphorylation of the third tyrosyl residue fully activates the phosphotransferase during in vitro assay; 3) in vivo, the 2Tyr(P) form predominates, suggesting that progression of the autophosphorylation cascade to the 3Tyr(P) form is regulated during insulin stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta.

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of the inner nuclear membrane, appears to be involved in the spatial organization of the interface between nucleoplasma, lamina, and nuclear envelope. Its ability to interact with other proteins and the structural integrity of the nuclear envelope is probably regulated by phosphorylation. Here, we report nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in the native protein when purified from nuclear envelopes of mouse neuroblastoma Neuro2a cells. Five phosphorylation sites were detected by nano-electrospray mass spectrometric analysis of tryptic LAP 2 beta peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing of these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a region known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases. Ser 179 is part of a consensus site for protein kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Additionally, we identified for the first time at the protein level the LAP 2 splice variant LAP 2 epsilon in nuclear envelopes.     

Sites of in vivo phosphorylation of histone H5

Previous studies have suggested that the phosphorylation and dephosphorylation of histone H5 play an important role in controlling the condensation of avian erythrocyte chromatin. The present work locates in the polypeptide chain the major sites at which H5 is phosphorylated in vivo. The majority of the radioactivity in 32P-labeled H5 is clustered in two regions of the molecule. Nearly 50% of the 32P is found in the amino-terminal N-bromosuccinimide (NBS) peptide (residues 1-28); the remainder is confined to three phosphopeptides arising from the C-terminal half of the molecule (residues 100-200). All phosphopeptides are found in a tryptic digest of monophosphorylated H5, indicating the phosphorylation of a given site is a random event. Automatic Edman degradation of the amino-terminal fragment shows that the radioactivity is equally divided between serines at positions 3 and 7. The C-terminal phosphorylated tryptic peptides share some features with the C-terminal phosphorylation sites in H1. If, as has been postulated, the sites of phosphorylation are in or near DNA combining regions, then H5 may have two DNA combining sites. The location of the phosphorylation sites is discussed in relation to a possible mechanism for controlling chromatin condensation.     

Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.

The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.     

Phosphorylation of high mobility group 14 protein by cyclic nucleotide-dependent protein kinases

Chromosomal high mobility group (HMG) proteins have been examined as substrates for cGMP-dependent and cAMP-dependent protein kinases. Of the four HMG proteins only HMG 14 contained a major high affinity site which could be phosphorylated by both enzymes, preferentially by cGMP-dependent protein kinase. One mol of 32P was incorporated/mol of HMG 14. Kinetic analysis revealed apparent Km and Vmax of 40.5 microM and 14.7 mumol/min/mg, respectively, for cGMP-dependent protein kinase, and 123 microM and 11.1 mumol/min/mg, respectively, for cAMP-dependent protein kinase. Tryptic maps of 32P-labeled phosphopeptides of HMG 14 demonstrated phosphorylation of the same site by both enzymes. The tryptic fragment containing the major phosphorylation site was identified by amino acid composition and sequence as HMG 14 (residues 4-13): H-Lys-Val-Ser(P)-Ser-Ala-Glu-Gly-Ala-Ala-Lys-OH. HMG 14 and HMG 17 also contained minor sites which could be phosphorylated by cGMP-dependent protein kinase. Tryptic phosphopeptides mapping suggested that the same minor site was phosphorylated on both HMG 14 and 17. On the basis of amino acid composition, the tryptic peptides carrying the minor phosphorylation sites were identified as H-Leu-Ser(P)-Ala-Lys representing residues 23-26 and 27-30 of HMG 14 and HMG 17, respectively.     

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).     

Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment

Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of approximately 120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47Dco breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the 32P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed A- and B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed A- and B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of approximately 43 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 43 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.     

Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein

To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.     

Hormone-dependent phosphorylation of the avian progesterone receptor

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.     

Identification of phosphopeptides by MALDI Q-TOF MS in positive and negative ion modes after methyl esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.     

Proteolytic processing by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin-A, the major globulin of fetal calf serum

Bovine fetuin-A is a member of a glycoprotein family with a wide spectrum of functions. Until now the bovine protein has been thought to be a single-chain protein. Recently we have shown that native bovine plasma fetuin-A partially exists as a disulfide-bridged two-chain protein with a heavy N-terminal and a lighter C-terminal chain similar to the structure of human fetuin-A homologue (alpha2HS glycoprotein), and also is partially phosphorylated at residues Ser120, Ser302, Ser305 and Ser306 (Wind et al., Anal. Biochem. 317 (2003) 26-33). Both fetuin-A modifications, the phosphorylation at the four sites as well as the proteolysis which causes longer or shorter light chains (termed lc-1 and lc-2, respectively), are probably brought about by targeted enzymatic activities which still need to be defined. In this study we show that authentic bovine fetuin-A disulfide-bridged two-chain forms, which include the original C-terminus, were liberated from the single-chain precursor by metalloproteinases MMP-3 (stromelysin-1) and MMP-7 (matrilysin), but not by elastase, cathepsin E and cathepsin G. Peptide sequencing suggested cleavage sites chiefly at the Pro277-Ser278 or Arg294-His295 peptide bonds. Fetuin-A radioactive phosphorylation in vitro by protein kinase CK2 caused (32)P incorporation into the fetuin-A light chain lc-1 but not lc-2 or the fetuin-A heavy chain, as revealed by MMP assisted proteolysis. Analysis by nanoESI-MS pinpointed phosphorylation at the native phospho-residues Ser302, Ser305 and Ser306 by increased relative abundance following in vitro phosphorylation. Moreover, CK2 phosphorylation of synthetic C-terminal fetuin-A peptides, used as effective controls to the native protein, strongly implies that CK2 is involved in the in vivo phosphorylation of fetuin-A. The phosphorylation of N-terminally truncated peptide homologs seemed highly dependent on the sequence context N-terminal of the phosphorylation sites, thus providing a likely explanation for the non-phosphorylation of the light chain lc-2 in native fetuin-A.     

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.     

EGF, PGF2 alpha and insulin induce the phosphorylation of identical S6 peptides in swiss mouse 3T3 cells: effect of cAMP on early sites of phosphorylation

Epidermal growth factor (10(-9)M), prostaglandin (8.5 X 10(-7)M), F2 alpha, and insulin (10(-9)M), each of which only leads to a partial phosphorylation of 40S ribosomal protein S6, generate the same first eight phosphopeptides induced by 10% serum, suggesting all three activate a common regulatory pathway for the phosphorylation of S6. Added together, they induce almost maximal S6 phosphorylation and a phosphopeptide pattern nearly equivalent to that of serum. Unlike the agents above, 8-Br-cAMP or PGE1 has no significant effect on protein synthesis, but does induce a small increase in S6 phosphorylation. Surprisingly, the three peptides that become phosphorylated are identical with insulin-induced phosphopeptides 10b, 11, and 9, based on either comigration, limited acid hydrolysis, or V8 protease digestion. Incubation of 40S subunits with cAMP-dependent protein kinase induces the phosphorylation of these same three phosphopeptides. The in vitro and in vivo studies described here raise the possibility that cAMP could, in part, be responsible for mediating the phosphorylation of S6 during the mitogenic response.