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PubMed1997

Specific stimulation of c-Fgr kinase by tyrosine-phosphorylated (poly)peptides--possible implication in the sequential mode of protein phosphorylation.

Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2002

SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities.

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.

Intracellular Signaling Peptides and Proteins↗

PubMed1995

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes.

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.

Animals↗

PubMed2005

Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.

The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found.

Chromatography, High Pressure Liquid↗

PubMed1989

Differential phosphorylation of the nucleoprotein of influenza A viruses.

An analysis of the nucleoprotein (NP) of 29 different influenza A viruses by phosphopeptide fingerprinting revealed three prototype patterns. The first, which was a complex pattern consisting of six to seven phosphopeptides, another which was relatively simple consisted of two or three phosphopeptides, and a third one which was complex but was missing the main phosphopeptide shared by the two other patterns. Phosphoserine was the only labelled phosphamino acid detected. A tentative deduction of two of the phosphate attachment sites (serine residues at positions 3 and 473) could be made by comparison of the known amino acid sequences of the NPs of 25 strains. No correlation was found between species specificity or subtype or year of isolation of the strains. During the infectious cycle the fingerprint underwent significant changes, indicating subtle phosphorylation and dephosphorylation of the NP at various stages during viral multiplication. Most of the phosphopeptides were metabolically stable; however one major phosphopeptide, which was not found in the NP of mature virions, exhibited a high turnover (presumably serine at position 3). The phosphopeptide fingerprint could be significantly influenced in vivo by the specific stimulation of cellular protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate or by its inhibition with the isoquinoline sulphonamide H7.H7 specifically inhibited the replication of influenza A viruses by deregulation of viral protein synthesis without interfering with the multiplication of a parainfluenza virus (Newcastle disease virus), an alphavirus (Semliki Forest virus) or a flavivirus (West Nile). Therefore the correct phosphorylation of the NP of influenza viruses appears to be essential for influenza virus replication.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2003

Fluorescent peptide probes for high-throughput measurement of protein phosphatases.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.

Enzyme Inhibitors↗

PubMed1995

Characterization of multiply phosphorylated peptides selectively precipitated from a pancreatic casein digest.

Anticariogenic phosphopeptides, released during the hydrolysis of casein with trypsin, contain the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu and have commercial potential as toothpaste, mouthwash, and food additives for the prevention of dental caries. To develop a commercial-scale process for the production of these peptides, we have comprehensively characterized casein phosphopeptides that were selectively precipitated using Ca2+ and ethanol from an acid-clarified (pH 4.6) pancreatic casein hydrolysate. Casein was hydrolyzed using pancreatin at 50 degrees C for 2 h. The precipitate contained a series of casein phosphopeptides that were slightly truncated relative to tryptic casein phosphopeptides. The major casein phosphopeptides released by pancreatin were beta-CN-4P(f7-24), alpha s1-CN-5P(f61-78), and alpha s1-CN-5P(f59-78), all containing the cluster sequence. The truncation of the tryptic peptides beta-CN-4P(1-25) and alpha s1-CN-5P(f59-79) resulted from the chy-motryptic and carboxypeptidase activities of the pancreatin. The peptides containing the cluster sequence constituted 77.8 +/- 6.7 mol/100 mol of the total peptides that were selectively precipitated. This composition was not significantly different from that of casein phosphopeptides produced under identical conditions using trypsin. In conclusion, pancreatin should be a suitable enzyme preparation for the production of anticariogenic casein phosphopeptides on a commercial scale.

Amino Acid Sequence↗

PubMed2000

Electron capture dissociation of singly and multiply phosphorylated peptides.

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.

Amino Acid Sequence↗

PubMed2005

A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation.

A new chemical strategy for phosphopeptide profiling is reported in this study. Phosphorylation represents one of the most important classes of posttranslational modifications of proteins. Here we report a generalized strategy that employs solid-phase capture and mass-encoding steps to selectively enrich phosphopeptides from complex mixtures. This method exploits conversion of phosphates into thiols and reactive compounds to selectively isolate products of phosphorylation. Selective isolation of phosphopeptides is achieved with a simple, novel, acid-cleavable, solid-support-bound maleimide reagent. Our chemistry efforts have focused on minimization of linker size and simplification of reagent production with incorporation of common solid-phase peptide synthesis steps. Relative quantitation was demonstrated by modifying phosphopeptides with incorporation of ethanedithiol and propanedithiol. We observed that appropriate normalization is necessary to utilize mass tag strategies for relative quantitation of posttranslational modifications. The utility of solid-phase capture was determined with model phosphopeptides, and the method was demonstrated with enriching phosphopeptides from beta-casein, alpha-casein and ovalbumin. The solid-phase capture and release methods were also demonstrated with unfractionated whole histone protein mixtures to show this compound applicability in real biological samples. The new chemical strategy will ultimately be utilized for high-throughput profiling of phosphorylation and possibly other posttranslational modifications.

Animals↗

PubMed2006

Selective sampling of multiply phosphorylated peptides by capillary electrophoresis for electrospray ionization mass spectrometry analysis.

The ionization of phosphorylated peptides in positive ion mode mass spectrometry is generally less efficient compared with the ionization of their non-phosphorylated counterparts. This can make phosphopeptides much more difficult to detect. One way to enhance the detection of phosphorylated proteins and peptides is by selectively isolating these species. Current approaches of phosphopeptide isolation are based on the favorable interactions of phosphate groups with immobilized metals. While these methods can be effective in the extraction, they can lead to incomplete sample recovery, particularly for the most strongly bound multiply phosphorylated components. A non-sorptive method of phosphopeptide isolation using capillary electrophoresis (CE) was recently reported [Zhang et al., Anal. Chem. 77 (2005) 6078]. The relatively low isoelectric points of phosphopeptides cause them to remain anionic at acidic sample pH. Hence, they can be selectively injected into the capillary by an applied field after the electroosmotic flow (EOF) is suppressed. The technique was previously coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this work, the exploitation of selective sampling in conjugation with electrospray ionization mass spectrometry (ESI-MS) is presented. The transition was not immediately straightforward. A number of major alterations were necessary for ESI interfacing. These adaptations include the choice of a suitable capillary coating for EOF control and the incorporation of organic solvent for efficient ESI. As expected, selective injection of phosphopeptides greatly enhanced the sensitivity of their detection in ESI-MS, particularly for the multiply phosphorylated species that were traditionally most problematic. Furthermore, an electrophoretic separation subsequent to the selective injection of the phosphopeptides was performed prior to analysis by ESI-MS. This allowed us to resolve the multiply phosphorylated peptides present in the samples, predominantly based on the number of phosphorylation sites on the peptides.

Caseins↗

PubMed2004

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry.

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.

Amino Acid Sequence↗

PubMed1998

Regulation of the p85/p110alpha phosphatidylinositol 3'-kinase. Distinct roles for the n-terminal and c-terminal SH2 domains.

Our previous studies on the p85/p110alpha phosphatidylinositol 3-kinase showed that the p85 regulatory subunit inhibits the p110alpha catalytic subunit, and that phosphopeptide activation of p85/p110alpha dimers reflects a disinhibition of p110alpha (Yu, J., Zhang, Y., McIlroy, J., Rordorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379-1387). We now define the domains of p85 required for inhibition of p110alpha. The iSH2 domain of p85 is sufficient to bind p110alpha but does not inhibit it. Inhibition of p110alpha requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptides increase the activity of nSH2/iSH2-p110alpha dimers, demonstrating that the nSH2 domain mediates both inhibition of p110alpha and disinhibition by phosphopeptides. In contrast, phosphopeptides did not increase the activity of iSH2/cSH2-p110alpha dimers, or dimers composed of p110alpha and an nSH2/iSH2/cSH2 construct containing a mutant nSH2 domain. Phosphopeptide binding to the cSH2 domain increased p110alpha activity only in the context of an intact p85 containing both the nSH2 domain and residues 1-322 (the SH3, proline-rich and breakpoint cluster region-homolgy domains). These data suggest that the nSH2 domain of p85 is a direct regulator of p110alpha activity. Regulation of p110alpha by phosphopeptide binding to the cSH2 domain occurs by a mechanism that requires the additional presence of the nSH2 domain and residues 1-322 of p85.

Animals↗

PubMed1993

Selective dephosphorylation of the subunits of skeletal muscle calcium channels by purified phosphoprotein phosphatases.

Multiple sites on the alpha 1 and beta subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both alpha 1 and beta subunits at similar rates, whereas calcineurin dephosphorylated beta subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the alpha 1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the alpha 1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the beta subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn2+ increased the rate and extent of dephosphorylation of all sites by calcineurin so that > 80% dephosphorylation of both alpha 1 and beta subunits was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the alpha 1 and beta subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.

Animals↗

PubMed2004

Comparative phosphorylation site mapping from gel-derived proteins using a multidimensional ES/MS-based approach.

Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. This is often a rather difficult task in that most phosphoproteins contain multiple substoichiometric sites of phosphorylation. Here we describe the multidimensional electrospray (ES) mass spectrometry (MS)-based phosphopeptide-mapping strategy developed in our laboratory. In the first dimension of the process, phosphopeptides present in a protein digest are selectively detected and collected into fractions during on-line liquid chromatography (LC)/ES/MS, which monitors for phosphopeptide-specific marker ions. This analysis generates a phosphorylation profile that can be used to assess changes in the phosphorylation state of a protein pointing to those phosphopeptides that require further investigation. The phosphopeptide-containing fractions are then analyzed in the second dimension by nano-ES with precursor-ion scan for the marker ion m/z 79. As the final step, direct sequencing of the phosphopeptides is performed by LC/ES/MS/MS. Merits and limitations of the strategy, as well as experimental details and suggestions, are described here.

Chromatography, High Pressure Liquid↗

PubMed1992

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.

Animals↗

PubMed1990

Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.

In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase C in glycerinated muscles to levels at least 40 times higher than those present during contraction of intact, phorbol ester-stimulated muscles does not activate contraction nor does it significantly modify the contraction of smooth muscle which occurs in response to the Ca2+/calmodulin-dependent phosphorylation of Ser19 by myosin light chain kinase.

Animals↗

PubMed1986

Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C.

Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain.

Amino Acid Sequence↗

PubMed1983

Protease-activated kinase II mediates multiple phosphorylation of ribosomal protein S6 in reticulocytes.

Ribosomal protein S6 was phosphorylated in vitro by the cAMP-independent protein kinase, protease-activated kinase II. The enzyme incorporated up to four phosphates into S6 as shown by analysis of the phosphorylated derivatives by two-dimensional gel electrophoresis. When tryptic digests of S6 phosphorylated by the enzyme were examined by two-dimensional peptide mapping, five phosphopeptides were observed. In contrast, only two phosphopeptides were identified with the cAMP-dependent protein kinase. One phosphopeptide was common to both peptide maps; the others were unique for each enzyme. The physiological significance of the phosphorylation by protease-activated kinase II was verified with phosphopeptide maps of S6 phosphorylated in intact cells. Highly phosphorylated forms of S6 were obtained in rabbit reticulocytes incubated in slightly acidified medium (pH 7.2 to 6.8). The phosphopeptide pattern contained the same five phosphopeptides observed with protease-activated kinase II in vitro indicating the pH-dependent phosphorylation of S6 was mediated by protease-activated kinase II.

Animals↗