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PubMed1988

Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A.

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.

Animals↗

PubMed1990

Identification of two cAMP-dependent phosphorylation sites on erythrocyte protein 4.1.

In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem. 260, 9073-9076). The 10 kDa domain also contains the spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase inhibits the binding of protein 4.1 to spectrin and actin (Ling, E., Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In this study, we have identified two sites on protein 4.1 which account for 80% of the phosphate incorporated into protein 4.1 during metabolic labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More than 95% of the 32P incorporated into protein 4.1 was in the form of phosphoserine. Reverse-phase HPLC of the peptides generated by digestion of the isolated protein with trypsin or endoproteinase lysine C produced two major radioactive peaks. The phosphorylation sites, identified by gas phase sequencing of the purified phosphopeptides and confirmed by determining the residues converted to S-ethylcysteine by reacting the phosphopeptides with ethanethiol under alkaline conditions, were Ser-331, in the 16 kDa domain and Ser-467, in the 10 kDa domain.

Amino Acid Sequence↗

PubMed1991

Insulin and phorbol ester stimulate phosphorylation of a 15,000 dalton membrane protein in rat diaphragm in a similar manner.

The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.

Adenosine Triphosphate↗

PubMed1991

In vitro phosphorylation and inactivation of spinach leaf sucrose-phosphate synthase by an endogenous protein kinase.

(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.

Adenosine Triphosphate↗

PubMed1993

Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-dependent protein kinase in vitro.

The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the alpha 4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the alpha 4 subunits. The 32P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32P-labeled alpha 4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.

Animals↗

PubMed1996

Peptide degradation: effect of substrate phosphorylation on aminopeptidasic hydrolysis.

The effect of substrate phosphorylation on the susceptibility to exopeptidasic attack by leucyl aminopeptidase of swine kidney, alanyl aminopeptidase from human liver and aminopeptidase N of Escherichia coli was investigated using a synthetic heptapeptide (L-R-R-A-S-L-G) and its phosphorylated derivative. The enzyme-catalyzed products were analyzed by thin layer chromatography and electrophoresis. The sensitivities of peptide and phosphopeptide to leucyl aminopeptidase digestion were then compared. Data obtained indicated that when phosphopeptide was used as substrate one main product accumulated, which corresponded to the fragment A-S(P)-L-G, while unphosphorylated peptide was completely degraded to its constituent amino acids. Identical results were obtained using aminopeptidase N of E. coli. Using alanyl aminopeptidase as enzyme, the results obtained were essentially similar, since the exopeptidasic activity on the phosphorylated peptide was strongly hampered in the vicinity of phosphoseryl residue leading to accumulation of the same phosphorylated product, although this enzyme could not completely degrade the unphosphorylated peptide. It was concluded that phosphorylation of substrates does effect enzymic degradation of proteins.

Amino Acid Sequence↗

PubMed2004

Structure of human Ki67 FHA domain and its binding to a phosphoprotein fragment from hNIFK reveal unique recognition sites and new views to the structural basis of FHA domain functions.

Recent studies by use of short phosphopeptides showed that forkhead-associated (FHA) domains recognize pTXX(D/I/L) motifs. Solution structures and crystal structures of several different FHA domains and their complexes with short phosphopeptides have been reported by several groups. We now report the solution structure of the FHA domain of human Ki67, a large nuclear protein associated with the cell-cycle. Using fragments of its binding partner hNIFK, we show that Ki67-hNIFK binding involves ca 44 residues without a pTXX(D/I/L) motif. The pThr site of hNIFK recognized by Ki67 FHA is pThr234-Pro235, a motif also recognized by the proline isomerase Pin1. Heteronuclear single quantum coherence (HSQC) NMR was then used to map out the binding surface, and structural analyses were used to identify key binding residues of Ki67 FHA. The results represent the first structural characterization of the complex of an FHA domain with a biologically relevant target protein fragment. Detailed analyses of the results led us to propose that three major factors control the interaction of FHA with its target protein: the pT residue, +1 to +3 residues, and an extended binding surface, and that variation in the three factors is the likely cause of the great diversity in the function and specificity of FHA domains from different sources.

Binding Sites↗

PubMed2006

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS.

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Inhibition of phosphorylation of the oxysterol binding protein by brefeldin A.

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol that localizes to a Golgi/vesicular compartment, migrated on SDS-PAGE as a doublet of 96 and 101 kDa. The reduced mobility of the upper band of this doublet is the result of phosphorylation on multiple serine residues. Phosphorylation of rabbit OSBP stably overexpressed in CHO-K1 cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors such as TPA, sphingosine and bis-indolylmaleimide were without affect. Treatment of overexpressing and control cells with brefeldin A (BFA) caused dephosphorylation of OSBP that coincided with disruption of the Golgi apparatus. [32P]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of dephosphorylation. Phosphopeptide maps of OSBP from overexpressing and control CHO-K1 cells were similar, and BFA promoted dephosphorylation of all five peptides. Compared to overexpressing cells, one tryptic phosphopeptide was more abundant in control CHO-K1 cells and was preferentially dephosphorylated by BFA treatment. OSBP was phosphorylated in vitro by the Golgi enriched fraction of CHO-K1 cells or rat liver by a staurosporine- and BFA-insensitive kinase. The phosphorylation status of OSBP was not affected by 25-hydroxycholesterol and did not alter in vitro 25-[3H]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphorylation of OSBP requires interaction with the Golgi apparatus and an associated kinase. (c) 1998 Elsevier Science B.V.

Animals↗

PubMed2002

High-affinity Src-SH2 ligands which do not activate Tyr(527)-phosphorylated Src in an experimental in vivo system.

The Src-SH2 domain has been determined to play a key role in many signaling pathways, especially in osteoclast-mediated bone resorption. Therefore, non-peptidic small molecules, mimicking the natural pYEEI peptide ligand, have been designed, to inhibit SH2-mediated protein-protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis. However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521)-phosphorylated C-terminal tail and the SH2 domain, thereby inducing a change from a "closed" inactive to an "open" active conformation of Src. Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding, or would activate the enzyme as do phosphopeptides. To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation, both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe. We found that, in the peptide series, the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2. By contrast such correlation is not valid for non-peptidic ligands, some high-affinity SH2 binders showing no detectable Src activation in vivo. These results have significant implications for the design of SH2 binders, as they allow a way to inhibit Src-SH2-mediated signal transduction in target cells, without activating Src in non-target cells, thereby reducing the possibility of side effects.

Animals↗

PubMed1988

Nucleolin (C23), a physiological substrate for casein kinase II.

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated by purified casein kinase II with about two moles phosphate per one mole of nucleolin.

Animals↗

PubMed2001

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3beta identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes.

In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3beta (GSK3beta) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild-type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3beta but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long-range conformational effects on the structure of tau.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Na+-ATPase from the plasma membrane of the marine alga Tetraselmis (Platymonas) viridis forms a phosphorylated intermediate.

Plasma membranes isolated from the marine unicellular alga Tetraselmis (Platymonas) viridis were phosphorylated by [gamma-32P]ATP, and membrane proteins were then analyzed by PAGE in SDS, under acidic conditions. Three radioactive components with apparent molecular masses of 100 kDa, 76 kDa, and 26 kDa were detected. The phosphorylation of one of them, the 100 kDa polypeptide, was specifically stimulated by Na+. Vanadate almost completely inhibited the Na+-mediated phosphorylation of the peptide. The phosphate bound to this peptide underwent rapid turnover and was discharged by hydroxylamine. The 100 kDa phosphopeptide was sensitive to ADP. The conclusion is drawn that the 100 kDa phosphopeptide is a phosphorylated intermediate of the Na+-transporting ATPase in the T. viridis plasma membrane.

Adenosine Diphosphate↗

PubMed1999

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin.

Animals↗

PubMed2005

Genome-wide surveys for phosphorylation-dependent substrates of SCF ubiquitin ligases.

The SCF (Skp1-Cullin-F-box) family of ubiquitin ligases target numerous substrates for ubiquitin-dependent proteolysis, including cell cycle regulators, transcription factors, and signal transducers. Substrates are recruited to an invariant core SCF complex through one of a large family of substrate-specific adapter subunits called F-box proteins, each of which binds multiple specific substrates, often in a phosphorylation-dependent manner. The identification of substrates for SCF complexes has proven difficult, especially given the requirement of often complex phosphorylation events for substrate recognition. The archetype for such interactions is the binding of the yeast F-box protein Cdc4 to its various substrates by means of multiple motifs that weakly match an optimal consensus called the Cdc4 phosphodegron (CPD), which is phosphorylated by cyclin-dependent kinases (CDKs) and possibly other kinases. Provided phosphodegron recognition motifs and/or the targeting kinases for SCF substrates are delineated, it is possible to use genome-wide methods to identify new substrates. Here we describe two methods for the systematic retrieval of SCF substrates based on membrane arrays of synthetic phosphopeptides and on genome-wide kinase substrate profiles. In the first approach, which identifies substrates with strong matches to the CPD, a search of the predicted yeast proteome with the optimal CPD motif identified approximately 1100 matches. A phosphopeptide membrane array corresponding to each of these sequences is then probed with recombinant Cdc4, thereby identifying potential substrates. In the second approach, which identifies substrates that lack strong CPD motifs, a genome-wide set of recombinant CDK substrates is phosphorylated and directly assayed for binding to Cdc4. The proteins corresponding to these hits from each approach can then be subjected to the more stringent criteria of phosphorylation-dependent binding to Cdc4, ubiquitination by SCF(Cdc4)in vitro, and Cdc4-dependent protein instability in vivo. Both methods have identified novel substrates of Cdc4 and may, in principle, be used to identify numerous new substrates of other SCF and SCF-like complexes from yeast to humans.

Amino Acid Sequence↗

PubMed1998

Crystal structure of the tyrosine phosphatase SHP-2.

The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Crystallography↗

PubMed1999

Catalytic activation of the membrane distal domain of protein tyrosine phosphatase epsilon, but not CD45, by two point mutations.

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.

Amino Acid Sequence↗

PubMed2002

Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B beta in primary rat adipocytes.

The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells. In this study, we have characterized the insulin-induced phosphorylation and activation of PKB beta in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKB beta from cytosol to membranes, and phosphorylation and activation of PKB beta. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKB beta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKB beta mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in human embryonic kidney (HEK) 293 cells demonstrating, in addition, phosphorylation of Thr-309.

Adipocytes↗