Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

Identification of the threonine phosphorylation sites on the polyomavirus major capsid protein VP1: relationship to the activity of middle T antigen.

Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32P labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threonine-to-glycine change at residue 63 (mutant G63) and a threonine-to-alanine change at residue 156 (mutant A156) resulted in viruses defective in phosphorylation of the respective peptides after in vivo labeling. Growth of the mutant G63 virus was similar to that of the wild-type virus, but the mutant A156 was inefficient in assembly of 240S viral particles. Polyomavirus nontransforming host range (hr-t) mutants are defective in VP1 threonine phosphorylation when grown in nonpermissive cells (R. L. Garcea, K. Ballmer-Hofer, and T. L. Benjamin, J. Virol. 54:311-316, 1985). Proteolytic mapping of VP1 peptides after in vivo labeling from hr-t mutant virus infections demonstrated that both residues T-63 and T-156 were affected. These results suggest that the block in virion assembly in hr-t mutant viruses is associated with a defect in phosphorylation of threonine 156.     

Phosphorylation of the human cell proliferation-associated nucleolar protein p120

The human cell proliferation-associated nucleolar protein p120 was found in a variety of human cancer specimens but not in most normal resting cells. Polyclonal antibodies raised against bacterially expressed p120 were used to immunoprecipitate the p120 protein isolated from 32P-labeled HeLa cells. The p120 protein was phosphorylated at serine, threonine and tyrosine residues. A tryptic peptide map showed it contained three labeled peptides. One of these peptides comigrated with a p120 peptide phosphorylated in vitro by casein kinase II. This peptide was phosphorylated in vitro both at Ser-181 and Thr-185. This region is juxtaposed to the epitope site recognized by the anti-p120 monoclonal antibody.     

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.     

The roles of autophosphorylation and phosphorylation in the life of osteopontin

Osteopotin is a secreted glycosylated phosphoprotein found in bone and other normal and malignant tissues. Osteopontin can be autophosphorylated on tyrosine residues and can also be phosphorylated on serine and threonine residues by several protein kinases. Autophosphorylation of osteopontin may generate sites for specific interactions with other proteins on the cell surface and/or within the extracelluar matrix. These interactions of osteopontin are thought to be essential for bone mineralization and function. The polyaspartic acid motif of osteopontin, in combination with neighboring sequences that include serine residues phosphorylated by protein kinases, could fold and assemble into a molecular structure that participates in the mineralization of the bone matrix.     

Phosphorylation of P1, a high mobility group-like protein, catalyzed by casein kinase II, protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II

P1, a high mobility group-like nuclear protein, phosphorylated by casein kinase II on multiple sites in situ, has been found to be phosphorylated in vitro by protein kinase C, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II on multiple and mostly distinct thermolytic peptides. All these enzymes phosphorylated predominantly serine residues, with casein kinase II and protein kinase C also labeling threonine residues. Both casein kinase II and second messenger-regulated protein kinases, particularly protein kinase C, might therefore be involved in the physiological regulation of multisite phosphorylation of P1.     

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.     

Identification of a phosphorylation site of the rat insulin receptor catalyzed by protein kinase C in an intact cell

In two-dimensional tryptic phosphopeptide mapping, the beta-subunit of the insulin receptor phosphorylated by 12-O-tetradecanoylphorbol-13-acetate in rat hepatoma cells (H-35) was separated into one phosphothreonine-containing peptide and several phosphoserine-containing peptides. The synthetic peptide coding residues 1327-1343 in the C-terminal region of the rat insulin receptor was phosphorylated at the threonine residue by protein kinase C in a phosphatidylserine and oleoylacetylglycerol dependent manner. Tryptic digest of this phosphopeptide migrated to the same position as the phosphothreonine containing peptide obtained from the beta-subunit in two-dimensional phosphopeptide mapping. These data suggested that Thr 1336 of the insulin receptor is the site of phosphorylation by protein kinase C in intact cells.     

Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues

A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities.     

Hyperphosphorylation of tau induces local polyproline II helix

Alzheimer's disease is characterized by two protein precipitates, extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). The primary constituent of NFTs is a hyperphosphorylated form of the microtubule-binding protein tau. Hyperphosphorylation of tau on over 30 residues, primarily within proline-rich sequences, is associated with conformational changes whose nature is poorly defined. Peptides derived from the proline-rich region of tau (residues 174-242) were synthesized, and the conformations were analyzed for the nonphosphorylated and phosphorylated peptides. CD and NMR data indicate that phosphorylation of serine and threonine residues in proline-rich sequences induces a conformational change to a type II polyproline helix. The largest phosphorylation-dependent conformational changes observed by CD were for tau peptides incorporating residues 174-183 or residues 229-238. Phosphoserine and phosphothreonine residues exhibited ordered values of (3)J(alphaN) (3.1-6.2 Hz; mean = 4.7 Hz) compared to nonphosphorylated serine and threonine. Phosphorylation of a tau peptide consisting of tau residues 196-209 resulted in the disruption of a nascent alpha-helix. These results suggest that global reorganization of tau may occur upon hyperphosphorylation of proline-rich sequences in tau.     

Molecular approaches to examine the phosphorylation state of the C type natriuretic peptide receptor

The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST‐NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine‐5'‐triphosphate (ATP). Western blot analysis using a monoclonal phospho‐Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross‐react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation. J. Cell. Biochem. 110: 985–994, 2010. © 2010 Wiley‐Liss, Inc.     

Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.     

Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.     

Phosphorylation of threonine and serine residues of native and partially dephosphorylated caseins by a rat liver cyclic AMP-insensitive protein kinase.

The preferential phosphorylation of threonine residues of native casein fractions by a rat liver cyclic AMP-independent protein kinase (EC 2.7.1.37) is abolished by preliminary limited dephosphorylation of the substrates, which promotes a fall in the phosphothreonine/phosphoserine ratios from values higher than 1 to much less than 0.1. This finding and the identification of the threonine residues phosphorylated support the view that the liver protein kinase affects threonine residues only when suitable serine residues, which fulfil the structural requirements for attack by the enzyme but which are not yet phosphorylated, are not available.     

The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase.

The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.     

Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.     

Myelin-associated glycoprotein, a cell adhesion molecule of oligodendrocytes, is phosphorylated in brain.

Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.     

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.     

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.