Selective enrichment in phosphopeptides for the identification of phosphorylated mitochondrial proteins.

In vivo protein phosphorylation is a reversible and dynamic process controlled by protein kinases and phosphatases for the addition and removal of the phosphate group to serine, threonine, and tyrosine residues. In addition to other regulation events, increasing evidence indicates that reversible protein phosphorylation plays an important role in regulating mitochondrial function. Detecting changes in the state of protein phosphorylation is a difficult task since phosphorylation on a specific protein is typically transient and usually presents in substoichiometric concentration. Moreover, what makes mass spectrometry (MS)-based phosphopeptide analysis difficult is that ionization efficiency of phosphopeptides is lower than their nonphosphorylated analogues. Different strategies have been proposed for the selective enrichment of low abundance phosphoproteins and phosphopeptides present in biological samples. As a result of recent advances, phosphoproteomics has become one of the most rapidly developing areas of proteomics. In the last few years, a number of studies have focused on the analysis of phosphoproteome purified from yeast, liver and heart mitochondria, as well as on the identification of endogenously phosphorylated subunits of mitochondrial oxidative phosphorylation system complexes. This chapter describes methods for the selective enrichment of phosphoserine, phosphothreonine, and phosphotyrosine containing peptides and proteins and phosphate-specific MS strategy generally applicable to phosphoprotein analysis but focusing specifically on mitochondrial samples.

Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides.

Alkaline-induced beta-elimination of phosphate from phosphoserine and phosphothreonine residues followed by addition of an affinity tag has recently been pursued as a strategy for enriching phosphorylated species from complex mixtures. Here we report the use of an introduced thiol tag as the ligand for affinity purification via disulfide exchange with an activated thiol resin and the development of a protocol to improve the sensitivity considerably over previous reports (i.e., to subpicomole levels.) During our experiments, we observed a side reaction in which water was eliminated from unmodified serine residues. This side reaction resulted in the introduction of the affinity tag into unphosphorylated proteins, confounding attempts to specifically purify phosphoproteins from mixtures. Unchecked, this side reaction will also prevent application of the beta-elimination strategy to phosphopeptide samples where the phosphorylated species are minor components (i.e., most current phosphoproteomics applications). Quantitation of the side reaction products using three synthetic unphosphorylated peptides showed varying conversion efficiencies; at maximum, 1.7% of unphosphorylated peptide was converted to the affinity-tagged form. Inclusion of EDTA into the reaction reduced the side reaction but also greatly reduced the conversion efficiency of one of the phosphoserine residues of ovalbumin, suggesting a role for trace metal ions in the beta-elimination chemistry. Despite the presence of the side reaction, the affinity strategy was shown to be effective at enriching phosphopeptides from fairly complex peptide mixtures. The strategy was applied to the analysis of in vitro phosphorylation of bovine synapsin I by Ca(2+)/calmodulin-dependent kinase II, resulting in the identification of four phosphorylation sites, two of which have not been previously reported.

Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates moesin at Thr558 in vitro. Here, using a site- and phosphorylation state-specific antibody, we found that the expression of dominant active RhoA in COS7 cells induced moesin phosphorylation and the formation of microvilli-like structures at apical membranes where the Thr558-phosphorylated moesin accumulated, whereas the expression of dominant negative Rho-kinase inhibited both of these processes. The expression of dominant active Rho-kinase also induced moesin phosphorylation. When COS7 cells expressing moesin or moesinT558A (substitution of Thr by Ala) were cultured under serum-depleted conditions, there were few microvilli-like structures, whereas microvilli-like structures remained in the cells expressing moesinT558D (substitution of Thr by Asp). The expression of moesinT558A inhibited the dominant active RhoA-induced formation of microvilli-like structures. These results indicate that Rho-kinase regulates moesin phosphorylation downstream of Rho in vivo and that the phosphorylation of moesin by Rho-kinase plays a crucial role in the formation of microvilli-like structures.

Protein kinases A and C phosphorylate purified Ca2+-ATPase from rat cortex, cerebellum and hippocampus.

The plasma membrane Ca2+-ATPase (PMCA), the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several independent mechanisms. In this paper we report that the protein kinases A and C differentially activate the Ca2+-ATPase purified from synaptosomal membranes of rat cortex, cerebellum and hippocampus. The effect of protein kinases was more pronounced for the cortical enzyme, whereas cerebellar and hippocampal Ca2+-ATPases were activated to a lesser degree. The preparation of Ca2+-ATPase contained the phosphoamino acids, i.e., P-Ser and P-Thr, indicating that the enzyme was purified in phosphorylated state. The phosphorylation of Ca2+-ATPase by PKA and PKC increased the amount of phosphoamino acids, but in a region-dependent manner. Using the specific antibodies against N-terminal portion of four main PMCA isoforms we have characterized the isoforms composition of Ca2+-ATPase purified from the nervous endings of examined brain areas. Our results indicate that the activity of calcium pump is related to its phosphorylated state, and that the phosphorylation is region-dependent. Moreover, the differences observed could be related to the composition of PMCA isoforms in the different brain areas. Phosphorylation of the plasma membrane Ca2+-ATPase appears to be a mechanism to control its activity. The results support also the possible involvement of PKA and PKC.

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.

Soluble glycosylated phosphoproteins of cementum.

Ethylenediaminetetraacetate and hydrochloric acid (EDTA) (HCl) extracts of cementum were fractionated by molecular sieving, ion exchange chromotography, and reverse phase high precision liquid chromatography (HPLC). Nine fractions were isolated, all of which contained serine phosphate, threonine phosphate, and high concentrations of aspartic acid (asp) and glutamic acid (glu). Five of the fractions obtained by repeated HPLC consisted of a single band by SDS-PAGE; the others contained at least one other minor component. All of the protein bands stained with both Rhodamine B and alcian blue, the latter consistent with analytical determinations that demonstrated that the phosphoprotein component contained a significant amount of carbohydrate, including neuraminic acid.

Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria.

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.

Resolution and identification of O-phosphoserine, O-phosphothreonine, O-phosphotyrosine, and gamma-carboxyglutamic acid as their fluorescent o-phthalaldehyde derivatives by high performance liquid chromatography.

High performance liquid chromatography was used to resolve O-phosphoserine, O-phosphothreonine, and O-phosphotyrosine as their fluorescent o-phthalaldehyde derivatives. By adjusting the buffer system, very small amounts of O-phosphothreonine could be detected and quantitated in the presence of very large amounts of O-phosphoserine. In addition, gamma-carboxyglutamic acid and glutamic acid were also separated and quantitated. Depending on the buffer used, various combinations of these amino acids could be resolved in a single run.

Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives.

Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.

Structure of component B (7-mercaptoheptanoylthreonine phosphate) of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum.

Component B, the heat-stable low-molecular-weight cofactor required for methane production by dialyzed cell-free extracts of Methanobacterium thermoautotrophicum, has been purified to homogeneity and its structure assigned. Results of low-resolution fast-atom-bombardment and field-desorption mass spectrometry indicated a molecular weight of 419, and high-resolution fast-atom-bombardment mass spectrometry agreed with the molecular formula C13H26NO8PS2. Evidence from fast-atom-bombardment and field-desorption mass spectrometry and 360-MHz 1H NMR in deuterium oxide argued that the compound was isolated as a mixed disulfide with 2-mercaptoethanol; so the proposed elemental formula of the free acid, free thiol would be C11H22NO7PS (molecular weight, 343). The proposed structure for an active form of the coenzyme is 7-mercaptoheptanoylthreonine phosphate.

Rapid separation of phosphoamino acids including the phosphohistidines by isocratic high-performance liquid chromatography of the orthophthalaldehyde derivatives.

Amino acids were derivatized with orthophthalaldehyde and separated by high-performance liquid chromatography on a polymer-based reverse-phase column (Hamilton PRP-1) at pH 7.2 using isocratic elution with 14.3 mM sodium phosphate, 1.1% tetrahydrofuran, 6.6% acetonitrile. Phosphorylated amino acids were eluted with baseline resolution in the following order: 1-phosphohistidine, phosphoserine, 3-phosphohistidine, phosphotyrosine, phosphothreonine, and phosphoarginine. Each of the phosphoamino acids was separated from its parent amino acid but aspartate and glutamate eluted in the same region as the phosphoamino acids. The sensitivity is in the picomole range and the separation time, injection to injection, is 15 min. The linearity for phosphothreonine extends at least from 30 pmol to 30 nmol. Quantitation by radioactivity is good for each of the phosphoamino acids except in the case of [1-32P]phosphohistidine, which coelutes with inorganic phosphate.

Threonine phosphorylation of rat liver glycogen synthase.

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.

Polyamide thin-layer chromatography of phosphorylated tyrosine, threonine, and serine.

One-dimensional thin-layer chromatography on polyamide plates offers an easy and rapid identification of O-phosphotyrosine. The thin-layer plate is developed for 30 min in 5% propionic acid containing 0.013%-0.025% sodium dodecyl sulfate. O-Phosphotyrosine, with Rf = 0.54, can be well separated from O-phosphothreonine and O-phosphoserine, which comigrate at Rf = 0.72.

A rapid microdetermination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins by automatic cation exchange on a conventional amino acid analyzer.

A cation-exchange chromatographic method for the separation and determination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins after partial acid hydrolysis is described. The short column (0.6 X 8 cm) of an automatic amino acid analyzer was used and elution was carried out isocratically with 10 mM trifluoroacetic acid. The method is highly sensitive and each of the three O-phosphoamino acids can be accurately determined down to the 50-pmol level. Higher sensitivity may be obtained by the use of [32P]phosphate-labeled proteins. A correction factor for the decomposition of phosphoserine or phosphothreonine during acid hydrolysis can be deduced from the amount of inorganic phosphate recovered at the column void volume. The method is sensitive enough to be used for 32P-labeled proteins isolated by two-dimensional gel electrophoresis.