Phosphoamino Acid Residue Research Articles

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser(41) of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca(2+) release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser(41). This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca(2+) homeostasis.

Antibody-Free Detection of Phosphoserine/Threonine Containing Peptides by Homogeneous Time-Resolved Fluorescence

Protein phosphorylation is a critical signaling mechanism in cellular regulation and stress response, and more than 95% of the phosphorylations are targeted toward Ser or Thr amino-acid residues. The classical techniques for analyzing phospho-amino acid residues use radioisotopes or sequence-specific antibodies. However, both practical and economical limitations have prevented their development, and we here propose an original approach for the detection of phospho-Ser/Thr residues. It requires no antibody and exploits the patented homogeneous time-resolved fluorescence (HTRF) technology, in association with a 3-step chemical transformation of phospho-amino acids into fluorescent derivatives. The process involves: (i) alkaline β-elimination of the phosphorylated group, (ii) Michael addition of a bifunctional group, and then (iii) introduction of cyanin-5 as fluorescent acceptor for HTRF. The donor fluorescent moiety at the N-terminus of the phosphorylated peptide is a streptavidin europium cryptate conjugate. After its development, the detection system has been validated on synthetic peptide substrates of Chk2, a key protein kinase activated in response to DNA damage and involved in cell cycle arrest. The results showed a good correlation with known specificity profiles. Interestingly, the detection system is versatile, easy to implement, and suitable for multiple parallel analyses.

A manual sequence method of peptides and phosphopeptides using 4-(1′-cyanoisoindolyl)phenylisothiocyanate

A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1′-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1′-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.

Characterization of O-Phosphohydroxyproline in Rat α-Crystallin A

Post-translational modifications have major importance for the structure and function of a multiplicity of proteins. Phosphorylation is a widespread phenomenon among eukaryotic proteins. Whereas O-phosphorylation on the side chains of serine, threonine, and tyrosine in proteins is well known and has been studied extensively, to our knowledge the endogenous phosphorylation of hydroxyproline has not previously been reported. In the present work, we provide evidence for the first time that O-phosphohydroxyproline (Hyp(P)) is a proteinogenic amino acid. To detect Hyp(P) in proteins we generated a Hyp(P)-specific polyclonal antibody. We could identify Hyp(P) in various proteins by Western blot analysis, and we characterized the sequence position of Hyp(P) in the protein α-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in α-crystallin A in the eye as well as in heart tissue of rat.

Phosphorylation of a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase, is regulated physiologically and developmentally in rosette leaves of Arabidopsis thaliana.

The phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (EC 2.7.1.105/ EC 3.1.3.46) in rosette leaves of Arabidopsis was examined. Immunoblotting with specific antisera detected 96-kDa and 92-kDa bands in the crude protein extracts from rosette leaves of Arabidopsis. Incubation of protein samples with alkaline phosphatase before SDS-PAGE reduced the 96-kDa band with concomitant increase of the 92-kDa band, suggesting that the former is a phosphorylated form of the latter. In accordance with this result, 96-kDa and 92-kDa bands were immuno-precipitated from the crude protein extracts from [(32)P]orthophosphate-labeled rosettes of Arabidopsis; and, the former was heavily labeled, the latter faintly labeled. Analysis of phospho-amino acid residues derived from the [(32)P]-labeled 96-kDa band revealed that the phosphorylation occurred on serine and threonine residues, excluding the possibility that the phosphorylated band represent a phospho-histidine intermediate that is known to form in the phosphatase reaction. The relative level of the 96-kDa band over the 92-kDa band in whole rosette extracts changed diurnally and was highest at the beginning of nighttime. Furthermore, the 96-kDa band was highly enriched in the extracts of very young rosette leaves, suggesting that the phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase is regulated physiologically and developmentally in Arabidopsis.

Mitotic Reorganization of the Intermediate Filament Protein Nestin Involves Phosphorylation by cdc2 Kinase

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

We have adressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.Ca++ (0.1–10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca++- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.

Influence of Peroxisome Proliferators on Phosphoprotein Levels in Human and Rat Hepatic‐Derived Cell Lines

To elucidate the effect of peroxisome proliferators on the signal-transduction pathway, we have compared the effect of ciprofibrate, an hypolipaemic agent, on the overall phosphoprotein level between rat and human well differentiated hepatic derived cell lines. The phosphorylation status of several phosphoproteins in the rat Fao cell line was increased by the drug while no changes were observed in the human HepG2 cell line. In rat Fao cells, this increase, which is concentration and time dependent, can be as much as eightfold for 20-kDa and 22-kDa proteins. Wy-14,643, a non-fibrate molecule and a more potent peroxisome proliferator than ciprofibrate, increased the phosphorylation status of the same phosphoproteins. Peroxisome proliferators may act by activating kinases inactive in control cells, by amplifying kinases already active in control cells or by inactivating phosphatases. The phosphoamino acid residues affected are essentially serine and threonine. This modification of the signal-transduction pathway by the peroxisome proliferators in rodent cells appears to be an early event or an independent mechanism of the peroxisome proliferation. These results support the accumulating evidence that the perturbation of this pathway may be a major cause of the hepatomegaly and the hepatocarcinogenesis induced by peroxisome proliferators in rodent species. In contrast, the lack of phosphorylation changes in the human HepG2 cell line supports the non-toxic effect of peroxisome proliferators also used as hypolipaemic agents in humans.

Hyperphosphorylation of keratins by treatment with okadaic acid of BALB/MK-2 mouse keratinocytes.

Protein hyper- or hypophosphorylation induced by okadaic acid (OA) treatment was examined using quiescent cultures of the BALB/MK-2 mouse epidermal keratinocytes. Treatment with OA enhanced the phosphorylation of five proteins with molecular weights of 65,000, 55,000, 50,000, 28,000 and 15,000 (p65, p55, p50, p28, and p15, respectively) and decreased that of two proteins with molecular weights of 22,000 and 20,000 (p22 and p20, respectively). The two major phosphorylated proteins, p65 and p55, were identified as type II and type I keratins, respectively, by immunoblotting and immunoprecipitation with keratin specific antibodies. Serine was the only phosphoamino acid residue in hydrolysates of the 32P-labeled keratins purified from OA-treated cells. Two-dimensional tryptic peptide maps of the phosphorylated keratins showed that the hyperphosphorylation was largely due to phosphorylation at several additional sites in both keratins. The hyperphosphorylation of keratins induced by OA treatment resulted in a drastic change in their solubility. This change closely correlated with reorganization of the keratin filament network, which finally collapsed into large perinuclear aggregates. Concomitantly the cells changed from a typical epithelial shape to a round shape. Of several protein kinase inhibitors tested, only staurosporine interfered with this OA-induced morphological change and reorganization of the keratin network.

Rapid phosphorylation of 28-kDa heat-shock protein by treatment with okadaic acid and phorbol ester of BALB/MK-2 mouse kerationocytes

Protein phosphorylation by okadaic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined using quiescent cultures of BALB/MK-2, a cell line derived from mouse epidermal keratinocytes. Treatment with okadaic acid caused rapid phosphorylation of five proteins with molecular masses of 65, 55, 50, 28 and 15 kDa (p65, p55, p50, p28, p15, respectively) while TPA caused rapid phosphorylation of five proteins with molecular masses of 80, 70, 40, 34 and 28 kDa (p80, p70, p40, p34, p28, respectively). In the present study, we examined p28, a common target protein of okadaic acid and TPA. The phosphorylation of p28 increased depending on time of exposure and doses of okadaic acid and TPA. Combined treatment with okadaic acid and TPA resulted in an additive effect. Its position on two-dimensional gel electrophoresis suggested that p28 is the 28-kDa heat-shock protein (HSP28). This possibility was confirmed by migration of p28 with HSP28 and comparative peptide mapping of the two proteins. The phosphoamino-acid residue of phosphorylated HSP28 was serine. In two-dimensional tryptic peptide maps, the same peptides were phosphorylated after treatment with both okadaic acid and TPA.

Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.

Phosphorylation of Escherichia coli enolase

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivies of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.

Protein phosphorylation in Bacillus thuringiensis during growth and delta-endotoxin production.

At least 14 phosphopolypeptides in which the phosphate groups were present as mono-esters were detected by pulse labelling of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel with [32P]orthophosphate at different stages of growth and differentiation. Marked changes in the profile of phosphopolypeptides were observed primarily during the late exponential phase of growth. Several phosphopolypeptides co-purified with the endotoxin crystal of this subspecies and the phosphoamino acid residue of the most abundant (Mr 25,000) phosphopolypeptide was identified as phosphothreonine. Comparison of the phosphopolypeptides in endotoxin crystals from several subspecies suggested that Mr 25,000 species might be a common component.

Phosphorylation Sites of Bovine Brain Myelin Basic Protein Phosphorylated with Ca2+-Calmodulin-Dependent Protein Kinase from Rat Brain1

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr.

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.

Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity.

We have examined human and rabbit blood platelets for expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp60c-src kinase activity was determined by an immune-complex kinase assay that uses enolase as the substrate, and pp60c-src protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-src-specific kinase activity and pp60c-src protein compared to the levels found in other tissues. pp60c-src was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp60c-src were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented approximately equal to 80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells.

An unexpected effect of divalent cations on the adenovirus endogenous protein kinase: Alteration in the specificity of phosphorylation

The adenovirus endogenous protein kinase specifically phosphorylates capsid protein IIIa in the presence of Mg 2+, utilizing either ATP or GTP as a phosphate donor. When Mn 2+ is substituted for Mg 2+, in the presence of ATP, phosphorylation of IIIa is enhanced by 2–3 fold. However, in addition to IIIa phosphorylation, the core proteins V and VII are now phosphorylated. A similar finding is made when Co 2+ is used instead of Mg 2+. Further, when Mn 2+ or Co 2+ is substituted for Mg 2+, the phosphoamino acid residue profile is changed, viz., instead of only phosphoserine being labeled, phosphothreonine is now extensively labeled. These results are specific for the above endogenous viral proteins, since when the viral protein kinase is used to phosphorylate added exogenous substrates such as casein, no enhancement of phosphorylation nor any change in phosphoamino acid profile is achieved by substituting Mn 2+ for Mg 2+. It is thus likely that MnATP or CoATP somehow interacts differently with adenovirus structural proteins than does MgATP and this facilitates their accessibility to the enzyme.

Epidermal growth factor-stimulated phosphorylation of tyrosine residues on a 120 000 dalton protein in mouse liver plasma membrane subfractions

Summary The addition of Epidermal growth factor (EGF) to mouse liver sinusoidal plasma membrane subfractions, isolated from the blood face of hepatocytes, resulted in a two to threefold increase of 32 P incorporation from [γ- 32 P ]ATP into a 120 000 dalton protein component. The EGF-stimulation of the phosphorylation of this component was also evidenced in plasma membranes derived from the lateral and biliary faces of hepatocytes. A major phosphoamino acid residue after EGF stimulation was identified as tyrosine. An EGF-enhancement of the phosphorylation of threonine and serine residues also appears to be detectable.

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.