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PubMed2006

Analytical methodologies for the detection and structural characterization of phosphorylated proteins.

Phosphorylation of proteins is a frequent post-translational modification affecting a great number of fundamental cellular functions in living organisms. Because of its key role in many biological processes, much effort has been spent over the time on the development of analytical methodologies for characterizing phosphoproteins. In the past decade, mass spectrometry-based techniques have emerged as a viable alternative to more traditional methods of phosphorylation analysis, providing accurate information for a purified protein on the number of the occurring phosphate groups and their exact localization on the polypeptide sequence. This review summarizes the analytical methodologies currently available for the analysis of protein phosphorylation, emphasizing novel mass spectrometry (MS) technologies and dedicated biochemical procedures that have been recently introduced in this field. A formidable armamentarium is now available for selective enrichment, exaustive structural characterization and quantitative determination of the modification degree for phosphopeptides/phosphoproteins. These methodologies are now successfully applied to the global analysis of cellular proteome repertoire according a holistic approach, allowing the quantitative study of phosphoproteomes on a dynamic time-course basis. The enormous complexity of the protein phosphorylation pattern inside the cell and its dynamic modification will grant important challenges to future scientists, contributing significantly to deeper insights into cellular processes and cell regulation.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

Effects of insulin on protein phosphorylation and protein kinase C activity in human malignant gliomas.

Modulation of protein phosphorylation activities by insulin was investigated in glioma and normal glial cells. Insulin suppressed the in vitro protein phosphorylation of glioma cells in a dose-dependent manner while it stimulated that of meningiomas, neurilemmomas and glial cells. Although gliomas and glial cells contained different species of tyrosyl phosphoproteins before treatment, they expressed similar kinds of tyrosyl phosphoproteins in response to insulin. Insulin increased the activities of casein kinase II and total protein kinase C (PKC) in glioma and normal glial cells. The membrane-bound PKC activity in U373-MG cells was elevated by insulin. The PKC isozymes, including subtypes alpha, beta, delta, epsilon and gamma, were detected in gliomas, but few were found in glial cells. Insulin down regulated the cytosolic PKC-gamma and the membrane-bound PKC-epsilon proteins in gliomas. These results indicate that an altered insulin signaling pathway exists in human gliomas, which might involve differential regulation of PKC isozymes.

Casein Kinase II↗
PubMed1988

Inhibitory effects of aminoglycosides on renal protein phosphorylation by protein kinase C.

Aminoglycosides such as neomycin are commonly prescribed antibiotics; however, there is associated serious damage to the kidney. We examined the effect of aminoglycoside antibiotics on renal protein phosphorylation and found that neomycin selectively inhibited Ca++-activated, phospholipid-dependent phosphorylation of 88-kDa protein in cell lysates of the rabbit kidney. Fifty percent inhibition of phosphorylation of this protein occurred with 5 X 10(-5) to 1 X 10(-4) M neomycin. In living PtK2 cells, neomycin dose-dependently inhibited 12-O-tetradecanoyl-phorbol-13-acetate-induced phosphorylation of 88 K Da protein. This drug also inhibited phosphorylation of exogenous protein catalyzed by protein kinase C, isolated from rabbit kidney in vitro. In contrast, neomycin had little or no inhibitory effect on cyclic GMP-dependent protein kinase, cyclic AMP-dependent protein kinase, casein kinase I, casein kinase II and Ca++-calmodulin-dependent myosin light chain kinase. Whereas activity of protein kinase C was inhibited 65% by neomycin (0.1 mM) at pH 5 to 7, inhibition decreases to 33% at pH 8 and to zero at pH 9. The potencies of a series of aminoglycoside antibiotics to inhibit the kinase agreed well with number of ionizable amino groups of compounds (gamma = 0.99) and this also approximates their known nephrotoxic potential; amikacin less than or equal to kanamycin less than gentamycin less than or equal to tobramycin less than neomycin. As aminoglycoside antibiotics present in the kidney after administration of toxicological doses (10(-2) M) will inhibit the effects of protein kinase C, the aminoglycoside antibiotics-induced nephrotoxicity is discussed in relation to inhibition of intracellular protein kinase C.

Aminoglycosides↗
PubMedDate not supplied

Increased 19 kDa protein phosphorylation and protein kinase C activity in pressure-overload cardiac hypertrophy.

The aim of the study was to determine the role of protein kinase C (PKC) in protein phosphorylation in hypertrophied C. myocytes, particularly the phosphorylation of the 19 kDa protein which corresponds to myosin light chains. In myocardial hypertrophy the PKC activity in the cytosolic fraction of tissue homogenate was increased up to 253% of control hearts, and in membrane fraction up to 140% of the control value. Phorbol ester (TPA), the specific activator of protein kinase C, stimulated phosphorylation of the 19 kDa protein obtained from isolated myocytes to 181 +/- 9% of control value in normal and to 248 +/- 66% in hypertrophic myocytes. Taken together, these data suggest that protein kinase C might be involved in the increased phosphorylation of cardiac myosin light chain protein in myocardial hypertrophy.

Animals↗
PubMed2001

Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins.

Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins. The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon. Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins. Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase. Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains. This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins. In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins.

Animals↗
PubMed1996

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents.

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K(+)-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with 35S-methionine or 32P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K(+)-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K+ or serotonin (12-15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes.

Adenine↗
PubMed1994

Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane.

The unusual Ca(2+)-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62,000, is activated by Ca2+ (K0.5 = 15 microM) in the presence of Mg2+ or Mn2+, and can associate with 45Ca2+. The activation by Ca2+ could be partially replaced by Mn2+, but not by Zn2+ or Mg2+. PfCPK preferentially phosphorylated casein and histone H1. The Km and Vmax for Mg2+ ATP were 26 microM and 70 nmol min-1 mg-1, respectively, with casein as substrate; and 34 microM and 143 nmol min-1 mg-1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.

Animals↗
PubMed1979

Variations in some molecular events during the early phases of the Reuber H 35 cell cycle. II.-Chromatin protein phosphorylation and protein kinases.

Reuber H 35 hepatoma cells were synchronized by transfer in a serum free medium. Growth was re-initiated by addition of serum. Under these conditions DNA synthesis exhibited a maximum after 24 hours. Chromatin non-histone proteins prepared from cells at various phases of the cell cycle were incubated with [gamma-32P] ATP and the radioactive pattern of protein bound 32P was analysed by electrophoresis on polyacrylamide gels. No radioactive peak was observed in G0. Several peaks appeared 3 hours after the addition of serum. The radioactivity progressively increased until the cells reached the S phase. When most of the cells were in the S phase the radioactivity strongly decreased. Chromatin protein kinase activities were found to increase in late G1 and continued to increase in the S phase. The increase was 65% when phosvitin was the substrate, 100% with casein and histone H1. It is suggested that chromatin phosphorylated proteins could be involved in the mechanism which initiates DNA synthesis in G1 phase cells.

Animals↗
PubMed1991

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors.

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.

Animals↗
PubMed1991

Protein phosphorylation during activation of surf clam oocytes.

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.

Animals↗
PubMed1999

Activated protein kinase C isoforms target to cardiomyocyte caveolae : stimulation of local protein phosphorylation.

Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4alpha-PMA, which does not activate PKC) recruited calcium-sensitive PKCalpha and novel PKCdelta and PKCepsilon to this compartment. The subcellular localization of the phorbol ester-insensitive PKClambda isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKCalpha and PKCepsilon (but not PKCdelta or PKClambda) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a approximately 36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates.

Animals↗
PubMedDate not supplied

Compromised sperm protein phosphorylation after capacitation, swim-up, and zona pellucida exposure in teratospermic domestic cats.

Tyrosine phosphorylated proteins recently have been found in mouse and human spermatozoa. Our objectives were to (1) determine if domestic cat spermatozoa also express tyrosine phosphorylated proteins, and (2) examine the changes in protein phosphorylation between normospermic and teratospermic domestic cats following sperm capacitation, swim-up separation and exposure to zona pellucida (ZP). Membranes from cat spermatozoa contained two phosphorylated proteins of molecular weights 160 kDa and 95 kDa (designated as p160 and p95) that immunoreacted with monoclonal antibodies to tyrosine phosphate. The p95 protein was distinct from sperm-specific hexokinase. Following capacitation, the extent of phosphorylation of p95 was increased (P < 0.05) 3-fold in normospermic cats compared to only 1.75-fold in teratospermic cats. Similarly, phosphorylation of p160 also increased (P < 0.05) 2.4-fold in normospermic compared to 1.84-fold in teratospermic cats. Although swim-up separation increased the percentage of normal spermatozoa in teratospermic ejaculates, phosphorylation of p95 in swim-up, aliquots was increased (P < 0.05) only 1.95-fold in teratospermic cats compared to 2.9-fold in normospermic counterparts. Likewise, phosphorylation of p160 was lower (P < 0.05) in teratospermic (1.5-fold) compared to normospermic cats (2.0-fold) cats. Phosphorylation also was influenced by exposure to cat ZP proteins (P < 0.05). Solubilized cat ZP bound to the sperm proteins of apparent molecular mass 120, 95, 50, 42, 30, 27, 23 and 20 kDa, suggesting a direct binding interaction between p95 and the ZP. Overall, these findings (1) indicate the presence of tyrosine phosphorylated proteins in the domestic cat spermatozoon that directly interact with homologous ZP glycoproteins; (2) demonstrate that cat sperm hexokinase is not phosphorylated on tyrosine residues; and (3) suggest that the diminished phosphorylation efficiency of sperm from teratospermic cats may result in a compromise in capacitation and the acrosome reaction.

Animals↗
PubMed2004

Identification of peptides mimicking the ligands of proteins phosphorylated by protein kinase CK2.

Synthetic peptides containing a phosphorylation site for protein kinase CK2 were used to investigate their binding properties to other peptides/proteins. The aim of this work was to find an efficient procedure to search for these peptide/protein ligands. The goal was successfully achieved through screening of random peptide libraries displayed on phage. Peptides corresponding to the amino terminal region of topoisomerase I were synthesized in both phosphorylated and unphosphorylated form and used to screen the libraries. Four of the selected sequences were also tested for their reactivity with synthetic peptides corresponding to the carboxy terminal region of the largest subunit of RNA polymerase II. The positive reaction detected supports the hypothesis that the isolated sequences may represent mimics of ligands of proteins phosphorylated by protein kinase CK2.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of phosphorylated proteins.

Reversible protein phosphorylation is of crucial importance in regulatory mechanisms and signaling pathways. Novel and efficient tools and strategies are being actively developed to allow, beyond the primary identification of phosphorylated proteins, the identification of phosphorylation sites and ultimately their quantification. These approaches are being used at various scales, from studies that have dedicated and functional goals to work with more exploratory and cataloguing objectives. The information thus generated now makes it possible to use bioinformatics to revisit previous knowledge about protein phosphorylation in plants and the pertinence of available prediction models. Although the analysis of phosphorylated proteins remains a challenging task, recent success and current developments are likely to mark the transition towards the introduction of phosphoproteomics as one of the main integration levels in post-genome plant biology.

Computational Biology↗
PubMed1986

Light-modulated ADP-ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes.

Rhodopsin (P, lambda max 480 nm) of blowfly photoreceptors R1-6 is converted by light into a thermally stable metarhodopsin (M, lambda max 565 nm). In isolated blowfly rhabdoms photoconversion of P to M affects bacterial toxin-catalyzed ADP-ribosylation of a 41-kDa protein, activates phosphorylation of opsin and induces the binding of a 48-kDa phosphoprotein to the rhabdomeric membrane. ADP-ribosylation of the 41-kDa protein is catalyzed by cholera toxin and is inhibited by P----M conversion. The 41-kDa protein might represent the alpha-subunit of the G-protein, proposed to be part of the phototransduction mechanism [Blumenfeld, A. et al. (1985) Proc. Natl Acad. Sci. USA 82, 7116-7120]. P----M conversion leads to phosphorylation of opsin at multiple binding sites: up to 4 mol phosphate are bound/mol M formed. Dephosphorylation of the phosphate binding sites is induced by photoconversion of M to P. High levels of calcium (2 mM) inhibit phosphorylation of M and increase dephosphorylation of P. Protein patterns obtained by sodium dodecyl sulfate gel electrophoresis of irradiated retina membranes show an increased incorporation of label from [gamma-32P]ATP also into protein bands of 48 kDa, 68 kDa and 200 kDa. Binding studies reveal that in the case of the 48-kDa protein this effect is primarily due to a light-induced binding of the protein to the photoreceptor membrane. The binding of the 48-kDa phosphoprotein is reversible: after M----P conversion the protein becomes extractable by isotonic buffers. These data suggest that in rhabdomeric photoreceptors of invertebrates light-activation of rhodopsin is coupled to an enzyme cascade in a similar way as in the ciliary photoreceptors of vertebrates, although there may be differences, e.g. in the type of G-protein which mediates between the activated state of metarhodopsin and a signal-amplifying enzyme reaction.

Adenosine Diphosphate Ribose↗
PubMed2007

Identification of phosphorylated proteins.

Reversible protein phosphorylation is crucially involved in all aspects of plant cell physiology. The highly challenging task of revealing and characterizing the dynamic protein phosphorylation networks in plants has only recently begun to become feasible, owing to application of dedicated proteomics and mass spectrometry techniques. The experimental methodology that identified most of the presently known proteins phosphorylated in vivo is based on protein cleavage with trypsin, following chromatographic enrichment of phosphorylated peptides and mass spectrometric fragmentation and sequencing of these phosphopeptides. This procedure is most efficient when it is limited to the tryptic digestion of proteins in distinct isolated fractions or compartments of plant cells. Immobilized metal affinity chromatography (IMAC) is most useful for phosphopeptide enrichment after methylation of the peptides in the complex protein digests. The following tandem mass spectrometry of the isolated phosphopeptides results in both identification of phosphorylated proteins and mapping of the in vivo phosphorylation sites. The relative quantitation of the extent of phosphorylation at individual protein modification sites may be accomplished by either stable isotope labeling technique or dedicated liquid chromatography-mass spectrometry measurements.

Cell Fractionation↗
PubMed1990

Protein phosphorylation in developing and regenerating rat kidney.

Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with gamma-[32P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [3H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.

Animals↗