Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Introduction of Impermeant Molecules into Synaptosomes Using Freeze/Thaw Permeabilization

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.     

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

Background  

Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.     

Phosphorylation of myelin proteins: Recent advances

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translational modification of myelin protein. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P₀), myelin-associated glycoprotein and 2′3′ cyclic nucleotide 3′-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites, of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed. Special issue dedicated to Dr. Marion E. Smith.     

Current technologies to identify protein kinase substrates in high throughput

Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.     

Signal transduction pathways through cytoprotective, apoptotic and hypertrophic stimuli: a comparative study in adult cardiac myocytes

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen‐activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress‐ activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro‐apoptotic concentration of H₂O₂ activates strongly both MAPKs and PI3K/Akt pathways. Insulin‐like growth factor‐1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro‐survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

蛋白质磷酸化作用的结构基础

蛋白质可逆磷酸化涉及到几乎所有细胞活动的调节.着重探讨了影响蛋白激酶作用专一性的几个因素和磷酸化影响蛋白质功能的结构基础及作用机制.     

Role of phosphorylation of porin (VDAC) in regulation of mitochondrial outer membrane under normal conditions and alcohol intoxication

The present work is an overview of the factors regulating permeability of the outer membrane of mitochondria and the state of the channels formed by porin (voltage-dependent anion channels, VDAC). According to the accumulated data, modulation of the outer membrane permeability can be induced by endogenous phosphorylation of VDAC channels. Different protein kinases, such as protein kinase A, protein kinase C, tyrosine protein kinase, hexokinase, glycogen synthetase kinase-3β (GSK-3β), Akt and p38 kinases, were shown to be involved in VDAC phosphorylation. Among these protein kinases, alcohol-induced stress-kinases, GSK-3β, Akt, and p38 identified in mitochondria may participate in phosphorylation of porin, modulation of VDAC conductance, and regulation of the outer membrane permeability.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Sphingosine activation of protein kinases in Jurkat T cells. In vitro phosphorylation of endogenous protein substrates and specificity of action.

Sphingosine displays multiple biochemical and biological effects, in particular inhibition and activation of protein kinases. To determine the predominant interaction of sphingosine with cellular kinases, the effects of sphingosine on endogenous protein phosphorylation in Jurkat T lymphoblastic cells were investigated in vitro. Sphingosine was found to cause prominent phosphorylation of a number of cytosolic proteins ranging in molecular mass from 18 to 165 kDa. Phosphorylation was calcium-independent. Phosphorylation of substrates was increased in response to concentrations of sphingosine as low as 10 microM and peaked at concentrations of 20-200 microM. Multiple lines of evidence suggested that sphingosine activated more than one protein kinase: 1) the concentration dependence on sphingosine differed from substrate to substrate, 2) phosphorylation of one group of substrates required ATP as the phosphate donor, whereas a second group showed no preference between ATP and GTP, and 3) phosphorylation of some substrates was inhibited by heparin, whereas other substrates were resistant. Activation of these kinases demonstrated a very specific requirement for D-erythro-sphingoid bases. DL-erythro-dihydrosphingosine was partially active, whereas DL-threo-dihydrosphingosine was not. Other related molecules such as stearylamine, sphingomyelin, and C2-ceramide were not active. Sphingosine-activated kinase(s) were distinct from protein kinase C, cyclic nucleotide-activated kinases, and calcium-dependent kinases. These observations demonstrate the existence of multiple sphingosine-activated protein kinases with high specificity for D-erythro-sphingosine, suggesting physiologic regulation of protein phosphorylation by sphingosine.     

The calcium‐dependent protein kinase CPK7 acts on root hydraulic conductivity

The hydraulic conductivity of plant roots (Lp_r) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post‐translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp_r of knockout Arabidopsis plants for four Ca²⁺‐dependent protein kinases. cpk7 plants showed a 30% increase in Lp_r because of a higher aquaporin activity. A quantitative proteomic analysis of wild‐type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp_r of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7‐dependent stability of specific membrane proteins.     

Cyclic nucleotide-dependent protein kinases of the rat pancreas

A cyclic GMP-dependent protein kinase, which catalyzes the phosphorylation of histones and protamine by ATP, was present together with a cyclic AMP-dependent protein kinase and a readily active protein kinase in the rat pancreas. These three protein kinases were separated by chromatography on DEAE-cellulose. The cyclic GMP-dependent protein kinase was relatively cationic and fragile. Upon activation by cyclic GMP, this kinase dissociated into a light catalytic subunit and a somewhat heavier cyclic GMP binding subunit. A crude 27,000 × g pancreas supernatant had two apparent K_a values for cyclic GMP of 2.10^?8 M and 3.10^?7 M. The possible relationships between protein kinases and enzyme secretion are discussed.     

Major Role of Epidermal Growth Factor Receptor and Src Kinases in Promoting Oxidative Stress-dependent Loss of Adhesion and Apoptosis in Epithelial Cells

A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H₂O₂-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H₂O₂ alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.     

2,3,7,8-tetrachlorodibenzo-P-dioxin induced alterations in protein phosphorylation in guinea pig adipose tissue

An in situ (explant tissue culture) model has been developed to study the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hormones, and growth factors either alone or in combination. In our model system, the effect of TCDD on protein phosphorylation was greatly affected by the presence or the absence of externally added D-glucose. In the presence of a physiologically relevant level of glucose (13.3 mM), TCDD clearly stimulated protein phosphorylation as in the case of in vivo data. However, in the absence of Dglucose TCDD clearly inhibited protein phosphorylation. On the other hand, TCDD reduced the glucose uptake activity in isolated adipose tissue either in the presence or absence of D-glucose (13.3 mM). Therefore, the TCDD-induced reduction of glucose transport does not appear to be related directly to the simultaneous rise in protein phosphorylation. For comparison, several agents which are known to affect protein phosphorylation were tested. These hormonal agents generally affected the TCDD-untreated adipose tissues in the manner expected from their known actions, indicating that this in situ model is an adequate system to study their independent actions. The TCDD-treated adipose tissue samples showed only mild or insignificant response to these hormonal stimuli. In terms of the changes in the pattern of protein phosphorylation activities, the action of TCDD appeared to resemble that of EGF and T3. Since under in situ conditions no agents such as EGF or T3 can be expected to be present, the observed TCDD-induced changes are suggestive of the basic intracellular changes in cellular activities. The types of TCDD-induced protein kinases appear to be protein tyrosine kinases and protein kinase C.     

Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes

Summary Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent α-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides. (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (K_c=0.2–0.3 mM Glc-6-P) were obtained when 1 P_i/subunit was introduced by cAMP dependent protein kinase, 0.5 P_i/subunit by synthase kinase and 0.8 P_i/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites. In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in K_c for Glc. 6-P to 4–5 mM at 1.85 P_i/subunit and to 20 mM at 3.3 P_i/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25). The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 P_i/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 P_i/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant. The rate of decrease in RI to 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and P_i. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.     

Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells.

Vasoconstrictors such as angiotensin II (ang II) stimulate vascular smooth muscle cell growth and share many signal transduction mechanisms with growth factors. Recently, growth factors have been shown to stimulate mitogen-activated protein (MAP) kinases, a family of serine/threonine protein kinases which phosphorylate pp90rsk, a cytosolic kinase that phosphorylates ribosomal S6 protein. We examined the effect of ang II on MAP kinase activity and phosphorylation. Ang II stimulated MAP kinase activity by 4-fold after 5 min exposure and also increased tyrosine phosphorylation of 42 kDa (74 +/- 41%) and 44 kDa (263 +/- 85%) proteins, shown to be pp42mapk and pp44mapk by Western blot analysis using a MAP kinase antibody. These results suggest that ang II-stimulated protein synthesis is mediated by a MAP kinase dependent pathway.     

Regulation of DNA fragmentation: the role of caspases and phosphorylation

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.