Protein phosphorylation in plant mitochondria

Reversible phosphorylation of proteins is one of the most common regulatory mechanisms in eukaryotic cells and it can affect virtually any property of a protein. We predict that plant mitochondria possess 50–200 protein kinases (PKs), at least as many target proteins and 10–30 protein phosphatases although all will not be expressed at the same time in the same cell type or tissue. Presently available high-throughput methods for the identification of phosphoproteins and their phosphorylation sites are first reviewed and a number of useful databases listed. We then discuss the known phosphoproteins, PKs and phosphatases in plant mitochondria and compare with yeast and mammalian mitochondria. Three case stories—respiratory chain complex I, pyruvate dehydrogenase and formate dehydrogenase—are briefly considered before a final treatment of mitochondrial protein phosphorylation in intracellular signal transduction and programmed cell death.     

Zea mays CCaMK: autophosphorylation-dependent substrate phosphorylation and down-regulation by red light.

The role of protein kinases has been extensively studied in various signal transduction pathways and they are one of the most important components that link the signal perception to the final response. However, not many studies have been reported, especially from the plant systems, that show the regulation pattern of the kinase itself under different conditions. A calcium/calmodulin-dependent protein kinase has already been purified and characterized from etiolated maize coleoptiles. In this paper a detailed study of how the kinase itself is regulated at the autophosphorylation level is provided. Evidence is also given that the autophosphorylation of kinase effects its activity towards substrate phosphorylation. It is further shown that the kinase is an important component of the light signalling pathway as the level of the kinase itself decreases by red light irradiation.     

蛋白质可逆磷酸化调节植物细胞离子跨膜运动研究进展

蛋白激酶和蛋白磷酸酶催化的可逆磷酸化是植物细胞中多种信号转导途径中重要的组成因子.本文对蛋白质可逆磷酸化通过调节多种离子跨膜运动而参与植物细胞激发子信号途径、毒性物质诱导的钙离子内流、盐胁迫适应、气孔运动以及蛋白质可逆磷酸化参与胞外与胞内之间Ca2 状况信息传递,调节花粉管顶端Ca2 离子通道活性进行综述,以揭示蛋白质可逆磷酸化在植物细胞离子跨膜运动中的调控作用,为蛋白质可逆磷酸化调节植物生长发育、响应逆境胁迫等机理的研究提供参考.     

Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.     

Protein phosphatases and DNA tumor viruses: transformation through the back door?

Cellular transformation by many oncogenic viruses is mediated by alterations in signal transduction pathways that control normal growth and proliferation. Common targets for many transforming viruses are pathways regulated by protein phosphorylation. The biochemical control of proteins in these pathways is a dynamic process that is regulated by the relative activities of protein kinases and phosphatases. Although there are numerous examples of viral oncogenes that encode protein kinases (Hunter, 1991), until recently there has been no evidence linking altered phosphatase activity to transformation. In this review we describe a novel mechanism, utilized by small DNA tumor viruses, in which viral oncogenes bind to and regulate a cellular protein serine/threonine phosphatase. The currently available evidence indicates that alteration of phosphatase activity and subsequent changes in phosphorylation levels is an important step in transformation by these viruses.     

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.     

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.     

Protein kinase activity in Cucumis sativus cotyledons: effect of calcium and light

Light signals received by phytochromes in plants may be transduced through protein phosphorylation. Ca(2+) as second messenger was involved in phytochrome-mediated cellular events. Our experiments with Cucumis sativus cotyledons, treated with red (R) and far-red (FR) light, showed a stimulatory effect on in vitro protein phosphorylation of histone, added as exogenous substrate to the cotyledon extracts, and also modified the phosphorylation of endogenous polypeptides. The effect of light treatments was mimicked by the addition of Ca(2+) to the phosphorylation buffer, indicating phytochrome- and Ca(2+)-dependence on activity of some protein kinases (PKs). In-gel kinase assays were performed to characterize the PKs involved at the cotyledon stage of cucumber plants. Three proteins of about 75, 57 and 47kDa with PK activity were detected between M(r) markers of 94 and 45kDa. All three were able to phosphorylate histone and undergo autophosphorylation. However, only the 75 and 57kDa proteins autophosphorylated and phosphorylated the substrate in a Ca(2+)-dependent manner, and were inhibited when calmodulin (CaM) antagonists were added to the incubation buffer. Western-blot analysis with polyclonal antibodies directed against calcium-dependent protein kinase of rice (OsCDPK11) or Arabidopsis (AtCPK2) recognised 57 and 75kDa polypeptides, respectively. These results indicate the presence in cucumber cotyledons of at least two proteins (ca. 75 and 57kDa) with activity of PKs that could be calcium-dependent protein kinases (CDPKs). Both CDPKs could be modulated by phytochromes throughout FR-HIR and VLFR responses.     

Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia

Bacterial LPS have the ability to act as modulators of the innate immune response in plants. Complex and largely unresolved perception systems exist for LPS on the plant cell surfaces that lead to the activation of multiple intracellular defense signaling pathways. The aim of the present study was to investigate the perception mechanism of cultured Nicotiana tabacum cells towards LPS from Burkholderia cepacia (LPS(B.cep.)), with regard to the role of protein phosphorylation during signal perception-related responses to gain a better understanding of the chemosensory perception of LPS elicitor signals in plant cells. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins with the hyperphosphorylation of two proteins of 28 and 2 kDa, respectively. Significant differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. The protein kinase inhibitor, staurosporine, totally inhibited the extracellular alkalinization response induced by LPS(B.cep.), while the oxidative burst was only partially inhibited by staurosporine. Inhibition of protein phosphatase activity by calyculin A intensified the LPS(B.cep.) responses. The results indicate that perception- and signal transduction responses during LPS(B.cep.) elicitation of tobacco cells require a balance between the actions of certain protein kinases and protein phosphatases.     

Structure and functional features of enzymes from the Ca2+-phospholipid-dependent protein kinase family

Present day scientific data about the Ca(2+)-phospholipid-dependent protein kinases structure and molecular mechanisms of their activity are summarized and analyzed in this review. Ca(2+)-phospholipid-dependent protein kinases family is well known to include a whole series of enzymes which are homologous by their structure. They play an important role in cell differentiation, growth and proliferation as well as signal transduction through the cytoplasmic membrane. They also take part in cell response realization by phosphorylation of target proteins. Now application of modern biochemical and biophysical methods provided for possibility of the clarification of these enzymes structure features. The great lot of experimental data about the molecular mechanisms of Ca(2+)-phospholipid-dependent protein kinases activity regulation by phosphatydyle serine, phorbol ethers, saturated and unsaturated fatty acids, calcium iones, autophosphorylation and holoferment phosphorylation by other kinases was obtained. As a model of Ca(2+)-phospholipide-dependent protein kinases regulation was their development on the base of the scientific data about this problem.     

Regulation of plant symbiosis receptor kinase through serine and threonine phosphorylation

We studied the biochemical properties of a plant receptor-like kinase to gain insights into the regulatory mechanism of this largest class of plant kinases. SYMRK (symbiosis receptor kinase) is required for early signal transduction leading to plant root symbioses with nitrogen-fixing rhizobia and phosphate-acquiring arbuscular mycorrhizal fungi. Amino acid substitutions in positions critical for activity of other related kinases cause a nonsymbiotic plant phenotype, suggesting that SYMRK kinase activity is required for symbiosis. SYMRK is capable of intermolecular autophosphorylation. Nonphosphorylated SYMRK is less active than the phosphorylated version, suggesting the phosphorylation status of SYMRK determines its activity. Three Ser/Thr residues were identified as residues required for full kinase activation through targeted mutagenesis. Using quadrupole time-of-flight mass spectrometry analysis, two of these were confirmed to be phosphorylated in vitro. These crucial phosphorylation sites are conserved among various plant receptor-like kinases as well as animal Pelle/interleukin-1 receptor associated kinase. Despite the distinct domain architecture of receptor-like kinases versus Pelle/interleukin-1 receptor associated kinase, our results suggest the existence of conserved activation mechanisms.     

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.     

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.     

In vivo regulation of the dsRNA-dependent protein kinase PKR by the cellular glycoprotein p67

Regulation of eIF2alpha phosphorylation is critical to the maintenance of cellular homeostasis, and eIF2alpha kinases are subject to complex and multidimensional controls. A cellular 67 kDa glycoprotein (p67) has been proposed to have an important role in regulating the activity of eIF2alpha kinases including the interferon-induced, dsRNA-stimulated protein kinase PKR. To dissect p67-PKR interactions and evaluate their significance in vivo, we have used a vaccinia virus (VV) expression system that successfully mimics PKR control pathways. Recombinant VV were constructed that constitutively express p67 and inducibly express PKR in BSC-40 cells. Stable expression of p67 reduced the PKR-mediated antiviral response and apoptosis. These effects correlated with decreased eIF2alpha phosphorylation, with rescue of PKR-mediated inhibition of protein synthesis, and with partial inhibition of PKR-triggered activation of NF-kappaB. The direct interaction between PKR and p67 was suggested by in vivo and in vitro analyses. These data demonstrate that in vivo p67 is an important modulator of PKR-mediated signal transduction pathways and may provide a useful tool to dissect the relative contributions of PKR to cell growth and stress response.     

Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet-derived growth factor (PDGF) beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta-receptor and downstream components in the signal transduction pathway.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.