Viral serine/threonine protein kinases

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.     

Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.

Xenopus MAP kinase activator, a 45 kDa protein, has been shown to function as a direct upstream factor sufficient for full activation and both tyrosine and serine/threonine phosphorylation of inactive MAP kinase. We have now shown by using an anti-MAP kinase activator antiserum that MAP kinase activator is ubiquitous in tissues and is regulated post-translationally. Activation of MAP kinase activator is correlated precisely with its threonine phosphorylation during the oocyte maturation process. It is a key question whether MAP kinase activator is a kinase or not. We have shown that Xenopus MAP kinase activator purified from mature oocytes is capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. Dephosphorylation of purified activator by protein phosphatase 2A treatment inactivates its autophosphorylation activity as well as its activator activity. Thus, Xenopus MAP kinase activator is a protein kinase with specificity for both serine/threonine and tyrosine. Partial protein sequencing of purified activator indicates that it contains a sequence homologous to kinase subdomains VI and VII of two yeast protein kinases, STE7 and byrl.     

Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity

Tyrosine phosphorylation in plants could be performed only by dual-specificity kinases. Arabidopsis thaliana dual-specificity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors manganese dependent serine/threonine kinase domain, COG3642. His248 and Ser265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr208 in the TEY motif and Thr293 of the activation loop were converted to alanine. The conversion of Thr284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the first identification of a manganese dependent dual-specificity kinase and the importance of Thr208, Thr284, and Thr293 residues in the regulation of kinase activity.     

Serine/threonine protein kinases and apoptosis

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.     

The serine/threonine kinase Pim-1

The human pim-1 gene encodes a serine/threonine kinase, which belongs to the group of calcium/calmodulin-regulated kinases (CAMK). It contains a characteristic kinase domain, a so-called ATP anchor and an active site. In mouse and human, two Pim-1 proteins are produced from the same gene by using an alternative upstream CUG initiation codon, a 44 kD and another, shorter 34 kD form that both contain the kinase domain. Expression of Pim-1 is widespread and ranges from the hematopoietic and lymphoid system to prostate, testis and oral epithelial cells. Two other proteins with significant sequence similarities exist, Pim-2 and Pim-3; both are also serine/threonine kinases and have largely overlapping functions. Pim-1 is able to phosphorylate different targets, most of which are involved in cell cycle progression or apoptosis. Pim-1 expression can be induced by several external stimuli in particular by a number of cytokines relevant in the immune system, which led to the labeling of Pim-1 as a "booster" for the immune response.     

Serine/threonine protein kinases in Drosophila

The study of serine/threonine kinases in Drosophila is coming of age. Recently several kinases have been identified and their role in cell determination has been established. This review discusses these recent findings and describes the potential for genetic analyses of kinase activity and signal transduction.     

Brain protein serine/threonine phosphatases.

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.     

Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations.

Mitogen-activated protein kinase (p42mapk) becomes transiently activated after treatment of serum-starved murine Swiss 3T3 cells or EL4 thymocytes with a diversity of mitogens. Similarly, a meiosis-activated protein kinase (p44mpk) becomes stimulated during maturation of sea star oocytes induced by 1-methyladenine. Both p42mapk and p44mpk have been identified as protein-serine/threonine kinases that are activated as a consequence of their phosphorylation. Because homologous protein kinases may play essential roles in both mitogenesis and oogenesis, we have compared in detail the biochemical properties of these two kinases. We find that these kinases are highly related based on their in vitro substrate specificities, sensitivity to inhibitors, and immunological cross-reactivity. However, they differ in apparent molecular weight and can be separated chromatographically, indicating that the two enzymes are distinct. Furthermore, in the course of this investigation, we have identified a 44-kDa protein kinase in mitogen-stimulated Swiss mouse 3T3 cells and EL4 thymocytes that co-purifies with p44mpk and thus appears to be a closer homolog of the sea star enzyme. Analysis of these protein kinases clarifies the relationships between a set of tyrosine-phosphorylated 41-45-kDa proteins present in mitogen-stimulated cells (Martinez, R., Nakamura., K. D., and Weber, M. J. (1982) Mol. Cell. Biol. 2, 653-655; Cooper, J. A., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37), two myelin basic protein kinases identified in epidermal growth factor-treated Swiss mouse 3T3 cells (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494), and p42mapk. Our work points to the existence of a group of related serine/threonine protein kinases, regulated by tyrosine phosphorylation and functioning at different stages of the cell cycle.     

Arabidopsis PPP family of serine/threonine phosphatases

Serine/threonine-specific phosphoprotein phosphatases (PPPs) are ubiquitous enzymes in all eukaryotes, but their regulatory functions are largely unknown in higher plants. The Arabidopsis genome encodes 26 PPP catalytic subunits related to type 1, type 2A and so-called novel phosphatases, including four plant-specific enzymes carrying large N-terminal kelch-domains, but no apparent homologue of the PP2B family. The catalytic subunits of PPPs associate with regulatory protein partners that target them to well defined cellular locations and modulate their activity. Recent studies of phosphatase partners and their interactions have directed attention again to functional dissection of plant PPP families, and highlight their intriguing roles in the regulation of metabolism, cell cycle and development, as well as their roles in light, stress and hormonal signalling.     

Enzymatic breakdown of threonine by threonine aldolase

1. The enzyme which splits threonine to acetaldehyde and glycine has been partially purified from rat liver (five- to sixfold purification) and the name threonine aldolase proposed for it. 2. The general properties of threonine aldolase have been studied. The enzyme is unstable to a pH below 5. The pH optimum of the enzyme reaction is at 7.5-7.7. The initial rate of production of acetaldehyde is proportional to the enzyme concentration, and when the enzyme concentration is constant, the production of acetaldehyde is proportional to the time, provided that the substrate is in excess. The enzyme is inhibited by the carbonyl group reagent, hydroxylamine. Attempts to demonstrate that pyridoxal phosphate is a cofactor were unsuccessful. 3. The enzyme splits only L-allothreonine and L-threonine and is inactive against the D-forms of these amino acids. 4. The enzyme reaction on DL-allothreonine follows first order kinetics. From the first order velocity constants and the initial rates of the rates of the reaction at various substrate concentrations the Michaelis constant, Ks, for this substrate has been evaluated. Michaelis constants have also been determined for threonine. 5. The optimum temperature for the enzymatic breakdown of DL-allothreonine at pH 7.65 was found to be 50 degrees C. in phosphate buffer and 48 degrees C. in tris-maleate buffer. The rate of thermal inactivation of the enzyme threonine aldolase obeys a first order reaction. The heat of thermal inactivation was calculated by the aid of the van't Hoff-Arrhenius equation to be 43,000 cal. per mole for the temperature range 41.2-46.6 degrees C. 6. Equivalent amounts of acetaldehyde and glycine were formed from DL-allothreonine and the enzymatic breakdown of DL-allothreonine was found to be irreversible.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.     

Protein serine/threonine phosphatases; an expanding family

Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57-69% identity) than PP1 (45-49% identity), while PPY and PPZ are more similar to PP1 (66-68% identity) than PP2A (44% identity). The cDNA for PP2Bw encodes a novel Ca2+/calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.     

Genetic analyses of yeast protein serine/threonine phosphatases

Abstract Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threoninc phosphatases in yeast.     

RAF protein-serine/threonine kinases: Structure and regulation

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.     

丝氨酸/苏氨酸蛋白磷酸酶与DNA损伤应答

DNA损伤应答(DNA damage response,DDR)是维持基因组稳定性的核心机制,对DDR的研究不仅有助于阐明癌症发生发展的机理,同时也为癌症治疗和抗癌新药开发提供生物学基础。蛋白质翻译后修饰,尤其是蛋白激酶介导的磷酸化修饰和蛋白磷酸酶介导的去磷酸化修饰,参与调控绝大多数的生命活动过程,包括DDR。对蛋白激酶ATM/ATR/CHK2/CHK1介导的DDR的研究已经比较透彻,但是对蛋白磷酸酶在DDR中的功能研究还有待加强和深入。比较全面地综述丝氨酸/苏氨酸蛋白磷酸酶在DDR中的功能并探讨在抗癌新药开发中的前景。     

Serine/threonine kinases in the nervous system.

Three principal serine/threonine kinases that catalyze protein phosphorylation in response to second messengers are: cAMP-dependent protein kinase, multifunctional Ca2+/calmodulin-dependent protein kinase, and protein kinase C. Studies are now focusing on the distinct isoforms of these kinases that may subserve specific functions in some systems, and on providing a more molecular understanding of kinase functions. Combined genetic and biochemical approaches are beginning to be used to define unique roles for these kinases.     

Serine/threonine phosphatases in the nervous system.

The cloning and sequence determination of cDNAs encoding different types of serine/threonine protein phosphatases has provided a molecular basis for the protein phosphatase classification proposed by Ingebritsen and Cohen. Each of the phosphatases, phosphatase-1, -2A, -2B and -2C, exists as multiple isozymes raising the possibility that isozymes selectively expressed in different tissues may perform specific functions. The recent discovery of potent toxin inhibitors specific for protein phosphatase-1 and -2A will undoubtedly play an important role in the elucidation of the role of these enzymes in neuronal function.     

Serine/threonine ligation for the chemical synthesis of proteins

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Modulation of human neutrophil responses to CD32 cross-linking by serine/threonine phosphatase inhibitors: cross-talk between serine/threonine and tyrosine phosphorylation

The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.     

Nitric oxide regulates prolidase activity by serine/threonine phosphorylation

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time-dependent and dose-dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S-methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8-Br-cGMP, a cGMP agonist, and found that 8-Br-cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp-8-Br-pCPT-cGMP, an inhibitor of cGMP, reduced NO donor-stimulated prolidase activity to control levels. To test whether the MAPK pathway is involved in this NO-dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG-cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation.