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.     

Origin of insulin receptor-like tyrosine kinases in marine sponges

One autapomorphic character restricted to all Metazoa including Porifera [sponges] is the existence of transmembrane receptor tyrosine kinases (RTKs). In this study we screened for molecules from one subfamily within the superfamily of the insulin receptors. The subfamily includes the insulin receptors (InsR), the insulin-like growth factor I receptors, and the InsR-related receptors--all found in vertebrates--as well as the InsR-homolog from Drosophila melanogaster. cDNAs encoding putative InsRs were isolated from the hexactinellid sponge Aphrocallistes vastus, the demosponge Suberites domuncula, and the calcareous sponge Sycon raphanus. Phylogenetic analyses of the catalytic domains of the putative RTKs showed that the sponge polypeptides must be grouped with the InsRs. The relationships revealed that all sponge sequences fall into one branch of this group, whereas related sequences from mammals (human, mouse, and rat), insects and molluscs, and polypeptides from one cephalochordate, fall together into a second branch. We have concluded that (i) the InsR-like molecules evolved in sponges prior to the "Cambrian Explosion" and contributed to the rapid appearance of the higher metazoan phyla; (ii) the sponges constitute a monophyletic taxon, and (iii) epidermal growth factor (EGF)-like domains are present in sponges, which allows the insertion of this domain into potential receptor and matrix molecules.     

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.     

Calcium-induced alterations in the functioning of protein serine/threonine and tyrosine kinases in Streptomyces fradiae cells

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate sporulation. Using in vivo labeling, we demonstrate that in S. fradiae phosphorylation of some proteins are also influenced by Ca2+ added exogenously. Calcium ions at physiological concentration increase phosphorylation of multiple proteins on serine/threonine residues and suppress modification of a 140-kDa protein on tyrosine residues. Assay of protein kinases in situ demonstrated that Ca2+-induced differences in the pattern of protein phosphorylation in vivo are accompanied by Ca2+-dependent cessation of autophosphorylation of 140-kDa tyrosine kinase and by increased autophosphorylation of three serine/threonine kinases with molecular masses of 127, 65, and 31.5 kDa.     

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases.

In addition to providing the framework for peptide presentation, major histocompatibility complex class I (MHC-I) molecules can act as signal transducing molecules in lymphoid cells. Here we show that the mobilization of intracellular calcium, which follows crosslinking of MHC-I molecules on human B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine/threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells.     

Interaction of Bruton's tyrosine kinase and protein kinase Ctheta in platelets. Cross-talk between tyrosine and serine/threonine kinases.

The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCtheta; in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCtheta; upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCtheta; in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCtheta; activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCtheta; in platelets, in which PKCtheta; positively modulates activity of Btk, which in turn feeds back negatively upon PKCtheta;.     

A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases

Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery.     

Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.     

Receptors for the TGF-beta superfamily: multiple polypeptides and serine/threonine kinases

Members of the transforming growth factor beta (TGF-beta) superfamily of peptide growth factors have profound effects on the growth and differentiation of many cell types. Insights into the poorly understood mechanisms of action of these ligands have come from the recent molecular cloning of two types of high-affinity receptors - type II and type III - for TGF-beta superfamily members. The cell surface expression of the type III receptor, a membrane-bound proteoglycan, appears to modulate the binding of ligand to the type II receptor, which is a transmembrane serine/threonine kinase. These results provide evidence for interactions between different receptor types, and suggest that serine/threonine phosphorylation is an important element in TGF-beta-induced signalling.     

丝氨酸/苏氨酸蛋白激酶在结核分枝杆菌致病机制中的作用

丝氨酸/苏氨酸蛋白激酶(STPK)是一类真核细胞样的蛋白激酶,是分枝杆菌生长和代谢的重要调节因子,参与其多种细胞活动(如细胞和菌落形态、葡萄糖和谷氨酰胺转运、吞噬体-溶酶体融合、转录因子活性等)的调节.结核分枝杆菌编码产生11种STPK( PknA、PknB、PknD～PknL),可分为5群(ABL群、HED群、FIJ...     

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.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.     

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.     

Pentosan polysulfate,a potent anti HIV and anti tumor agent,inhibits protein serine/threonine and tyrosine kinases

The effect of nicotinamide-adenine dinucleotides (NAD⁺ and NADP⁺) on Ca²⁺ transport in rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD⁺ (2mM) or NADP⁺ (1 and 2mM) caused a significant inhibition of Ca²⁺ uptake following addition of 2mM ATP. Ca²⁺, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD⁺ (0.5–2mM) or NADP⁺ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca²⁺ uptake and release clearly weakened in comparison with the effects of NAD⁺ and NADP⁺. Meanwhile, ryanodine (10M), thapsigargin (10M) or oxalate (0.5mM) had no effect on Ca²⁺ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD⁺ on Ca²⁺ uptake and release. Thus, NAD⁺ and NADP⁺ had a potent effect on Ca²⁺ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD⁺ (NADP⁺) is a factor in the regulation of the nuclear Ca²⁺ concentration. (Mol Cell Biochem121: 127–133, 1993)     

Plant receptor-like serine threonine kinases: roles in signaling and plant defense

Plants are hosts to a wide array of pathogens from all kingdoms of life. In the absence of an active immune system or combinatorial diversifications that lead to recombination-driven somatic gene flexibility, plants have evolved different strategies to combat both individual pathogen strains and changing pathogen populations. The receptor-like kinase (RLK) gene-family expansion in plants was hypothesized to have allowed accelerated evolution among domains implicated in signal reception, typically a leucine-rich repeat (LRR). Under that model, the gene-family expansion represents a plant-specific adaptation that leads to the production of numerous and variable cell surface and cytoplasmic receptors. More recently, it has emerged that the LRR domains of RLK interact with a diverse group of proteins leading to combinatorial variations in signal response specificity. Therefore, the RLK appear to play a central role in signaling during pathogen recognition, the subsequent activation of plant defense mechanisms, and developmental control. The future challenges will include determinations of RLK modes of action, the basis of recognition and specificity, which cellular responses each receptor mediates, and how both receptor and kinase domain interactions fit into the defense signaling cascades. These challenges will be complicated by the limited information that may be derived from the primary sequence of the LRR domain. The review focuses upon implications derived from recent studies of the secondary and tertiary structures of several plant RLK that change understanding of plant receptor function and signaling. In addition, the biological functions of plant and animal RLK-containing receptors were reviewed and commonalities among their signaling mechanisms identified. Further elucidated were the genomic and structural organizations of RLK gene families, with special emphasis on RLK implicated in resistance to disease and development.