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.     

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.     

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;.     

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus,a developmental bacterium exhibiting social behavior

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like protein serine/threonine kinases in the prokaryotes. © 1993 Wiley-Liss, Inc.     

LKB1 in lung cancerigenesis: a serine/threonine kinase as tumor suppressor

Lung cancer is featured with high mortality, with a 15% five-year survival rate worldwide. Genetic alterations, such as loss of function of tumor suppressor genes, frequently contribute to lung cancer initiation, progression and metastasis. Liver kinase B1 (LKB1), as a serine/threonine kinase and tumor suppressor, is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Recent studies have provided strong evidences that LKB1 loss promotes lung cancerigenesis process, especially lung cancer progression and metastasis. This review will summarize recent progress on how LKB1 modulates the process of lung cancerigenesis, emphasizing on LKB1 downstream signaling pathways and biological functions. We will further discuss the potential development of prognostic biomarkers or therapeutic targets in lung cancer clinic based on the molecular alteration associated with deregulated LKB1 signaling.     

The serine/threonine kinases SGK1, 3 and PKB stimulate the amino acid transporter ASCT2

The human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2/SLC1A5) plays an important role in the transport of neutral amino acids in epithelial cells. The serine and threonine kinases SGK1-3 and protein kinase B have been implicated in the regulation of several members of the SLC1 transporter family by enhancing their plasma membrane abundance. The present study explored whether those kinases modulate hASCT2. In Xenopus oocytes heterologously expressing hASCT2, coexpression of constitutively active (S422D)SGK1, (S419D)SGK3 or (T308DS473D)PKB upregulated the transporter activity. The stimulation requires the catalytical activity of the kinases since the inactive mutants (K127N)SGK1, (K191N)SGK3, and (T308AS473A)PKB failed to modulate the transporter. According to kinetic analysis and chemiluminescence assays, SGK1 and SGK3 modulate hASCT2 by enhancing the transporter abundance in the plasma membrane. As SGK1, 3 and PKB are activated by insulin and IGF1, the described mechanisms presumably participate in the hormonal stimulation of cellular amino acid uptake.     

Expression of raf serine/threonine protein kinases in the cell bodies of primary sensory neurons of the adult rat

We have used immunocytochemistry to examine the distribution of raf protein kinases in sensory neurons of the adult rat. In lumbar and trigeminal sensory ganglia, cells of all size ranges appeared to be raf immunoreactive and this was confirmed by double labeling using subpopulation specific markers. Almost all cells labeled with Griffonia simplicifolia IB4 (a small cell marker) or immunostained by using a large cell marker (RT97) showed raf immunoreactivity. These two markers label cells known to differ in their expression of neurotrophin receptors (trk). Thus raf kinases are not confined to cells expressing only certain trk subtypes. In the dorsal horn of the spinal cord, raf immunoreactivity was present in scattered neurons. However, sensory axons identified by IB4 histochemistry were devoid of raf immunostaining. Lectin-labeled nerve fibers in the cornea, lower lip and glans penis were also not immunoreactive. Ligation of the sciatic nerve did not produce any accumulation of raf immunoreactivity, confirming that raf kinases are not axonally transported to the peripheral processes of sensory neurons. Surgical dissection of the sciatic nerve caused the normal homogeneous cytoplasmic raf immunoreactivity to be replaced in some cells by a staining concentrated predominantly under the plasma membrane. One possibility is that this represents activation of raf in these cells. Received: 2 October 1995 / Accepted: 4 March 1996     

Advances,challenges and tools in characterizing bacterial serine,threonine and tyrosine kinases and phosphorylation target sites.

Introduction: The cellular response to infection by bacterial pathogens involves a complex and highly regulated series of pathways that carry messages to various parts of the cell. These messages are transferred using post-translational modifications including phosphorylation by kinases. Understanding the host’s signaling pathways is valuable in identifying potential treatment targets, but the bacterial signaling pathways and host-pathogen crosstalk are equally important to the development of therapeutics.

Areas covered: This review summarizes some of the recent findings related to the bacterial phosphoproteome and especially serine/threonine/tyrosine sites, including methods and considerations for identifying novel phosphosites. We also consider the bioinformatics tools that have been developed to sift through the large volume of data in these studies and connect them to biologically relevant knowledge about pathways and function. Literature databases used include PubMed and Google Scholar from April 2018 to December 2018.

Expert opinion: While the field has developed significantly in the past decade of research, high-quality experimental sequence data remains the limiting factor to future research into bacterial phosphoproteomics. As more proteomes are explored, it will be easier to tailor tools and techniques to prokaryotes. It will be necessary to consider the phosphoproteome in the broader biological context, through interdisciplinary collaborations.     

Mr 25 000 protein,a substrate for protein serine/threonine kinases,is identified as a part of Xenopus laevis vitellogenin B1

A phosphorylated protein with a molecular mass of 25 000 (pp25) previously purified from the cytosolic fraction of Xenopus laevis oocytes is an effective phosphate acceptor for casein kinases and protein kinase C. In this study, based on the partial amino acid sequence of pp25, a cDNA was isolated that encodes a new yolk precursor protein, Xenopus vitellogenin B1, which contained the sequence encoding pp25. Both mRNA and protein of vitellogenin B1 were expressed in all of the female organs examined. In agreement with a previous report, the amount of vitellogenin B1 protein in the liver increased after stimulation with estrogen. These results suggest that pp25 is a cytosolic non-crystallized yolk protein nutrient source, but it might also play a role in rapid development.     

Effect of serine/threonine protein kinases and protein phosphatases inhibitors on mitosis progression in a synchronized tobacco BY-2 culture

In order to investigate the role of various serine/threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells, the influence of cyclin(olomoucine) and Ca²⁺/calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine), and a protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin-dependent protein kinases and protein kinase C caused a prophase delay, reduced the mitotic index, and displaced the mitotic peak as compared with control cells. Inhibition of Ca²⁺/calmodulin-dependent protein kinases enhanced the cells entry into prophase and delayed their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances synchronized BY-2 cells entering into all phases of mitosis.     

The role of serine/threonine and tyrosine protein kinases in the depression of cholinosensitivity in Helix lucorum neurons in the cellular correlate of habituation

Inhibitor ofadenylate cyclase (SQ 22,536) and inhibitors ofserin/threonine protein kinases A (PKA -Rp-cAMPS), G (PKG - H-Arg-Lys-Arg-Ala-Arg-Lys-Glu-OH), calcium/calmodulin-dependent kinase II (CaMKII - KN-93), p38mitogen-activated (MAPK - PD 169316), and tyrosine protein kinases (genistein), including their Src-family (PP2), weaken the depression of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior under conditions of rhythmical local acetylcholine applications to the soma in the cellular analogue of habituation. Selective inhibitor of protein kinase C (PKC - chelerythrine) does not change the depression of the ACh-current. Mathematical simulation of the influence of the inhibitors applied on a number of membrane-connected acetylcholine receptors made it possible to obtain the design curves consistent with the experimental curves of the ACh-current depression. The experimental data and the results of calculations allowed us to make the following assumptions. The reversible depression of sensitivity to ACh of command Helix neurons of defensive behavior in the cellular correlate of habituation depends on the decrease in the number of membrane-connected ACh receptors as a result of activation of several serine/threonine protein kinases: A, G, CaMKII, p38 MAPK (without the participation of PKC), and tyrosine protein kinases including the family of Src-kinases. The main targets of all protein kinases under study (excluding PKC) in command neurons are the proteins of cytoskeleton (actin microfilaments and microtubules). Phosphorylation of these proteins evokes polymerization and stabilization ofactin microfilaments, stabilization of the main microtubule protein tubulin, a change in the activity of motor proteins responsible for the speed of receptor endocytosis and exocytosis. The PKG action is indirect via the modification of actin-myosin interaction. Protein kinase A, CaMKII, and tyrosine Src-kinase phosphorylate also proteins activating receptor translocation into clathrin-coated membrane invaginations during endocytosis.     

Regulation of phenobarbital-induction of CYP2B and CYP3A genes in rat cultured hepatocytes: involvement of several serine/threonine protein kinases and phosphatases

We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3′:5′ cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca²⁺/calmodulin-dependent protein kinases II (independently of Ca²⁺) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.     

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1(PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC₅₀ = 0.16 μM) at a lower concentration than that of PPI (IC₅₀ = 1.7 μM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose-inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.