Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco

A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.     

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.     

Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco

A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain. The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis. The spatial expression of the NTHK1 protein was then investigated. We found that NTHK1 was abundant during flower and ovule development. It was also expressed in glandular hairs, stem, and in leaves that had been wounded. The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees. Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants.     

Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.     

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.     

Recombinant human pim-1 protein exhibits serine/threonine kinase activity.

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.     

Modulation of serine/threonine protein kinase activity in chloramphenicol-resistant mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cmlr) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in early stationary growth. By labeling in vivo, the Cmlr mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cmlr mutant, and a Cmls revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K PK as pkn 24, pkn 32, pkn 13, pkn 12, and pkn 5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca2+-dependent PK 85K in the Cmlr mutant. It was assumed that PK 85K is involved in regulating the avermectin production.     

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.     

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39 °C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

G protein coupled receptor transactivation: extending the paradigm to include serine/threonine kinase receptors

The current paradigm of G protein coupled receptor signaling involves a classical pathway being the activation of phospholipase C and the generation of 1,4,5-inositol trisphosphate, signaling through β-arrestin scaffold molecules and the transactivation of tyrosine kinase growth factor receptors. Transactivation greatly expands the range of signaling pathways and responses attributable to the receptor. Recently it has been revealed that G protein coupled receptor agonists can also transactivate the serine/threonine kinase cell surface receptor for transforming growth factor-β (Alk5). This leads to the generation of carboxyl terminal phosphorylated Smad2 which is the immediate downstream product of the activated Alk5. Thus, the current paradigm of G protein coupled signaling can be expanded to include the transactivation of the serine kinase receptor Alk5. These insights expand the possibilities for outcomes of therapeutically targeting GPCRs where more substantive and prolonged actions such as the synthesis of extracellular matrix may be affected.     

Expression and characterization of the Streptomyces coelicolor serine/threonine protein kinase PkaD

We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.     

Neuronal functions of the novel serine/threonine kinase Ndr2

We have identified a novel member of the Ndr subfamily of serine/threonine protein kinases, Ndr2, as a gene product that is induced in the mouse amygdala during fear memory consolidation and examined a possible function of this kinase in neural differentiation. Expression of Ndr2 mRNA was detected in various cortical and subcortical brain regions, as well as non-neuronal tissues. Its expression in the amygdala was increased 6 h after Pavlovian fear conditioning training and returned to control levels within 24 h. To study intracellular localization and functions of Ndr2, EGFP::Ndr2 fusion proteins were expressed in rat pheochromocytoma (PC12) cells and acutely isolated cortical neurons, thereby revealing an association of Ndr2 with the actin cytoskeleton in somata, neurites and filopodia, in spines and at sites of cell contact. Co-precipitation and pull-down experiments support this finding. Evidence for an involvement of Ndr2 in actin-mediated cellular functions further comes from the observation of decreased cell spreading and changes in neurite outgrowth that were associated with protein serine phosphorylation in transfected PC12 cells. Together, our data suggest that Ndr2 is an interesting candidate gene for the regulation of structural processes in differentiating and mature neuronal cells.     

pp54 microtubule-associated protein-2 kinase requires both tyrosine and serine/threonine phosphorylation for activity.

pp54 microtubule-associated protein-2 (MAP-2) kinase, a recently discovered protein serine/threonine kinase (Kyriakis, J., and Avruch, J. (1990) J. Biol. Chem. 265, 17355-17363), is shown to contain immunoreactive phosphotyrosine residues. Treatment with recombinant rat brain protein tyrosine phosphatase-1 deactivates pp54 MAP-2 kinase, concomitant with the removal of phosphotyrosine residues. Protein (serine/threonine) phosphatase-1 also deactivates pp54 MAP-2 kinase in a specific fashion. pp54 MAP-2 kinase joins pp42 MAP-2 kinase and cdc2/maturation-promoting factor as one of only three serine/threonine protein kinases known to be regulated by phosphorylation at both tyrosine and, independently, at serine/threonine residues. In view of these shared regulatory properties, a role for pp54 MAP-2 kinase in the control of cell division is likely.     

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.