Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.     

The Drosophila orthologue of xPlkk1 is not essential for Polo activation and is necessary for proper contractile ring formation

Polo-like kinases (Plks) are essential for progression through mitosis. The activity of these kinases peak during M phase and this activation has been attributed to phosphorylation. Kinases capable of activating Plks in vitro have been previously identified both in mammalian cells and in Xenopus laevis oocytes (SLK and xPlkk1, respectively), although an in vivo correlation has not been clearly established. In order to study the regulation of Polo activity, we identified and cloned a Drosophila melanogaster kinase belonging to the ste20 ser/thr family that presents a close sequence homology with xPlkk1 and SLK. We termed this kinase dPlkk and showed that dPlkk associates with and phosphorylates Polo in vitro, resulting in the activation of the latter. On the other hand, when dPlkk is depleted from S2 cells, Polo activation does not seem to be impaired, suggesting that other kinases are involved in regulating Polo activity in vivo. Additionally, we found that a percentage of dPlkk-depleted cells fail to form a proper actin ring at the end of mitosis, leading to a failure in the assembly of the cleavage furrow and to the formation of binucleated cells. The detected accumulation of dPlkk in the contractile ring late in anaphase reinforces the idea that this kinase plays a role in cytokinesis.     

Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain

The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein.     

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac IKs

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K⁺ current (I_Ks) via unknown mechanisms. In the present study, I_Ks was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 μM H89; 200 μM H8; 50 μM H7; 1 μM bisindolylmaleimide I; 10 μM LY294002; 50 μM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 μM forskolin; 0.1 μM phorbol-12-myristate-13-acetate; 10 μM acetylcholine; 0.1 μM angiotensin II; 20 μM ATP), (iii) suppress G-protein activation (10 mM GDPβS), or (iv) disrupt the cytoskeleton (10 μM cytochalasin D), had little effect on the stimulation of I_Ks by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 μM) strongly attenuated both the hyposmotic stimulation of I_Ks in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.     

Aminoglycoside 2'-phosphotransferase IIIa (APH(2')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2' kinases

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2'-phosphotransferase IIIa (APH(2')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.     

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.     

Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.     

Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine

Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.     

SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.     

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca²⁺ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr⁶⁹⁷ and/or Thr⁸⁵⁵ (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser⁶⁹⁶ prevents phosphorylation at Thr⁶⁹⁷. However, the effects of Ser⁸⁵⁴ and dual Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵), Ser phosphorylation events (Ser⁶⁹⁶/Ser⁸⁵⁴) and dual Ser/Thr phosphorylation events (Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵). Dual phosphorylation at Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr⁶⁹⁷ and Thr⁸⁵⁵ by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵ in rat caudal artery, whereas U46619 induced Thr⁶⁹⁷ and Thr⁸⁵⁵ phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.