phosphothreonine and microcystin

A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.

Topics: Animals; Erythrocytes; Ferrets; Genistein; Magnesium; Marine Toxins; Microcystins; Molecular Weight; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Protein Phosphatase 1; Pyrazoles; Pyrimidines; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Staurosporine; Threonine

Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca(2+)](i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcium; Chickens; Enzyme Inhibitors; Gizzard, Avian; Humans; Kinetics; Microcystins; Models, Biological; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Myosin-Light-Chain Kinase; Peptide Fragments; Peptides, Cyclic; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Sequence Alignment; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

In this study, we report that the Src substrate Cas (p130 Crk-associated substrate) associates with protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We investigated this interaction in cells expressing a temperature-sensitive mutant form of v-Src. v-Src activation (by shifting cells from the nonpermissive to the permissive temperature) led to an increase in the tyrosine phosphorylation of v-Src and Cas, as well as in the association between v-Src and Cas. v-Src has previously been shown to bind to PP2A and to phosphorylate the catalytic subunit of PP2A, resulting in inhibition of phosphatase activity. We found that the association between v-Src and PP2A decreased as cells were shifted to the permissive temperature. In contrast, the levels of PP2A that co-immunoprecipitated with Cas increased when v-Src was activated. We obtained similar results in pull-down experiments with immobilized Microcystin, a PP2A inhibitor. Serine/threonine phosphorylation of Cas has previously been shown to occur in a cell cycle regulated matter. Treatment of NIH3T3 cells with okadaic acid, a PP2A inhibitor, augments the serine/threonine phosphorylation of Cas that occurs at mitosis. Furthermore, PP2A dephosphorylates serine residues on Cas in vitro. Taken together, our results suggest that PP2A may be involved in the cell cycle-specific dephosphorylation of Cas.

Topics: 3T3 Cells; Animals; Crk-Associated Substrate Protein; Enzyme Inhibitors; Mice; Microcystins; Mutation; Okadaic Acid; Oncogene Protein pp60(v-src); Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Phosphatase 2; Proteins; Retinoblastoma-Like Protein p130; Signal Transduction; Temperature

The product of the retinoblastoma susceptibility gene, pRB, is a demonstrated substrate for the type 1 serine/threonine protein phosphatases (PP1). Curiously, there has been a paucity of data supporting the idea that phosphorylated pRB can be found in a complex with PP1. To more fully characterize the association between these two proteins, we utilized a PP1-affinity chromatography approach to increase our ability to capture from mammalian cell lysate populations of pRB capable of binding to PP1. Western blot analysis of the bound proteins indicates that both faster migrating, hypophosphorylated pRB, as well as slower migrating, hyperphosphorylated pRB can bind. Phosphorylated pRB binding was confirmed by immunoprecipitation of eluted 32P-labeled pRB. In addition, Western blotting of eluted proteins with pRB phosphorylated-site-specific antibodies revealed select phosphorylated forms of pRB binding to PP1. Similar binding studies performed with toxin-inhibited PP1 indicate that catalytic activity of PP1 is not required for pRB binding. The significance of this finding with respect to the functional importance of this interaction is discussed.

Topics: Animals; Catalysis; Cell Line; Chlorocebus aethiops; Chromatography, Affinity; Cloning, Molecular; Enzyme Inhibitors; Microcystins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Phosphothreonine; Recombinant Fusion Proteins; Recombinant Proteins; Retinoblastoma Protein