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1.
FEBS Lett ; 377(2): 123-7, 1995 Dec 18.
Article in English | MEDLINE | ID: mdl-8543033

ABSTRACT

We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51-55) from the same species. The encoded cDNA was expressed as a soluble GST-fusion protein in E. coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.


Subject(s)
Muscle, Smooth/enzymology , Peptide Fragments/metabolism , Phosphoprotein Phosphatases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , DNA, Complementary , Gene Expression Regulation, Enzymologic , Kidney/enzymology , Molecular Sequence Data , Muscle Contraction , Myosin-Light-Chain Phosphatase , Peptide Fragments/genetics , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 1 , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity , Swine
2.
J Biol Chem ; 270(41): 24540-7, 1995 Oct 13.
Article in English | MEDLINE | ID: mdl-7592672

ABSTRACT

We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 +/- 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carrier Proteins/metabolism , Muscle, Skeletal/enzymology , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/isolation & purification , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Glutathione Transferase/biosynthesis , Glutathione Transferase/metabolism , Kinetics , Mitogen-Activated Protein Kinase Kinases , Models, Structural , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , Phosphorylation , Plasmids , Point Mutation , Protein Kinases/biosynthesis , Rabbits , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Trypsin
3.
J Physiol ; 487 ( Pt 2): 283-9, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-8558463

ABSTRACT

1. Recombinant, activated mitogen-activated protein kinase (3.3 microM; p42mapk) phosphorylated caldesmon in phasic (rabbit portal vein) and tonic (rabbit femoral artery) smooth muscle strips permeabilized with Triton X-100. 2. Phosphorylation of caldesmon by p42mapk neither induced contraction of relaxed smooth muscle nor affected the Ca2+ sensitivity of submaximally contracted permeabilized phasic or tonic smooth muscle.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium/metabolism , Calmodulin-Binding Proteins/metabolism , Muscle Contraction/physiology , Muscle, Smooth, Vascular/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Enzyme Activation , Femoral Artery/physiology , In Vitro Techniques , Male , Mitogen-Activated Protein Kinase 1 , Octoxynol , Phosphorylation , Portal Vein/physiology , Rabbits
4.
J Biol Chem ; 269(37): 23185-91, 1994 Sep 16.
Article in English | MEDLINE | ID: mdl-8083223

ABSTRACT

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.


Subject(s)
Adipocytes/metabolism , Carrier Proteins , Insulin/pharmacology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Adipocytes/drug effects , Amino Acid Sequence , Animals , Intracellular Signaling Peptides and Proteins , Male , Mitogen-Activated Protein Kinase 1 , Molecular Sequence Data , Phosphorylation , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism
5.
J Biol Chem ; 269(17): 12804-8, 1994 Apr 29.
Article in English | MEDLINE | ID: mdl-8175693

ABSTRACT

Treatment of adipocytes with insulin or phorbol 12-myristate 13-acetate (PMA) results in transient activation of mitogen-activated protein kinase kinase (MEK) (Tmax = 90 s) and mitogen-activated protein kinase (MAPK) (Tmax = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid phasic kinetics that temporally precede that of MEK. Maximal activation of I-MEKK occurs within 20 +/- 5 s (S.D., n = 3) followed by complete inactivation by 30 +/- 10 s (S.D., n = 3). I-MEKK was characterized by anion-exchange and gel filtration chromatography and separated into two distinct activities of approximately 56 kDa that phosphorylated and activated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Protein phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. These results suggest activation of I-MEKK involves phosphorylation on serine or threonine residues. I-MEKK was not activated by PMA, suggesting that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.


Subject(s)
Adipocytes/enzymology , Insulin/pharmacology , MAP Kinase Kinase Kinases , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Enzyme Activation , Kinetics , Male , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase Kinases , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2 , Protein Serine-Threonine Kinases/isolation & purification , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-raf , Rats , Rats, Wistar , Tetradecanoylphorbol Acetate/pharmacology
6.
Eur J Biochem ; 214(2): 459-67, 1993 Jun 01.
Article in English | MEDLINE | ID: mdl-8513796

ABSTRACT

Recently, Sowadski and colleagues [Knighton, D.R., Zheng, J., Eyck, L.F.T., Ashford, V.A., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1991) Science 407, 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D.R., Eyck, L.F.T., Karlsson, R., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the alpha, beta and gamma phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [gamma-32P]adenosine-5'-(gamma-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its gamma phosphate. In the presence of 20 microM ATP, adenosine-5'-(gamma-4-aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 microM against cyclic A kinase, mitogen-activated protein kinase (p42mapk), mitogen-activated protein kinase kinase and p60c-src, respectively. To illustrate the effectiveness of adenosine-5'-(gamma-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity.


Subject(s)
Chromatography, Affinity , Muscles/enzymology , Phosphates/chemistry , Protein Kinases/isolation & purification , Sepharose/analogs & derivatives , Amino Acid Sequence , Animals , Binding Sites , Mitogens/pharmacology , Molecular Sequence Data , Molecular Structure , Molecular Weight , Protein Kinases/chemistry , Protein Kinases/metabolism , Rabbits , Sepharose/chemistry
7.
FEBS Lett ; 317(1-2): 12-6, 1993 Feb 08.
Article in English | MEDLINE | ID: mdl-8428620

ABSTRACT

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated by dual tyrosine and threonine phosphorylation in vivo. Both MAPKs are phosphorylated and activated in vitro by an activator recently identified as a protein-tyrosine/threonine kinase. We have isolated a putative cDNA for a MAP kinase kinase (MAPKK) and determined its structure [Proc. Natl. Acad. Sci. USA, in press]. The protein encoded by this cDNA shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this protein amplifies MAPK activator activity markedly. The increased activity co-migrates during chromatography with the expressed 45 kDa protein, recognized by an anti-STE7/byr1 antibody, and is abrogated by treatment with phosphatase 2A. Thus, this cDNA encodes a functional MAPKK. The anti-STE7/byr1 antibody also recognized a 46 kDa COS cell protein that was resolved from the expressed MAPKK by anion-exchange chromatography. This immunoreactive protein co-eluted with endogenous MAPKK activity, suggesting identification of the immunoreactive band as monkey MAPKK.


Subject(s)
Protein Kinases/genetics , Amino Acid Sequence , Animals , Antibodies/immunology , Cells, Cultured , Cloning, Molecular , DNA , Kidney/enzymology , Mitogen-Activated Protein Kinase Kinases , Molecular Sequence Data , Phosphorylation , Protein Kinases/immunology , Rats
8.
Proc Natl Acad Sci U S A ; 90(1): 173-7, 1993 Jan 01.
Article in English | MEDLINE | ID: mdl-8380494

ABSTRACT

MAP kinases p42mapk and p44mapk participate in a protein kinase cascade(s) important for signaling in many cell types and contexts. Both MAP kinases are activated in vitro by MAP kinase kinase, a protein-tyrosine and threonine kinase. A MAP kinase kinase cDNA was isolated from a rat kidney library by using peptide sequence data we obtained from MAP kinase kinase isolated from rabbit skeletal muscle. The deduced sequence, containing 393 amino acids (predicted mass, 43.5 kDa), is most similar to byr1 (Bypass of ras1), a yeast protein kinase functioning in the mating pathway induced by pheromones in Schizosaccharomyces pombe. An unusually large insert is present in MAP kinase kinase between domains IX and X and may contribute to protein-protein interactions with MAP kinase. Major (2.7 kilobases) and minor (1.7 kilobases) transcripts are widely expressed in rat tissues and appear to be derived from a single gene.


Subject(s)
Kidney/enzymology , Muscles/enzymology , Protein Kinases/genetics , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Brain/enzymology , Calcium-Calmodulin-Dependent Protein Kinases , Cloning, Molecular , DNA/biosynthesis , DNA/genetics , DNA/isolation & purification , Female , Gene Library , Male , Mitogen-Activated Protein Kinase Kinases , Molecular Sequence Data , Oligodeoxyribonucleotides , Organ Specificity , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Rats , Sequence Homology, Amino Acid
9.
FEBS Lett ; 306(1): 17-22, 1992 Jul 13.
Article in English | MEDLINE | ID: mdl-1628739

ABSTRACT

Preparation of milligram amounts of [32P]p42mapk, phosphorylated at Tyr185 or diphosphorylated at Tyr185/Thr183, for use as specific protein phosphatase substrates is described. Tyr- but not Thr-phosphorylated p42mapk, accumulates when ATP is limiting. Furthermore, Tyr185-phosphorylated p42mapk exhibits an apparent 10-fold decrease in apparent Km (46.6 +/- 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (approximately 476 nM). We conclude that Tyr185 precedes Thr183 phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42mapk for MAP kinase kinase.


Subject(s)
Protein Kinases/metabolism , Animals , Autoradiography , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Kinetics , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase Kinases , Muscles/enzymology , Phosphorylation , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Rabbits , Recombinant Proteins/metabolism , Substrate Specificity , Threonine/metabolism , Tyrosine/metabolism
10.
EMBO J ; 10(4): 877-83, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1826254

ABSTRACT

Molecular steps in endocytosis and degradation of the c-fms protein were analyzed by following the fate of mutated c-fms molecules after M-CSF binding. A mutant c-fms protein lacking tyrosine kinase activity was rapidly internalized after M-CSF binding but not degraded. Another mutant c-fms molecule that lacked most of the kinase insert region was similarly internalized after M-CSF binding and also not degraded. This indicates that the signal for internalization is separate from that directing degradation of the receptor. It has been shown previously that a c-fms mutant in which the kinase insert domain is deleted retains tyrosine kinase activity but lacks two major sites of autophosphorylation. The degradation step therefore requires both kinase activity and the kinase insert region whereas the internalization step is independent of these factors. The major sites of tyrosine autophosphorylation within the kinase insert region were next mutated to determine whether autophosphorylation in the kinase insert region of c-fms might be the signal that triggers degradation of internalized receptors. These mutant receptors were still rapidly degraded in response to M-CSF. Therefore, ligand-induced degradation of c-fms may require tyrosine phosphorylation of a protein other than the c-fms receptor itself and the kinase insert region may be necessary for recognition of this substrate.


Subject(s)
Macrophage Colony-Stimulating Factor/pharmacology , Protein-Tyrosine Kinases/metabolism , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Kinetics , Ligands , Mice , Phosphorylation , Receptor, Macrophage Colony-Stimulating Factor/genetics , Substrate Specificity
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