Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B.

Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by Galphai/o subunits.

A membrane-associated form of Raf-1 in v-Ras transformed NIH 3T3 cells can be inactivated by protein phosphatases regulated by GTP. Herein, a distinct protein-tyrosine phosphatase (PTPase) in membrane preparations from v-Ras transformed NIH 3T3 cells was found to be activated by guanyl-5'-yl imidodiphosphate (GMPPNP) and was identified as an effector for pertussis toxin (PTx)-sensitive G-protein alpha subunits. PTPase activation was blocked by prior treatment of cells with PTx.

Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not.

Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases.

The Raf-1 protein kinase participates in transduction of mitogenic signals, but its mechanisms of activation are incompletely understood. Treatment of human Raf-1 purified from insect Sf9 cells co-expressing c-H-Ras and Src(Y527F) (in which phenylalanine replaces tyrosine at residue 527) with either serine-threonine or tyrosine phosphatases resulted in enzymatic inactivation of Raf-1. Inactivation of purified Raf-1 was blocked by addition of either the 14-3-3 zeta protein or heat shock protein 90.

Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain.

Suppression of growth factor-mediated MAP kinase activation by v-raf in macrophages: a putative role for the MKP-1 phosphatase.

Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.

The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk.

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms.

Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.

The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase. Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown. The ability of preparations of plasma membranes to activate exogenous (His)6-Raf-1 was studied.

zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro.

The zeta isotype of protein kinase C (zeta PKC), a distinct PKC unable to bind phorbol esters, is required during NF-kappa B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by zeta PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es) with zeta PKC. The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained.

Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf.

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.

Methotrexate for rheumatoid arthritis. Suggested guidelines for monitoring liver toxicity. American College of Rheumatology.

Methotrexate (MTX) has become an important drug in the treatment of rheumatoid arthritis (RA). The American College of Rheumatology convened a committee to assess the risks of development of clinically significant liver disease (CSLD) during MTX treatment, to evaluate the risk and role of surveillance liver biopsies, and to provide recommendations about monitoring patients for liver toxicity. The committee recommends obtaining liver blood tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase, albumin, bilirubin), hepatitis B and C serologic studies, and other standard tests including complete blood cell count and serum creatinine tests prior to starting treatment with MTX.

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292.

Development of a self-report functional status index for juvenile rheumatoid arthritis.

Objective: There are few functional indices available for juvenile rheumatoid arthritis (JRA). Our goal was to develop a reliable, valid and responsive self-report physical functional status index for individuals with JRA, ages 8-18 years.

Methods: Activity (item) generation by interview of children, parents, teachers, clinicians yielded 280 items.

An innovative model of system-linked community care: home-based traction as an alternative to institutional treatment.

Home-based traction is an alternative treatment to conventional hospital-based traction for children with orthopedic conditions such as congenital dislocated hip and Legg-Perthes disease. The application of a proposed theoretical model is used to describe the process of the home-based traction innovation. Creativity and innovation occasions a reconceptualization of stresses.

Dual phosphorylation and autophosphorylation in mitogen-activated protein (MAP) kinase activation.

p42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro.

Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate.

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated in cells stimulated with epidermal growth factor (EGF) and other agents. A principal pathway for MAP kinase (MAPK) activation by EGF consists of sequential activations of the guanine nucleotide exchange factor Sos, the guanosine triphosphate binding protein Ras, and the protein kinases Raf-1, MAPK kinase (MKK), and MAPK. Because adenosine 3',5'-monophosphate (cAMP) does not activate MAPK and has some opposing physiologic effects, the effect of increasing intracellular concentrations of cAMP with forskolin and 3-isobutyl-1-methylxanthine on the EGF-stimulated MAPK pathway was studied.

Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity.

Activation of the mitogen-activated protein kinase pathway in Triton X-100 disrupted NIH-3T3 cells by p21 ras and in vitro by plasma membranes from NIH 3T3 cells.

We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (MAPKs) are capable of direct biochemical manipulation in vitro. MAPKs p42mapk and p44mapk are activated in signal transduction cascade(s) initiated by occupancy of plasma membrane receptors for peptide growth factors, hormones, and neurotransmitters. One likely activation pathway for MAPKs consists of sequential activations of c-ras, c-raf-1, and a protein-tyrosine/threonine kinase, MAP kinase kinase.

A myofibrillar protein phosphatase from rabbit skeletal muscle contains the beta isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin.

A form of protein phosphatase-1 (PP1M), which possesses 25-fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase-1, was purified over 200,000-fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase-1 (PP1C) and was identified as the beta isoform of PP1C by amino acid sequencing.

Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.

Mitogen-activated protein (MAP) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors. MAP kinase and the protein kinase that activates it (MAP kinase kinase) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms (p35EC12, p3722W) of the c-Raf-1 protein kinase. The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro.