Activation of MAP kinase by a dual specificity Tyr/Thr kinase.
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Amino Acid Sequence↗
Biomedical subjects
P Dent
Publications and source records attributed to P Dent.
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. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal-muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase. The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035] and the previously characterised glycogen-associated form of protein phosphatase-1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.
Amino Acid Sequence↗
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. Furthermore, a bacterially expressed v-Raf fusion protein (glutathione S-transferase-p3722W) also activated MAP kinase kinase in vitro. These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase.
3T3 Cells↗
Ordered phosphorylation of p42mapk by MAP kinase kinase.
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.
Animals↗
Dissection of the protein kinase cascades involved in insulin and nerve growth factor action.
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Animals↗
Safety and immunogenicity of subcutaneous H. influenzae vaccines in 15-17 month-old children.
To evaluate the safety and immunogenicity of subcutaneously administered PRP and PRP-D, one hundred and one healthy 15 to 17 month-old children were randomly assigned to receive two subcutaneous doses, 2 months apart, of either vaccine. The incidence of local and systemic reactions did not differ significantly between the two groups. PRP antibody levels in the pre-immunization sera of 95% of subjects were below the level associated with immediate protection from Hib disease (less than 0.15 microgram/ml). After the first dose, 32% of PRP and 70% of PRP-D (p less than 0.001) recipients achieved antibody levels of greater than or equal to 0.15 microgram/ml, and 8% and 26% (p = 0.003) respectively achieved levels greater than or equal to 1 microgram/ml, a level believed to predict long-term protection. After the second dose, antibody levels greater than or equal to 0.15 microgram/ml were reached in 38% of PRP and 88% of PRP-D (p less than 0.001) recipients and levels of greater than or equal to 1 microgram/ml were reached in 22% and 78% respectively (p less than 0.001). The geometric mean levels of PRP antibody after immunization were significantly higher in the PRP-D group after both the first and second doses (p less than 0.001). These results suggest that subcutaneous administration of both vaccines is safe, but PRP-D results in significantly higher antibody levels compared to PRP. The levels were lower than what has been reported following intramuscular administration.
Antibodies, Bacterial↗
The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle.
The ability of insulin to promote the phosphorylation of some proteins and the dephosphorylation of others is paradoxical. An insulin-stimulated protein kinase is shown to activate the type-1 protein phosphatase that controls glycogen metabolism, by phosphorylating its regulatory subunit at a specific serine. Furthermore, the phosphorylation of this residue is stimulated by insulin in vivo. Increased and decreased phosphorylation of proteins by insulin can therefore be explained through the same basic underlying mechanism.
Amino Acid Sequence↗
Targetting of protein phosphatase 1 to the sarcoplasmic reticulum of rabbit skeletal muscle by a protein that is very similar or identical to the G subunit that directs the enzyme to glycogen.
The amount of protein phosphatase 1 (PP1) activity in rabbit skeletal muscle associated with membranes (predominantly sarcoplasmic reticulum) is similar to that bound to glycogen-protein particles. Membrane-vesicle-associated (sarcovesicular) PP1 can be solubilised with 0.5% Triton X-100 (but not 0.5M NaCl) and is complexed to a protein that is structurally and functionally very similar or identical to the G subunit which targets PP1 to glycogen-protein particles. This conclusion is based on immunoblotting and immunotitration experiments using two different preparations of G-subunit-specific antibodies, binding of Triton-solubilised sarcovesicular enzyme to glycogen, stimulation of phosphorylase phosphatase activity by glycogen, phosphorylation of the same tryptic peptides by cyclic-AMP-dependent protein kinase (A-kinase) and release of catalytic subunit following phosphorylation by A-kinase. Membrane-association is not mediated via glycogen because sarcovesicular PP1 is (1) not released by digestion with alpha-amylase or at dilutions which fully dissociate the glycogen-bound enzyme, and (2) is solubilised by Triton X-100 (whereas glycogen-associated PP1 is not). These findings demonstrate that sarcovesicular PP1 is highly homologous to, or the same as, glycogen-associated PP1G and raises the possibility that a common targetting subunit may direct PP1 to different subcellular locations.
Animals↗
Identification of three in vivo phosphorylation sites on the glycogen-binding subunit of protein phosphatase 1 from rabbit skeletal muscle, and their response to adrenaline.
The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.
Amino Acid Sequence↗
Medical staffing in Ontario neonatal intensive care units.
Advances in technology have improved the survival rates of infants of low birth weight. Increasing service commitments together with cutbacks in Canadian training positions have caused concerns about medical staffing in neonatal intensive care units (NICUs) in Ontario. To determine whether an imbalance exists between the supply of medical personnel and the demand for health care services, in July 1985 we surveyed the medical directors, head nurses and staff physicians of nine tertiary level NICUs and the directors of five postgraduate pediatric residency programs. On the basis of current guidelines recommending an ideal neonatologist:patient ratio of 1:6 (assuming an adequate number of support personnel) most of the NICUs were understaffed. Concern about the heavy work pattern and resulting lifestyle implications has made Canadian graduates reluctant to enter this subspecialty. We propose strategies to correct staffing shortages in the context of rapidly increasing workloads resulting from a continuing cutback of pediatric residency positions and restrictions on immigration of foreign trainees.
Health Services Needs and Demand↗
Multisite phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic AMP-dependent protein kinase and glycogen synthase kinase-3.
The glycogen-binding (G) subunit of protein phosphatase-1G is phosphorylated stoichiometrically by glycogen synthase kinase-3 (GSK3), and with a greater catalytic efficiency than glycogen synthase, but only after prior phosphorylation by cyclic AMP-dependent protein kinase (A-kinase) at site 1. The residues phosphorylated are the first two serines in the sequence AIFKPGFSPQPSRRGS-, while the C-terminal serine (site 1) is one of the two residues phosphorylated by A-kinase. These findings demonstrate that (i) the G subunit undergoes multisite phosphorylation in vitro; (ii) phosphorylation by GSK3 requires the presence of a C-terminal phosphoserine residue; (iii) GSK3 can synergise with protein kinases other than casein kinase-2.
Amino Acid Sequence↗
The concentration of IgG in the serum is a major determinant of Fc-dependent reticuloendothelial function.
Defective Fc receptor-specific reticuloendothelial (RE) function has been reported in certain patients with a variety of immunologic and nonimmunologic diseases. The mechanism responsible for the impaired RE function is uncertain, but it could be caused by immune complexes that are present in many of these disorders. Alternatively, the impaired RE function could be a secondary effect of the high concentrations of monomeric IgG in the serum of these patients, since monomeric IgG can compete with complexed IgG for macrophage receptors in vitro. We studied the Fc-dependent RE function in 30 healthy control subjects and 27 patients using IgG-coated radiolabeled autologous red cells. There was a significant relationship between the concentration of IgG in the serum and the rate of clearance of antibody-sensitized cells (r = 0.51, P less than .01). Patients with hypergammaglobulinemia had the slowest Fc-dependent clearance, whereas those with hypogammaglobulinemia had the most rapid clearance. Immune complexes (Raji or polyethylene glycol) could not be shown to contribute to Fc-dependent RE clearance above the effect of the IgG in the serum. The unusually rapid clearance in a patient with hypogammaglobulinemia could be returned to normal by raising the concentration of IgG in the serum. This study supports the concept that serum (monomeric) IgG competes with immune complexed IgG for macrophage Fc receptors in vivo. The competition for Fc receptors determines the level of competence of Fc-dependent RE function. Based on the results of this study, one can predict that a number of disorders characterized by hypergammaglobulinemia also will have impaired Fc-dependent RE function.
Agammaglobulinemia↗
Differentiation therapy of human cancer: basic science and clinical applications.
Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed 'differentiation therapy.' This approach is based on the tacit assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment, results in tumor reprogramming and a concomitant loss in proliferative capacity and induction of terminal differentiation or apoptosis (programmed cell death). Laboratory studies that focus on elucidating mechanisms of action are demonstrating the effectiveness of 'differentiation therapy,' which is now beginning to show translational promise in the clinical setting.
Animals↗
Nurses 'n labour: education covers many areas.
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British Columbia↗