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Biomedical subjects

P H Lin

Publications and source records attributed to P H Lin.

7 recordsLinked to original sources
PubMed1992

Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis.

Phosphorylation of pp60c-src by p34cdc2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosis-specific decrease in Tyr-527 phosphorylation and half the increase in pp60c-src kinase activity. We conclude that p34cdc2 partially activates pp60c-src by a two-step process in which its serine/threonine phosphorylations either sensitize pp60c-src to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase. Furthermore, additional events, independent of these p34cdc2-mediated phosphorylations, participate in mitotic activation of pp60c-src.

3T3 Cells↗
PubMed1992

c-Src and mitosis.

The transforming potential and by inference the physiological function of the proto-oncoprotein pp60c-src closely correlate with the level of its protein tyrosine kinase activity. We have investigated the cell cycle-dependent regulation of this activity using mouse fibroblasts overexpressing chicken or mouse pp60c-src as a model system. During mitosis pp60c-src becomes phosphorylated at specific serine and threonine residues by p34cdc2. At the same time its tyrosine kinase activity, assayed in vitro, is increased approximately twofold and accessibility of its SH2 domain for binding relevant phosphotyrosine-containing ligands increases by about 15-fold. A kinase-defective mutant of pp60c-src exhibits a substantial (50-70%) decrease in phosphorylation at Tyr527 during mitosis. Phosphorylation of this residue negatively regulates kinase activity. Indirect evidence indicates a lesser decrease in wild-type pp60c-src Tyr527 phosphorylation during mitosis. Coordinate mutation of the mitosis-specific phosphorylation (MSP) sites in kinase-defective pp60c-src greatly reduces, though does not abolish, its mitosis-specific tyrosine dephosphorylation. Similarly, coordinate mutation of the three MSP sites in chicken pp60c-src or the corresponding two sites in mouse pp60c-src does not completely block mitotic stimulation of kinase activity. Thus, additional events beyond p34cdc2-mediated phosphorylation are involved in cell-cycle dependent regulation of pp60c-src activity. This is also suggested by the stimulation of pp60c-src kinase activity and decrease in phosphorylation of Tyr527 observed following treatment of fibroblasts with okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The potential role of cell cycle-dependent regulation of phosphatases and kinases acting on the regulatory tyrosine residue of pp60c-src is discussed.

Animals↗
PubMed1992

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Amino Acid Sequence↗
PubMed1991

Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins.

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.

Animals↗
PubMed1991

Isolation of cell membrane for epidermal growth factor receptor studies.

The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the purified cell membranes. The membrane purification procedure has been adapted by others in assessing EGF kinase activity and has been used for the purification of cell membranes from other types of cultured cells.

Animals↗
PubMed1991

Plasma uptake of manganese as affected by oral loads of manganese, calcium, milk, phosphorus, copper, and zinc.

Six adult subjects were administered a series of manganese (Mn) tolerance tests to investigate the influence of various minerals on Mn plasma uptake. Oral loads given to all six subjects included 40 mg manganese alone, or with 800 mg calcium (Ca) as either calcium carbonate (CaCO3) or 545 ml 2% milk. Four of the subjects also received loads of 800 mg phosphorus (P), 2 mg copper (Cu), and 50 mg zinc (Zn) with the 40 mg Mn. Baseline Mn tolerance tests for all subjects produced a rapid increase in plasma Mn, followed by return to baseline. The addition of Ca as either CaCO3 or 2% milk to the oral Mn essentially blocked the plasma uptake of Mn. No significant differences were found between the source of Ca in its inhibitory effect. Plasma Ca uptake was lower when Mn was simultaneously administered, but the results were not significantly different. Ionized levels of plasma Ca did not change significantly. The addition of Cu to the Mn load decreased the area under the curve for plasma Mn by about half, but it was not significantly different in the four subjects. In contrast, the addition of Zn to the Mn produced a significant increase in plasma Mn. Phosphorus has no influence on plasma uptake of Mn. These results indicate that the plasma uptake of Mn is greatly reduced by concomitant ingestion of Ca but may be increased by an oral load of Zn.

Administration, Oral↗