Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts

Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1992-11, Vol.267 (32), p.22951-22956
Main Authors: SUSA, M, KEELER, M, VARTICOVSKI, L
Format: Article
Language:English
Subjects:
3T3 Cells
activation
Animals
Biochemistry & Molecular Biology
Biological and medical sciences
Cell Membrane - enzymology
Cell physiology
Cells, Cultured
Cytosol - enzymology
Detergents
Enzyme Activation
fibroblasts
Fundamental and applied biological sciences. Psychology
Kinetics
Life Sciences & Biomedicine
mediation
Mice
Molecular and cellular biology
phosphatidylinositol 3-kinase
Phosphatidylinositol 3-Kinases
Phosphatidylinositols - isolation & purification
Phosphatidylinositols - metabolism
Phosphotransferases - metabolism
platelet-derived growth factor
Platelet-Derived Growth Factor - pharmacology
Responses to growth factors, tumor promotors, other factors
Science & Technology
translocation
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)50039-9