Evidence that protein tyrosine kinase p56-Lck regulates the activity of phosphatidylinositol-3'-kinase in interleukin-2-dependent T-cells.
The relative levels of phosphatidylinositol-3'-kinase (PI3K) activity were measured in interleukin-2 (IL-2)-dependent helper (HT-2) and cytolytic (CTLL-2) T-cell clones that had been stably transfected with expression plasmids encoding either the normal p56-Lck kinase or a mutant version of this kinase, p56-Lck(Y505F), that has constitutively high levels of kinase activity. Stimulation of untransfected T-cells or of transfected T-cells containing increased levels of normal p56-Lck resulted in an approximate doubling of the relative amounts of total cellular PI3K activity. In contrast, T-cells producing the activated version of p56-Lck contained levels of PI3K activity comparable to or slightly higher than those found IL-2-stimulated control cells, even in the absence of IL-2. Assessments of the relative levels of PI3K activity in immunoprecipitates prepared with the use of anti-phosphotyrosine-specific antibodies revealed constitutively high levels of anti-phosphotyrosine-immunoprecipitable PI3K activity in T-cells containing p56-Lck(Y505F), as opposed to T-cells containing normal p56-Lck where increases in anti-PY-immunoprecipitable PI3K activity were IL-2-inducible. IL-2 stimulation of T-cells containing the normal p56-Lck kinase resulted in marked increases in the relative amounts of PI3K activity and p85 that were coimmunoprecipitated when using anti-p56-Lck antibodies. In contrast, PI3K activity and the p85 subunit of PI3K could be coimmunoprecipitated from T-cells producing the activated p56-Lck(Y505F) kinase even in the absence of IL-2 stimulation, implying constitutive association of PI3K with the activated Lck kinase. Taken together with previous studies showing that IL-2 induces rapid increases in the activities of both p56-Lck and PI3K in T-cells, these findings suggest that p56-Lck lies immediately upstream of PI3K in a signal transduction cascade initiated by the binding of IL-2 to its specific receptor on T-lymphocytes.
Taichman
R
R
Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Merida
I
I
Torigoe
T
T
Gaulton
G N
GN
Reed
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D015151:Immunoblotting; D007376:Interleukin-2 / Q000494:pharmacology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D007376:Interleukin-2; D010455:Peptides; D011994:Recombinant Proteins; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)