Activation of mitogen-activated protein kinase/ERK-2 in phytohaemagglutin in blasts by recombinant interleukin-2: contrasting features with CD3 activation.
We investigated activation of mitogen-activated protein (MAP) kinase, also known as microtubule associated protein-2 kinase (MAP-2K), by recombinant interleukin-2 (rIL-2) in phytohaemagglutinin (PHA)-induced peripheral blood lymphoblasts (PBL). MAP-kinase activation has been implicated in growth of lymphocytes and other cell types. Enzyme activity was purified from cell lysates by ion-exchange chromatography and activity measured by the ability to phosphorylate the substrates MAP-2 and myelin basic protein peptide (APRTPGGRR) in vitro. Recombinant IL-2 stimulated a variable (two-to 10-fold) and evanescent MAP-2K response which was dose dependent over the range 0-50 U/ml. In contrast to MAP-kinase activation by the CD3 receptor, activation by the IL-2 receptor (IL-2R) proceeded independently from protein kinase C (PKC) and extracellular-free Ca2+. MAP-kinase activation by CD3 involves an activation cascade which depends on Ca2+ influx and PKC activation. These events culminate in tyrosine phosphorylation and activation of MAP kinase. Recombinant IL-2 induced tyrosine phosphorylation of several intracellular proteins, including a 40,000 MW substrate which co-electrophoresed with ERK-2 on SDS-PAGE. The ERK-2 gene encodes a 41,000 MW MAP-2K and is subject to regulation by a variety of mitogens and growth factors in lymphocytes and non-lymphoid cells. MAP-kinase activation by rIL-2 was abrogated when PHA blasts were pretreated with the tyrosine protein kinase (TPK) inhibitor, methyl-2,5-dihydroxy-cinnamate. Although the TPK, p56lck, has been implicated in the activation of MAP kinase and the function of IL-2R, we found no mobility shift from a 56,000 to a 60,000 MW position as seen during PKC activation. Together these data suggest that tyrosine phosphorylation is critical to IL-2-mediated signal transduction and that MAP kinase is one of the cellular intermediates involved in this pathway.
Department of Medicine, UCLA School of Medicine 90024-1680.
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Fairhurst
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Daeipour
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Amaral
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D017252:CD3 Complex / Q000276:immunology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000276:immunology*; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D010835:Phytohemagglutinins / Q000276:immunology; D011494:Protein Kinases / Q000097:blood*; D017346:Protein Serine-Threonine Kinases / Q000097:blood; D011994:Recombinant Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D014443:Tyrosine / Q000097:blood
D017252:CD3 Complex; D007376:Interleukin-2; D010835:Phytohemagglutinins; D011994:Recombinant Proteins; D014443:Tyrosine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1