Activation of mitogen-activated protein kinase/ERK-2 in phytohaemagglutin in blasts by recombinant interleukin-2: contrasting features with CD3 activation
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R M Fairhurst1, M Daeipour, M C Amaral
1Department of Medicine, UCLA School of Medicine 90024-1680.
Abstract:
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.
Insights
Recombinant interleukin-2 (rIL-2) activates mitogen-activated protein (MAP) kinase in lymphoblasts, independent of protein kinase C. This activation requires tyrosine phosphorylation, highlighting its critical role in IL-2 signaling pathways.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- Mitogen-activated protein (MAP) kinase activation is crucial for lymphocyte proliferation.
- Interleukin-2 (IL-2) is a key cytokine in immune responses, regulating lymphocyte growth.
- Understanding IL-2 receptor (IL-2R) signaling pathways is vital for immune modulation.
Purpose of the Study:
- To investigate the activation mechanism of MAP kinase by recombinant IL-2 (rIL-2) in phytohemagglutinin (PHA)-stimulated peripheral blood lymphoblasts (PBL).
- To elucidate the role of tyrosine phosphorylation and protein kinase C (PKC) in IL-2-mediated MAP kinase activation.
- To identify intracellular signaling intermediates involved in IL-2R signal transduction.
Main Methods:
- Purification of MAP kinase activity from PHA-induced PBL lysates using ion-exchange chromatography.
- In vitro kinase assays measuring phosphorylation of MAP-2 and a myelin basic protein peptide.
- Analysis of protein tyrosine phosphorylation and comparison with CD3 receptor-mediated signaling.
Main Results:
- Recombinant IL-2 induced a dose-dependent, transient activation of MAP kinase (MAP-2K) in PBL.
- IL-2R-mediated MAP kinase activation occurred independently of protein kinase C (PKC) and extracellular Ca2+, unlike CD3 receptor activation.
- Recombinant IL-2 induced tyrosine phosphorylation of intracellular proteins, including a 40 kDa substrate, and this activation was blocked by a tyrosine protein kinase (TPK) inhibitor.
Conclusions:
- Tyrosine phosphorylation is a critical event in IL-2-mediated signal transduction.
- MAP kinase is a key cellular intermediate in the IL-2 signaling pathway.
- IL-2R signaling diverges from CD3 signaling regarding the involvement of PKC and Ca2+ in MAP kinase activation.