Doreen A. Cantrell

Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.

The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)‐2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood‐derived T cells and the leukemic T cell line Kit225. An IL‐2‐induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL‐2. However, IL‐2 regulates the DNA binding and tyrosine phosphorylation of two STAT‐like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL‐2. The activation of STAT5 cannot be excluded, so the IL‐2‐activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR‐stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL‐2R‐mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL‐2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation.

INTERLEUKIN-2 ACTIVATION OF STAT5 REQUIRES THE CONVERGENT ACTION OF TYROSINE KINASES AND A SERINE/THREONINE KINASE PATHWAY DISTINCT FROM THE RAF1/ERK2 MAP KINASE PATHWAY

Interleukin‐2 (IL‐2) induces DNA binding of STAT5, a member of the family of cytokine‐regulated transcription factors termed ‘signal transducers and activators of transcription’. IL‐2‐stimulated STAT5‐DNA complexes include two tyrosine phosphoproteins which exhibit distinct mobilities in SDS‐PAGE gels. Our studies have shown that IL‐2 rapidly induces both tyrosine phosphorylation and serine phosphorylation of STAT5 and that the two STAT5 tyrosine phosphoproteins detected in IL‐2‐activated cells differ in their levels of phosphorylation on serine residues. The two different phosphoforms of STAT5 have identical in vitro DNA binding specificity and reactivity with tyrosine phosphopeptides, but differ in their cellular localization. As well, the present data indicate that the transcriptional activity of STAT5 is regulated by serine kinases in T lymphocytes. Two previously characterized serine kinases activated by IL‐2, MAP kinase/ERK2 and p70 S6 kinase, do not appear to be involved in STAT5 regulation by this cytokine. Accordingly, STAT5 activation in T cells requires the convergent action of tyrosine kinases and a distinct serine/threonine kinase which has not previously been implicated in IL‐2 signalling.

p21ras mediates control of IL-2 gene promoter function in T cell activation.

It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin‐2 (IL‐2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL‐2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL‐2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL‐2. Furthermore, using a dominant negative mutant of ras, Ha‐rasN17, we show that endogenous ras function is essential for induction of IL‐2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL‐2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.

Multiple pathways for the regulation of p21ras in T lymphocytes

Resumen del trabajo presentado al Keystone Symposia on Molecular and Cellular Biology, celebrado en Silverthorne, Colorado (US) del 26 de enero al 2 de febrero de 1992.