Activation of T lymphocytes to produce cytokines is

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine͞threonine phosphatases in STAT3 signaling in human antigen-specific CD4 ؉ T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1͞PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine͞threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine͞threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogenactivated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.STATs (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that upon activation translocate into the nucleus where they activate target genes (reviewed in ref. 1). At present, seven STATs have been cloned, all of which have an Src homology 2 domain near their carboxyl terminus and a tyrosine residue near position 700 (e.g., Y705 in STAT3). Upon ligation, cytokine and growth factor receptor-associated Janus kinases (JAKs) become activated, possibly by transphosphorylation and͞or autophosphorylation. Once activated, JAKs phosphorylate the receptor on key tyrosine residues, which leads to recruitment of STAT proteins, which in turn are tyrosine-phosphorylated by JAKs. Phosphorylated STAT proteins homodimerize or heterodimerize through reciprocal Src homology 2-phosphotyrosine interactions and translocate to the nucleus where they bind specific DNA elements and regulate transcriptional activity of target genes (reviewed in refs. 1-3).STATs also are serine-phosphorylated in response to ligation of many cytokine and growth factor receptors (reviewed in ref. 4). The major site for serine phosphorylation in STAT1 and STAT3 is residue 727 (5), allthough additional serine phosphorylation sites have been proposed (6). Serine phosphorylation of STAT proteins modulate the DNA binding and͞or transcri...

Studies with motheaten mice, which lack the SHP1 protein tyrosine phosphatase, indicate that this enzyme plays an important negative role in T cell antigen receptor (TCR) signaling. The physiological substrates for SHP1 in T lymphocytes, however, have remained unclear or controversial. To define these targets for SHP1 we have compared the effects of constitutively active and inactive mutants of SHP1 on TCR signaling. Expression of wild‐type SHP1 had a very small effect on the TCR‐induced tyrosine phosphorylation of ZAP‐70 and Syk, even when SHP1 was overexpressed 20 – 100‐fold over endogenous SHP1. Inactive SHP1‐D421A and wild‐type SHP2 were without effects. Constitutively active SHP1‐ΔSH2 had a more pronounced effect on ZAP‐70 and Syk, even when expressed at near physiological levels. SHP1‐ΔSH2 also inhibited events downstream of ZAP‐70 and Syk, such as activation of the mitogen‐activated protein kinase Erk2 and the transcriptional activation of the interleukin‐2 gene. In contrast, a constitutively active SHP2‐ΔSH2 had no statistically significant effect (although it caused a slight augmentation in some individual experiments). None of the constructs influenced the anti‐CD3‐induced tyrosine phosphorylation of the TCR ζ‐chain or phospholipase Cγ1, indicating that Src family kinase function was intact. Taken together, our findings support the notion that ZAP‐70 and Syk can be direct substrates for SHP1 in intact cells. However, the two SH2 domains of SHP1 did not facilitate its recognition of ZAP‐70 and Syk as substrates in intact cells. Therefore, we suggest that SHP1 is not actively recruited to inhibit TCR signaling induced by ligation of this receptor alone. Instead, we propose that ligation of a distinct inhibitory receptor leads to the recruitment of SHP1 via its SH2 domains, activation of SHP1 and subsequently inhibition of TCR signals if the inhibitory receptor is juxtaposed to the TCR.

Interleukin-4 (IL-4