The IL-2 Receptor Promotes Lymphocyte Proliferation and Induction of the c-myc, bcl-2, and bcl-x Genes Through the trans-Activation Domain of Stat5

Abstract

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R β-chain (IL-2Rβ). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rβ except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.

Abrogating Cbl-b in effector CD8+ T cells improves the efficacy of adoptive therapy of leukemia in mice

“…To determine whether the concentrations of the combined IL-2 blocking antibodies used in these studies were efficiently blocking physiological levels of IL-2 in these experiments, we cultured CFSElabeled CTLL-2 cells, an IL-2-responsive cell line (37,38), with 2 ng/ml recombinant mouse IL-2 (r-mIL-2), and with either blocking antibody alone or with r-mIL-2 and a combination of both blocking antibodies. Three days later, CFSE dilution was measured to quantify CTLL-2 cell proliferation.…”

Uncoupling of Promitogenic and Antiapoptotic Functions of IL-2 by Smad-Dependent TGF-β Signaling

“…That is, mutated forms of IL-2R␤ that activate Shc but not STAT5 induce expression of promitogenic genes such as c-myc, cyclin D2, and cyclin E, as well as robust T cell proliferation (13,14). Similarly, mutant receptors that activate STAT5 but not Shc can mediate these same effects (15). Finally, mutant receptors that activate JAK1 and JAK3 but fail to recruit Shc or STAT5 are absolutely nonmitogenic (9,15).…”

“…Similarly, mutant receptors that activate STAT5 but not Shc can mediate these same effects (15). Finally, mutant receptors that activate JAK1 and JAK3 but fail to recruit Shc or STAT5 are absolutely nonmitogenic (9,15). Thus, the cell cycle apparatus in T cells is dually regulated by the Shc and STAT5 pathways, which therefore represent potential targets for inhibition by TGF-␤.…”

“…Thymidine incorporation assays were performed as described (15). Cell counts and percent viability were determined by trypan blue exclusion and light microscopy.…”

“…The chimeric receptor chains ␣␥wt, ␤␤325Shc ⌬P , and ␤␤325-Y510 have been described (14,15,59). DN-Smad3 was generated by PCR using an oligonucleotide that converted serines 422, 423, and 425 of human Smad3 to alanine (60).…”