Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins.

Abstract

Human, murine and chicken c‐ets‐1 proteins migrate in SDS‐polyacrylamide gels as multiple species. We show here that most if not all of this heterogeneity is due to phosphorylation events occurring predominantly on serine and to a lesser extent on threonine residues. These phosphorylations can be specifically and rapidly stimulated by treatment with the calcium ionophore A23187 or abolished by lowering the extracellular calcium concentration to less than 0.1 microM. The products encoded by c‐ets‐2 are also phosphorylated in a Ca2+‐dependent manner, indicating that these modifications have been conserved in the products encoded by different members of the same gene family. In thymocytes, where the expression of c‐ets‐1 is elevated as compared with other cell types, c‐ets‐1 protein phosphorylation occurs after stimulation with mitogenic doses of concanavalin A, is short lived and is strictly dependent upon extracellular Ca2+ sources. This suggests that the c‐ets‐1 gene product may play a role in the Ca2+‐mediated early events linked to T‐cell activation.

IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells

“…These observations suggest integration between di erent T cell signaling pathways, mediated by the formation of Stat5 and Ets-1/2 protein complex in solution that can then form higher order oligomeric complexes on DNA. IL-2 activates Stat5 via activation of protein tyrosine kinases Jak1 and Jak3 associated with receptor components (Ihle et al, 1997;Lin and Leonard, 1997) whereas T cell activation results in calcium-dependent serine phosphorylation and activation of Ets-1/2 proteins (Bassuk and Leiden, 1997;Pognonec et al, 1988). Several studies have placed Ets-1 and Ets-2 in the Ras/MAPK signaling pathway (Co er et al, 1994;Miyazaki and Taniguchi, 1996;Rabault et al, 1996;Yang et al, 1996).…”

“…For competition experiments, unlabeled double-stranded oligonucleotides were pre-incubated with cell extracts for 10 min at RT prior to addition of the probe. Immunoassays were performed by incubating the binding reactions 30 min at 48C with antisera directed against Ets1/2 (#8), Fli-1 (#61), Tel (#69), Ets-1 (#49), Ets-2 (#36) (Bailly et al, 1994;Pognonec et al, 1988), GABPa, GABPb (de la Brousse et al, 1994), Stat3, Stat4, Stat6 (Transduction Laboratories), Elf-1, Sp1, Stat5a, Stat5b, Stat5 C17 which recognizes both Stat5a and Stat5b (Santa Cruz Biotechnology), as indicated in Figure legends. DNA binding proteins were isolated from whole cell extracts as described (Beadling et al, 1994).…”

Ets1 is an effector of protein kinase Cα in cancer cells

“…It disappeared when PKCa expression was fully re-established (lane 5). A decrease in the mobility of the Ets1 protein can be caused by phosphorylation (Pognonec et al, 1988;Ballschmieter et al, 2003). We, therefore, incubated SK-Mel protein extracts containing the slower migrating Ets1 form with alkaline phosphatase.…”

“…Ets1 is an effector of PKCa M Vetter et al Phosphorylation can have positive or negative effects on the activity of the Ets1 protein, depending on where phosphorylation on the Ets1 protein has taken place (Pognonec et al, 1988;Yang et al, 1996;Cowley and Graves, 2000;Seidel and Graves, 2002). Phosphorylation at the N-terminus by ERK1/2 leads to Ets1 superactivation (Yang et al, 1996;Seidel and Graves, 2002), whereas phosphorylation of the exon-VII domain by calcium or CaMKII results in inhibition of the Ets1 DNA-binding activity (Pognonec et al, 1988;Cowley and Graves, 2000). In previous studies, PKCa has been shown to interfere with the activities of ERK1/2 as well as with that of CaMKII.…”

“…Ras increases Ets1 transcriptional activity through ERK1/2 (Yang et al, 1996), which phosphorylate Ets1 at a single threonine within the N-terminal domain (Seidel and Graves, 2002). CaMKII is able to mediate calcium-dependent inactivation of Ets1 DNA-binding activity (Pognonec et al, 1988) by phosphorylating the exon VII domain of Ets1 (Cowley and Graves, 2000). DVII-Ets1 is devoid of this domain, rendering this natural splicing variant of Ets1 resistant to CaMKII.…”