Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity

Abstract

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates their signaling pathways. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by v-Src and Jak2, using a phospho-specific antibody against STAP-2 phosphorylated at Tyr250. Mutational analyses revealed that Tyr250 was involved in the STAT3-enhancing activity of STAP-2. We further found that leukemia inhibitory factor (LIF) or Jak2 expression stimulated STAP-2 Tyr250 phosphorylation in 293T and Hep3B cells. Moreover, endogenous STAP-2 was phosphorylated at Tyr250 following LIF stimulation of murine M1 cell line. Taken together, our findings demonstrate that endogenous STAP-2 is phosphorylated at Tyr250 and that this phosphorylation is involved in its function.4

STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation

“…Anti-STAP-2 antibody was purchased from Everest Biotech (Oxfordshire, UK). Anti-phosphoSTAP-2 Tyr250 was prepared as previously described [14].…”

“…To further probe the phosphorylation status of STAP-2 Tyr250, we also used a phosphospecific antibody against the Tyr250 site, designated anti-pSTAP-2 Tyr250, as described previously [14]. Myc-tagged wild-type STAP-2 (STAP-2 WT) or a series of STAP-2 YF mutants were expressed with or without Brk in 293T cells.…”

STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells

“…Expression vectors, c-Fms, epitope-tagged STAP-2 and its YF (substitution of Tyr to Phe) mutants were described previously [11,13,16]. Antibody against c-Fms, ERK1, and GST antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”

The protein content of an adaptor protein, STAP-2 is controlled by E3 ubiquitin ligase Cbl

“…Previously, we have shown that STAP-2 shows the characteristics of an adaptor protein that can interact with several other signaling molecules and that it regulates a variety of signaling pathways. However, only tyrosine-phosphorylation of STAP-2 has been demonstrated to modulate its functions [14]. In this study, we demonstrated, for the first time, a mechanism that regulates STAP-2 activity through Cbl-mediated proteasome-dependent degradation.…”

“…Human embryonic kidney carcinoma cell line 293T, human hepatoma cell line Hep3B and human cervix carcinoma cell line HeLa were maintained in DMEM containing 10% FCS. Stable transformants expressing Myc-tagged STAP-2 in Jurkat [3], HeLa [13] and Hep3B [14] were established as described previously and maintained in the above medium in the presence of G418 (0.5 mg/ml). 293T was transfected by the standard calcium precipitation protocol.…”