Ubiquitination of nonphosphorylated Stat5A in vitro and identification of the region between amino acid residues 751 and 762 within the Stat5A C-terminal domain as the Stat5A ubiquitination signal. (A) Nonphosphorylated Stat5AY-F694 forming a heterodimer with tyrosine-phosphorylated wild-type Stat5A. Flag-tagged wild-type Stat5A (Stat5AFL) or Flag-tagged mutant Stat5A with Tyr-694 mutated to Phe (Stat5AY-F694FL) was coexpressed with Jak2 in the presence or absence of wild-type Stat5A in COS-7 cells. Cell lysates were immunoprecipitated with anti-Flag antibodies. Precipitated proteins were subjected to Western blot analyses with anti–phospho-Stat5 (α-pStat5) (top blot) or anti-Flag (α-Flag) (middle blot) antibodies. Cell lysates were incubated with the biotin-labeled double-stranded Stat5-binding oligodeoxynucleotides and avidin-conjugated Sepharose beads. Bound proteins were subsequently subjected to Western blot analyses with anti-Flag antibodies (bottom blot). (B) Ubiquitination of nonphosphorylated Stat5AY-F694 in vitro. Stat5AFL or Stat5AY-F694FL was coexpressed with wild-type Stat5A and Jak2 kinase in COS-7 cells. Cell lysates were incubated with the biotin-labeled double-stranded Stat5-binding oligodeoxynucleotides and avidin-conjugated Sepharose beads. Bound proteins were subjected to cell-free ubiquitination in vitro and ubiquitinated forms (*) were identified as higher-molecular-weight species detected by Western blot analyses with anti-Flag antibodies. (C) A schematic diagram of N-terminal Flag-tagged full-length (Flag-Stat5A) and carboxyl-truncated Stat5A at amino acid 773 (Flag-Stat5AΔ773), 762 (Flag-Stat5AΔ762), 751 (Flag-Stat5AΔ751), or 740 (Flag-Stat5AΔ740). (D) The region between amino acid 751 and 762 within the Stat5A C-terminal domain in the control of Stat5A ubiquitination. 32D (EpoR wt) cells expressing N-terminal Flag-tagged wild-type (WT) or carboxyl-truncated Stat5A as shown in panel A (Δ773, Δ762, Δ751, or Δ740) were stimulated with IL-3. The wild-type or carboxyl-truncated Stat5A proteins were pulled down from cell lysates using biotin-labeled double-stranded Stat5-binding oligodeoxynucleotides and avidin-conjugated Sepharose beads. Following cell-free ubiquitination in vitro, N-terminal Flag-tagged wild-type or carboxyl-truncated Stat5A proteins were subjected to SDS-PAGE and ubiquitinated forms (*) were identified as higher-molecular-weight species detected by Western blot analyses with anti-Flag antibodies.
