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. 2015 Mar 13;43(7):3524–3545. doi: 10.1093/nar/gkv188

Figure 2.

Figure 2.

Structure of the STAT5A-1*6 lysine mutants. (A) Schematic representation of mouse wild-type and constitutively active (1*6) STAT5A proteins. STAT5A-1*6 presents two point mutations (H298R, S710F; *) responsible for its constitutive activity (66). STAT5A and STAT5A-1*6 cDNAs were subcloned into the pcDNA3 expression vector, in frame with a C-terminal FLAG tag (not shown), to allow protein detection by western blot. Specific lysine residues (K84, K359, K384, K675, K681, K689, K696, K700) were mutated to glutamine (Q) or to arginine (R) by site-directed mutagenesis. (B) Sequence alignment of the C-terminal SH2 and transactivation domains of mouse (m) and human (h) STAT5A and STAT5B highlighting conserved lysine residues (*). Lysine residues mutated within mouse STAT5A-1*6 C-terminal domain are specified (K675, K681, K689, K696, K700). Double (2xQ/R), triple (3xQ/R) and quintuple (5xQ/R) STAT5A-1*6 lysine mutants were also generated, as indicated. The essential and constitutively phosphorylated tyrosine Y694 of mouse STAT5A-1*6 was also mutated into phenylalanine (Y694F), as a control for STAT5A-1*6 inactivation. N-term., N-terminal domain; DBD, DNA binding domain; P-Tyr, phospho-tyrosine tail; TAD, transactivation domain.