Figure 1

Hck-YEEI models Csk-downregulated Hck in yeast. A) Domain organization of wild-type (WT) Hck and Hck-YEEI. Both kinases consist of an N-terminal unique domain (N), SH3 and SH2 domains, a kinase domain and a negative regulatory tail with a conserved tyrosine phosphorylation site. In wild-type Hck, the tail sequence is Tyr-Gln-Gln-Gln (YQQQ), and requires Csk for phosphorylation. In Hck-YEEI, this sequence was modified to Tyr-Glu-Glu-Ile (YEEI), which allows for Csk-independent downregulation in yeast. B) Yeast cultures were transformed with expression plasmids for wild-type Hck (WT), Hck-YEEI (YEEI) or the empty expression plasmid (−Hck). Cells were co-transformed with expression vectors for Csk (+) or the corresponding empty vector (−) as indicated. Cells were spotted onto agar selection plates containing galactose as the sole carbon source to induce kinase expression and incubated for 3 days at 30°C. Cultures were spotted in four-fold dilutions to enhance visualization of the growth suppressive phenotype. Plates were scanned and yeast patches appear as dark circles. C) Immunoblots from cultures shown in part B. Transformed cells were grown in liquid culture in the presence of galactose at 30°C for 18 h. Protein extracts were separated via SDS-PAGE, and immunoblotted for tyrosine-phosphorylated proteins (pTyr) as well as for Hck, Csk, and actin as a loading control.