Figure 3.

Residues 201–300 of Wis1 MAPKK are required for binding and phosphorylation of Spc1 MAPK. (A) Wis1ΔN300 is an active protein kinase. Bacterially produced GST-ΔN300 and GST-DDΔN300 proteins were tested for their autophosphorylation activity in the presence of [γ-³²P]ATP. The samples were subjected to SDS-PAGE, followed by autoradiography. (B) Wis1ΔN300 cannot phosphorylate Spc1 in vitro. Activities of bacterially produced GST, GST-ΔN200, GST-ΔN300, or GST-DDΔN300 proteins were examined using GST-Spc1 as substrate. Phosphorylation of GST-Spc1 was detected by immunoblotting with anti-phospho-p38 antibodies. (C) Wis1ΔN300 cannot bind Spc1. Bacterially produced GST or different GST-Wis1 proteins were immobilized onto glutathione-beads and incubated with cell lysates from a spc1:myc strain (CA839). After extensive washes, proteins bound to the beads were analyzed with anti-GST and anti-myc antibodies.