Figure 4.

Auto-phosphorylation of PAK is necessary for GC activation by PAK. A, PAK did not phosphorylate GC-E. Purified GST-PAK2, Rac1-GTPγS, and/or GC-E-intra-His₆ were mixed as indicated. In each lane, γ-³²P-ATP was included. The positions of GST-PAK2 and GC-E-intra-His₆ are indicated on the right. B, PAK did not phosphorylate phosphatase-pretreated GC-E. C, Kinase-dead PAK1 could not stimulate GC-E and GC-D in cells. D, Phosphorylation state of purified PAK proteins. Purified GST-fusion proteins treated with or without calf intestinal alkaline phosphatase (CIP). These protein samples were then separated on SDS-PAGE and Western blotted with anti-phospho-Thr423-PAK1/phospho-Thr402-PAK2 antibody. E, Dephosphorylation of PAK abolished its stimulatory effect on GC. F. Purified GC-D-intra activation by purified PAK2 in the presence of purified Rac1-GTPγS or Cdc42-GTPγS. Purified PAK1(K298A/T423E) and phosphorylated-PAK1(K298A) could also activate GC-D-intra. Data are shown as mean ± s.d. of three experiments. **, P < 0.001; *, P < 0.005 (Student’s t-test).