Figure 4.

PI3KC2β regulates Rac and JNK activity. (A) PI3KC2β up-regulates Rac activity. Parental A-431 cells, and A-431 expressing wild-type PI3KC2β and kinase-dead PI3KC2β (D1213A-17, D1213A-32) were serum-starved for 24 h and stimulated with 1 nM EGF (+) for 5 min. The cell lysates were equalized for protein content before immunoprecipitation with GST-PAK CRIB. Immunoprecipitated and total Rac was detected by immunoblotting with a Rac mAb. The blots were quantified by densitometry. Data are mean with SD from four experiments. (B–E) A-431, A-431 cells expressing wild-type or kinase-dead PI3KC2β (D1213A-17, D1213A-32) were made quiescent by culturing in serum-free medium for 24 h before stimulation with 1 nM (C–D) or 8 nM (E) EGF for the times indicated. The Triton-soluble extracts were equalized for protein content, resolved on duplicate gels, and probed with the indicated antibodies. The extent of JNK, Jun, Erk, and Akt activation was assessed in the respective cell lines by immunoblotting with phospho-specific antibodies directed against the activated forms of the proteins. The amount of total protein was established by blotting the duplicate blots with antibodies against JNK, c-Jun, Erk, and Akt. The positions of the bands corresponding to JNK1 (p46) and JNK2 (p54) are indicated by arrows.