Figure 6.

PKC-dependent phosphorylation of ARNO does not positively regulate its ability to induce lamellipodia. A, HeLa cells expressing myc-tagged wild-type ARNO or ARNO(S392A) were metabolically labeled with [³²P]orthophosphate for 4.5 h. During the final 30 min PMA (+) or DMSO (−) were added to culture medium. The cells were then lysed and immunoprecipitated with ARNO antiserum as described in MATERIALS AND METHODS. The resulting immune complexes were transferred to nitrocellulose membranes and analyzed by autoradiography (upper panel) followed by immunoblotting with anti-myc antibody (lower panel). B, Purified His₆-ARNO was incubated with or without purified PKC for 30 min at 37°C, as described in MATERIALS AND METHODS. The PKC inhibitor BIM was included in one reaction at 10 μM. C, HeLa cells transfected with plasmid encoding myc-tagged wild-type ARNO or ARNO(S392A) were incubated with PMA, fixed, and labeled for immunofluorescence as in Figure 1. BIM was added to some cultures 10 min before the addition of PMA.