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. 2001 Mar 1;15(5):535–553. doi: 10.1101/gad.855801

Figure 3.

Figure 3

Figure 3

RhoA stimulates the phosphorylation and kinase activity of ERK6 (p38γ). NIH-3T3 cells were transfected with expression vectors containing (A) HA-tagged ERK2, JNK, ERK5, or (B) HA-tagged p38α or ERK6 along with Ras V12, RhoA QL, Rac1 QL, or Cdc42 QL. After serum starvation, lysates were immunoprecipitated with anti-HA antibody and used for kinase reactions. 32P-labeled substrates are indicated. Histograms represent the kinase activity relative to that in cells transfected with empty expression vector (control), whose value was taken as 1. Data represent the means ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three additional experiments. (Bottom) Expression of HA-tagged kinases in lysates from control and each indicated transfectant upon analysis by Western blot using a specific anti-HA antibody. (C) NIH-3T3 cells were cotransfected with HA-tagged ERK6 together with RhoA QL, RhoA N19, or MKK6. Twenty-four hours later, lysates were immunoprecipitated with anti-HA antibody and used for Western blot. Gels show phosphorylated ERK6 (p38γ) (top) and expression of HA-tagged ERK6 (bottom) from the same samples recognized by anti-phospho ERK6 and anti-HA specific antibodies, respectively. (D) Cells were seeded on coverslips and transfected as above with HA-tagged ERK6, together with AU5-tagged-RhoA QL, RhoA N19, Ras V12, or GST-tagged MKK6, or HA-tagged ERK2 along with AU5-tagged Ras V12, as indicated. Twenty-four hours after transfection, cells were transferred to serum-free medium for an additional 24 h, fixed, and analyzed by immunofluorescence for nuclear staining (DAPI, top) as well as for ERK6, ERK2, AU5, GST, and Ras with specific antibodies, as indicated (middle and bottom).