Figure 6

Tyrosine phosphorylated Cas substrate domain blocks activation of JNK. a) NIH3T3 cells stably transfected with vector alone, the Src*/Cas(SD) chimera alone, v-crk alone or v-crk plus the Src*/Cas(SD) chimera were assayed for total cellular tyrosine phosphorylation (top panel). JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (middle and bottom panels). Lanes 1, 2 and 3, NIH3T3 vector transfected clones; lane 4 NIH3T3 transfected with the Src*/Cas(SD) chimera; lanes 5 and 6, NIH3T3 transfected with v-crk; lane 7, NIH3T3/v-crk cells transfected with the Src*/Cas(SD) chimera. Arrows are pointing to the Src*/Cas(SD) chimera (top panel), gst-c-jun (middle panel) and JNK (bottom panel). b) Cos-7 cells were transiently transfected with gst-JNK alone, or along with v-crk, or v-crk plus the Src*/Cas(SD) chimera. gst-JNK was immunoprecipitated and assayed for kinase activity against gst-c-jun in vitro (first panel). The expression levels of the gst-JNK, v-crk and the Src*/Cas(SD) chimera were determined as indicated. Arrows are pointing to the gst-c-jun substrate (first panel), gst-JNK (second panel), v-crk (third panel) and the Src*/Cas(SD) chimera (fourth panel).