Characterization of the MAP Kinase Inhibition Domain of ST5 p70

The ST5 gene encodes three proteins with molecular §masses of 126, 82 and 70 kDa. The 126 kDa protein §contains domains suggestive of a role in regulating §signal transduction pathways and activates ERK2 MAP §kinase in response to EGF stimulation. This activity §is suppressed by p70. More recently, p70 expression §has demonstrated the ability to suppress baseline §ERK2 activation. In order to identify the p70 domain §which mediates suppression of ERK2 activity, HA-§tagged p70 variants were constructed and used with §HA-tagged ERK2 to co-transfect COS-7 cells. The §effects of these variants on ERK2 activity were §determined by performing an in vitro kinase assay, §and comparing these results to the inhibitory §effects of full-length p70. The results suggest that §the critical ERK2 inhibition domain comprises not §only a MADD homology domain, but also includes amino §acid residues immediately downstream of this region. §This region contains no MAPK docking domains, which, §along with the inability of p70 to bind directly to §ERK2, suggest that p70 mediates ERK2 suppression §indirectly through a presently unidentified protein.