Multiple regions of MAP kinase phosphatase 3 are involved in its recognition and activation by ERK2

Mitogen-activated protein kinase phosphatase 3 (MKP3) is a specific regulat or of extracellular signal-regulated protein kinase 2 (ERK2). Association o f ERK2 with MKP3 results in a powerful increase in MKP3 phosphatase activit y. To determine the molecular basis of the specific ERK2 recognition by MKP 3 and the ERK2-induced MKP3 activation, we have carried out a systematic mu tational and deletion analysis of MKP3. Using activation-based and competit ion-based assays, we are able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP 3 activation. Our results show that recognition and activation of MKP3 by E RK2 involves multiple regions of MKP3. Thus, the kinase inter action motif (KIM; residues 61-75) in MKP3 plays a major role (135-fold) for high affini ty ERK2 binding. The most important residue in the KIM sequence of MKP3 is Arg(65), which probably interacts with Asp(319) in ERK2. In addition to KIM , a unique sequence conserved in cytosolic MKPs (residues 161-177 in MKP3) also contributes to ERK2 binding (15-fold). However, these two regions are not essential for ERK2-induced MKP3 activation. A third ERK2 binding site i s localized in the C terminus of MKP3 (residues 348-381). Although deletion of this region or mutation of the putative ERK specific docking sequence ( 364)FTAP(367) in this region reduces MKP3's affinity for ERK2 by less than 10-fold, this region is absolutely required for ERK2-induced MKP3 activatio n.