Substrate recognition domains within extracellular signal-regulated kinasemediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual spec ificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERR and the MKP-8 amino terminus with consequent phosphatase activation and dephos phorylation of the bound MAP kinase. We have used a series of p38/ERK chime ric molecules to identify domains within ERR necessary for binding and cata lytic activation of MKP-3. These studies demonstrate that ERK kinase subdom ains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK, p38/ERK chimeras possessing these regions display increased sensit ivity to inactivation by MKP-3. These data also reveal an overlap between E RR domains interacting with MKP-3 and those known to confer substrate speci ficity on the ERK RAP kinase. Consistent with this, we show that peptides r epresenting docking sites within the target substrates Elk-1 and p90(rsk) i nhibit ERR-dependent activation of MKP-3. In addition, abolition of ERR-dep endent phosphatase activation following mutation of a putative (k) under ba r inase (i) under bar nteraction (m) under bar otif (KIM) within the MKP-3 NH2 terminus suggests that key sites of contact for the ERK COOH-terminal s tructural lobe include residues localized between the Cdc25 homology domain s (CH2) found conserved between members of the DSP gene family.