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 regulator of extracellular signal-regulated protein kinase 2 (ERK2). Association of ERK2 with MKP3 results in a powerful increase in MKP3 phosphatase activity. To determine the molecular basis of the specific ERK2 recognition by MKP3 and the ERK2-induced MKP3 activation, we have carried out a systematic mutational and deletion analysis of MKP3. Using activation-based and competition-based assays, we are able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP3 activation. Our results show that recognition and activation of MKP3 by ERK2 involves multiple regions of MKP3. Thus, the kinase interaction motif (KIM; residues 61--75) in MKP3 plays a major role (135-fold) for high affinity 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 is 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 activation.
Zhou
B
B
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wu
L
L
Shen
K
K
Zhang
J
J
Lawrence
D S
DS
Zhang
Z Y
ZY
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D054642:Dual Specificity Phosphatase 6; D004789:Enzyme Activation; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism
D019950:Mitogen-Activated Protein Kinase 1; D054642:Dual Specificity Phosphatase 6; D017027:Protein Tyrosine Phosphatases