Mapping ERK2-MKP3 binding interfaces by hydrogen/deuterium exchange mass spectrometry.
ERK2, a prototypic member of the MAPK family, plays a central role in regulating cell growth and differentiation. MKP3, an ERK2-specific phosphatase, terminates ERK2 signaling. To understand the molecular basis of ERK2 recognition by MKP3, we carried out hydrogen/deuterium exchange mass spectrometry experiments to map the interaction surfaces between the two proteins. The results show that the exquisite specificity of MKP3 for ERK2 is governed by two distinctive protein-protein interactions. To increase the "effective concentration" of the interacting molecules, the kinase interaction motif in MKP3 ((64)RRLQKGNLPVR(74)) and an MKP3-specific segment ((101)NSSDWNE(107)) bind the common docking site in ERK2 defined by residues in L(16), L(5), beta(7)-beta(8), and alpha(d)-L(8)-alpha(e), located opposite the kinase active site. In addition to this "tethering" effect, additional interactions between the (364)FTAP(367) sequence in MKP3 and the ERK2 substrate-binding site, formed by residues in the activation lip and the P+1 site (beta(9)-alpha(f) loop), L(13) (alpha(f)-alpha(g) loop), and the MAPK insert (L(14)-alpha(1L14)-alpha(2L14)), are essential for allosteric activation of MKP3 and formation of a productive complex whereby the MKP3 catalytic site is correctly juxtaposed to carry out the dephosphorylation of phospho-Thr(183)/phospho-Tyr(185) in ERK2. This bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.
Zhou
Bo
B
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
Zhang
Jialin
J
Liu
Sijiu
S
Reddy
Sharanya
S
Wang
Fang
F
Zhang
Zhong-Yin
ZY
D000595:Amino Acid Sequence; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D054637:Dual-Specificity Phosphatases; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D054639:Mitogen-Activated Protein Kinase Phosphatases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006859:Hydrogen; D003903:Deuterium; D019950:Mitogen-Activated Protein Kinase 1; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054637:Dual-Specificity Phosphatases; C094132:Dusp4 protein, rat; D017027:Protein Tyrosine Phosphatases