Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry.
Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.
Lee
Thomas
T
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.
Hoofnagle
Andrew N
AN
Kabuyama
Yukihito
Y
Stroud
James
J
Min
Xiaoshan
X
Goldsmith
Elizabeth J
EJ
Chen
Lin
L
Resing
Katheryn A
KA
Ahn
Natalie G
NG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D013058:Mass Spectrometry / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment
D010455:Peptides; D020928:Mitogen-Activated Protein Kinases