Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism.
Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892. Electronic address: ying-xin.fan@fda.hhs.gov.
Ying-Xin
YX
Fan
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892.
Lily
L
Wong
Bioinformatics Section, Laboratory of Neurogenetics, NIA, National Institutes of Health, Bethesda Maryland 20892.
Jinhui
J
Ding
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892.
Nikolay A
NA
Spiridonov
Department of Neuroscience, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, Maryland 21205.
Richard C
RC
Johnson
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892. Electronic address: gibbes.johnson@fda.hhs.gov.
Gibbes R
GR
Johnson
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases