Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis.
The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.
Jan
A Y
AY
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8066, USA.
Johnson
E F
EF
Diamonti
A J
AJ
Carraway III
K L
KL
Anderson
K S
KS
D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009711:Nucleotides / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D009711:Nucleotides; D009842:Oligopeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C000715470:MAS1 protein, human