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 prot ein which has been implicated in the pathogenesis of several human malignan cies. Although much has been learned about the physiological significance o f this receptor tyrosine kinase, its catalytic mechanism remains poorly und erstood. We have expressed, purified, and characterized two recombinant pro teins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an opti mal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degene rate 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-me thylanthraniloyl)-nucleotide derivatives provided direct measurements of th e association and dissociation rate constants for these nucleotide interact ions with the HER-2 recombinant proteins, thereby enabling the determinatio n of nucleotide k(d) values. Moreover, the actual step of chemical catalysi s was isolated using rapid chemical quench techniques and shown to occur ap proximately 3-fold faster than the steady-state rate which corresponds to p roduct release. Evidence is also provided that suggests a conformational ch ange that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was ex amined. Studies carried out with pn-phosphorylated recombinant HER-2 protei ns suggest that while autophosphorylation is not a prerequisite for enzymat ic activity, this protein modification actually directly affects the cataly tic mechanism by enhancing the rate of ADP release and that of the rate-lim iting 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 inves tigation of a receptor tyrosine kinase. This work serves as a basis for com parison of these two important protein kinase families and in this report w e highlight these similarities and differences.