Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

Zhonghui Huang, Bo Zhou, Zhong-Yin Zhang
2004 Journal of Biological Chemistry  
The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr 183 and Tyr 185 in the activation loop. Tyr 185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a k cat /K m of 2.6 ؋ 10 6 M ؊1 s ؊1 . In
more » ... trast, the k cat /K m values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr 106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (ϳ20-fold increase in k cat /K m ) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (ϳ20-fold increase in k cat /K m ) by organizing the catalytic residues with respect to Tyr(P) 185 for optimal phosphoryl transfer.
doi:10.1074/jbc.m407820200 pmid:15466470 fatcat:zk46mqrck5fcln53xjvw5blx5q