PROTEIN TYROSINE PHOSPHATASES--MECHANISM OF CATALYSIS

Yeshiva University, New York NY

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Abstract

DESCRIPTION (provided by applicant): Protein tyrosine phosphorylation is a major post-translational modification mechanism that cells utilize to regulate various signal transduction pathways. The level of tyrosine phosphorylation, and thus the strength and duration of the signal transmitted, are balanced by the opposing action of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). This balance is perturbed in cancer cells, making the increased or altered activity of protein tyrosine kinases and PTPases a hallmark of tumor tissues. To understand the role of PTPase in signal transduction, it is necessary to have a detailed understanding of how they catalyze substrate dephosphorylation. The long-term goal is to elucidate the mechanisms of catalysis and regulation, and the structural basis for substrate recognition by PTPases. Understanding the molecular basis for protein dephosphorylation by PTPases will open doors to new experimental approaches (such as the creation o PTPases with altered catalytic and regulatory properties and the design and development of specific PTPase inhibitors) that will elucidate mechanisms by which these enzymes control cell functions. Knowledge of mechanism and structural features that govern substrate selectivity and regulation in a representative set of PTPases will establish general principles applicable to the study of all PTPases. The mitogen-activated protein (MAP) kinases are essential participants in signal transduction pathways from the cell membrane to the nucleus. While a significant effort has been placed on the mechanism of MAP kinase activation, much less is known about how MAP kinase activity is down regulated. Because activated MAP kinases contain both pThr and pTyr, they could serve as substrates for all classes of protein phosphatases. Thus, MAP kinase deactivation could occur through the action of serine/threonine protein phosphatases (e.g., PP2A), PTPases (e.g., HePTP), or dual specificity MAP kinase phosphatases (MKPs). The fact that multiple protein phosphatases are involved in MAP kinase inactivation suggests that phosphatases may play a crucial role in determining cellular responses to external stimuli. The MKPs belong to the PTPase superfamily and are capable of cleaving phosphoryl groups from both pTyr and pThr in the activation loop of MAP kinases, causing specific MAP kinase inactivation. MKPs differ from other well-characterized PTPases because they are inactive in the absence of their physiological substrates, the MAP kinases. Structural features of MKPs that mediate specific MAP kinase recognition and inactivation are not understood. The tyrosine specific HePTP inactivates MAP kinases by selective dephosphorylation of pTyr in activated MAP kinases. The molecular basis of specific MAP kinase inactivation by tyrosine specific PTPases is unknown. A multidisciplinary approach, involving a combination of mutagenesis, biochemical and cellular assays, steady state and pre-steady state transient kinetic analyses, protein interaction surface mapping by hydrogen/deuterium exchange and x-ray crystallography, will be used to 1) map the protein-protein interaction surface between extracellular signal-regulated protein kinase 2 (ERK2) and MKP3, 2) determine the mechanism of ERK2 dephosphorylation by MKP3, 3) define the role of MKP3-ERK2 protein-protein interactions in modulating MKP3 activity and specificity, and 4) reveal the molecular basis for ERK2 recognition and deactivation by the tyrosine specific PTPase HePTP.