Protein tyrosine phosphatase function: the substrate perspective

Abstract

It is now well established that the members of the PTP (protein tyrosine phosphatase) superfamily play critical roles in fundamental biological processes. Although there has been much progress in defining the function of PTPs, the task of identifying substrates for these enzymes still presents a challenge. Many PTPs have yet to have their physiological substrates identified. The focus of this review will be on the current state of knowledge of PTP substrates and the approaches used to identify them. We propose experimental criteria that should be satisfied in order to rigorously assign PTP substrates as bona fide. Finally, the progress that has been made in defining the biological roles of PTPs through the identification of their substrates will be discussed.

TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk

“…We addressed the issue of specificity of the TULA-2 effect on the GPVI-mediated signaling pathway utilizing [H380C/E490Q]TULA-2, a substrate-trapping mutant of this PTP (39), to pull down proteins bound to this form of TULA-2 from the lysate of CVX-stimulated human platelets, because such binding is considered a key criterion of the PTP substrate identification (40). The precipitated proteins were then analyzed using immunoblotting with the antibodies to several individual Tyr(P) sites that are relevant for GPVI signaling along with the corresponding total proteins.…”

PTPL1: a large phosphatase with a split personality

“…This process has become easier within the last decade by an approach for substrate identification called "substrate-trapping," first described by Flint et al [34]. Substrate trapping identification of putative substrates requires rigorous experimental criteria for confirmation, as proposed by Tiganis and Bennett [35], to avoid false positive results. Thus far, IκBα is the only PTPL1 binding partner [26] that is also a putative PTPL1 substrate, identified in vitro using a cys to ser trapping mutant [36].…”

“…Each molecule is placed on PTPL1 to where they are described to bind as in the text Putative substrates of PTPL1. According to the guidelines suggested by Tiganis and Bennett [35] for the identification of bona fide PTP substrates, the evidence of a substrate has to be confirmed by three experimental criteria. Criterion 1 states that the presence of an enzymesubstrate complex has to be demonstrated both in vivo and in vitro.…”

Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K + channel KCa3.1

“…Direct binding of phosphatases (PT) to their target is one mechanism that sometimes determines PT specificity (10). To determine whether PHPT-1 physically associates with KCa3.1, we expressed Flagtagged KCa3.1 with GFP-tagged PHPT-1 in HEK 293 cells and determined whether the two proteins coimmunoprecipitate (3).…”